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Proteolytic activity in plant tissue and cell suspension culture Nilsson, E. Kristina 1982

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PROTEOLYTIC ACTIVITY IN PLANT TISSUE AND CELL SUSPENSION CULTURE b y E. KRISTINA NILSSON B. Sc., The University of Western Ontario, 1 t A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES (Food Science Department) We accept this thesis as conforming to MASTER OF SCIENCE in the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1982 E. Kristina Nilsson, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of F~~ooE> AJOic The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date O^- 5 / ? f i IE-6 n/R-n ABSTRACT P r o t e o l y t i c enzymes are common i n p l a n t s but are u s u a l l y s p e c i f i to endogenous p r o t e i n . P l a n t proteases w i t h s p e c i f i c i t i e s a p p l i c a b l e to the food i n d u s t r y i n c l u d e papain, f i c i n and bromelain. Other p l a n t s have been used i n t r a d i t i o n a l methods of food p r e p a r a t i o n f o r t h e i r p r o t e o l y t i c a c t i o n on food components. The f o l l o w i n g species were i n v e s t i g a t e d f o r propagation i n t i s s u e c u l t u r e : • C a r i c a papaya, Ficu s c a r i c a , Cynara cardunculus, Galium verum, Circium arvense, D i e f f e n b a c h i a amoena, D. p i c t a and Ananas comosus. Tissues of the f i r s t f i v e of these demonstrated p r o t e o l y t i c a c t i v i t y by c l e a r i n g of m i l k t u r b i d i t y i n agar medium. Commercial papain and f i c i n preparations are c u r r e n t l y obtained from l a t e x of immature papaya and f i g f r u i t , r e s p e c t i v e l y . This i n v e s t i g a t i o n was conducted, i n p a r t , to determine the f e a s i b i l i t y of producing these two enzymes by the i n v i t r o c e l l c u l t u r e technique. Standard method of a s e p t i c seed germination and l e a f t i s s u e e x c i s i o n were employed f o r c a l l u s i n i t i a t i o n . C e l l suspension c u l t u r e s derived from c a l l u s were maintained i n B5 medium at 28 °C i n darkness. P r o t e o l y t i c a c t i v i t y was determined by a m o d i f i c a t i o n of the Food Chemicals Codex method f o r papain and p r o t e i n content was determined by Bradford's dye-binding, method. Production of p r o t e i n and protease v a r i e d among c e l l c u l t u r e s , but could be i n f l u e n c e d by changes to some n u t r i t i o n a l f a c t o r s . F i g c e l l s were grown i n medium supplemented w i t h s i n g l e amino acids i n the presence of e i t h e r n i t r a t e or ammonia as a source of inorganic n i t r o g e n . A l l ni t r a t e - b a s e d media produced higher y i e l d s of c e l l dry weight than ammonia-based media. Glutamic and a s p a r t i c a c i d s were most s t i m u l a t o r y i i i growth, protein accumulation and protease a c t i v i t y of f i g c e l l s . Skimmed milk, added at 3% (v/v), was a highly e f f e c t i v e growth stimulant, and also resulted i n higher protein and protease l e v e l s than the amino acids. Fresh casein and whey, added i n d i v i d u a l l y , produced s i m i l a r r e s u l t s to skimmed milk. C i t r i c a c i d , added at the l e v e l found i n the 3% milk supplement, also caused stimulation of f i g c e l l growth, protein synthesis and protease a c t i v i t y not s i g n i f i c a n t l y d i f f e r e n t from skimmed milk. It appears that nitrogen accumulation and reduction in f i g c e l l s may have been l i m i t e d by an energy requirement which could be s a t i s f i e d with the addition of c i t r i c acid or milk whey to the basal medium. i v CONTENTS page Abstract i i Table of Contents i v L i s t of Tables v L i s t of Figures v i Acknowledgements v i i I. Introduction 1 II . L i t e r a t u r e Review 1. Plant proteases: Description and use 6 2. Plant tissue and c e l l culture 14 II I . Materials and Methods 1. Callus culture of selected species 18 2. Suspension culture of f i g and papaya 23 3. Assay methods 24 4. Medium supplementation 30 IV. Results 1. Culture of plant tissues 33 2. Assessment of assay methods for biomass, protein and protease 40 3. Milk c l o t t i n g a c t i v i t y 53 4. Electrophoresis 57 5. Medium supplementation 58 V. Discussion 1. Tissue d e d i f f e r e n t i a t i o n 73 2. C e l l suspension cultures 75 3. Determination of biomass, protein and protease 76 4. Nitrogen n u t r i t i o n i n f i g c e l l cultures 86 5. Stimulatory e f f e c t s of skimmed milk and milk components 93 6. Applications, problems and po t e n t i a l of plant c e l l cultures 95 VI. Summary 101 References 104 Biographical information 114 V LIST OF TABLES page I. Products detected i n pla n t t i s s u e c u l t u r e 5 I I . C h a r a c t e r i s t i c s of the three major p l a n t proteases 7 I I I . Composition of B5 medium 19 IV. S u r v i v a l of pla n t species i n t i s s u e c u l t u r e 34 V. Drying methods f o r harvested f i g and papaya c e l l s 42 VI. C e l l e x t r a c t i o n methods 46 V I I . M i l k c l o t t i n g a c t i v i t y of papaya and f i g c e l l e x t r a c t s 56 V I I I . I n f l u e n c i n g p r o t e i n and protease synthesis w i t h a n t i b i o t i c s , detergent and s u l f u r compounds 70 v i LIST OF FIGURES page 1. Substrate residues i n papain active s i t e 10 2. Papain: t h i o l - d i s u l f i d e interchange 11 3. Establishment of papaya tissue and c e l l culture 20 4. Papaya c a l l u s and c e l l suspension culture 35 5. F i g explants and c a l l u s 36 6. Bedstraw tis s u e on agar and i n l i q u i d medium 37 7. Protein and protease v a r i a b i l i t y among papaya and f i g 39 8. H e r i t a b i l i t y of protein and protease p r o d u c t i v i t y 41 9. Settled c e l l volume as an in d i c a t o r of harvest weight 44 10. Growth of papaya and f i g i n B5: biomass and pH changes 45 11. Bradford's protein assay: standard curve 48 12. FCC protease assay: standard curve 52 13. E f f e c t of pH and temperature on p r o t e o l y t i c a c t i v i t y 54 14. Influence of some organic compounds on p r o t e o l y t i c a c t i v i t y 55 15. Inorganic nitrogen: e f f e c t on biomass, protein and protease 59 16. Amino acid supplements: e f f e c t on biomass 61 17. Amino acid supplements: e f f e c t on protein and protease a c t i v i t y 62 18. Proteins and peptides: e f f e c t on protease a c t i v i t y 64 19. Changes i n protein and protease of papaya and f i g c e l l suspension cultures over 3 weeks 65 20. Skimmed milk and components: e f f e c t on biomass 66 21. Skimmed milk and components: e f f e c t on protein and protease a c t i v i t y 68 22. Intra- and e x t r a - c e l l u l a r protein and protease i n f i g c e l l suspension cultures 72 v i i Acknowledgements The author wishes to express g r a t e f u l acknowledgement to the many friends and acquaintances who have d i r e c t l y or i n d i r e c t l y helped i n the planning, experimental, and w r i t i n g stages of t h i s work. Drs. P.M. Townsley and G.G. J a c o l i are responsible f o r f o s t e r -ing an i n t e r e s t i n , and enthusiasm f o r , plant ti s s u e and c e l l c u l t u r e . Drs. S. Nakai and I.E.P. Taylor were generous with t h e i r time to help with questions i n biochemistry and plant metabolism. Dr. B.J. Skura has h e l p f u l l y supplied ideas and new perspectives. Assistance of the following people was much appreciated: Mr. T. Kuwata and Mr. R. Yada for help with electrophoresis, Mr. D. Arm-strong of the UBC greenhouses f o r a q u i s i t i o n and i d e n t i f i c a t i o n of plant material, Miss L. Robinson f o r guiding me through data a n a l y s i s , and Mr. S. Yee for general technical and moral support. I w i l l always owe a great deal to my parents, Aino and Bjorn Nilsson, f o r teaching the value of education without misrepresent-a t i o n . I am also indebted to my husband, Robert Shadwick, who was la r g e l y responsible f o r my i n i t i a t i n g and completing t h i s p r o j e c t . He has t i r e l e s s l y provided assistance, consolation and d i s c i p l i n e as required. F i n a n c i a l assistance from an NSERC post-graduate scholarship, a UBC summer fellowship and the J.F. Morgan award was welcome, and a l l are g r a t e f u l l y acknowledged. 1 I. INTRODUCTION Many of the substances used i n the pharmaceutical, food, flavour and perfume in d u s t r i e s o r i g i n a t e from plants, and although the trend over the l a s t f i f t y years has been towards chemical synthesis, plants s t i l l remain an important source of many of these compounds, for a number of reasons. (1) The compounds are d i f f i c u l t and/or c o s t l y to synthesize. (2) Complex mixtures, eg. rose o i l , cannot be constituted s u c c e s s f u l l y by man. (3) I s o l a t i o n from a natural source circumvents many of the regulations which must be s a t i s f i e d before a chemically synthetic compound can be used commercially as a food or drink a d d i t i v e . (4) Chemical synthesis may, depending on the compound, r e s u l t i n a mixture of isomers which cannot be separated on a commercial s c a l e . This i s of obvious importance when the major desirable property resides with one isomer. The majority of commercially useful substances o r i g i n a t e from plants grown i n t r o p i c a l and sub-tropical regions of the world and the a v a i l a b i l i t y and cost of these materials i s frequently affected by p o l i t i c a l and economic considerations i n the countries of o r i g i n . These comments from a p u b l i c a t i o n by Yeoman et a l . (1980) concisely summarize the reasons for and j u s t i f i c a t i o n of research i n plant tissue and c e l l c u l t u r e . We have a dependence on plants to provide us with much more than energy and n u t r i e n t s . Most food systems are complex, including such minor components as pigments, gums, enzymes, e s s e n t i a l o i l s 2 and other flavour compounds, most of which are derived from plants. With the goal of s e l f - s u f f i c i e n c y , plant breeding programs are aimed at adaptation of plants to temperate climates or greenhouse growth (Bozzini, 1980). The time required for development of new c u l t i v a r s and hybrids was d r a s t i c a l l y reduced by tissue and organ c u l t u r e . The groundwork for t h i s approach had already been l a i d by researchers interested i n rapid methods of plant propagation, h y b r i d i z a t i o n , elimination of or resistance to pathogens (Reinert & Bajaj, 1977; Ingram & Helgeson, 1980). Several important compendia, containing a r t i c l e s dealing with these issues, have been edited by Barz et a l . (1977), Reinert and Bajaj (1977), Thorpe (1978), Sharp et a l . (1979) and Sala et a l . (1980). From plant development via tissue culture, the next l o g i c a l step i s to circumvent the t r a d i t i o n a l plant form and proceed d i r e c t l y to i s o l a t e the compounds of i n t e r e s t from plant c e l l s grown i n v i t r o (Klein, 1960). Commercial use of enzymes i s widespread: the food industry i s only one among many which also include photochemicals, tanning, cosmetics, pharmaceuticals, surgery, biochemical research and waste treatment. As a r e s u l t , research and development i n the enzyme f i e l d has been l a r g e l y di r e c t e d to improvements i n cost or e f f i c i e n c y of enzyme processes already i n use, rather than development of new enzyme sources or processes. In the food and beverage industry, approximately 12% of annual sales of enzymes are proteases derived from plants, the remainder being microbial i n o r i g i n . Plant enzymes may be recovered from by-products of plants harvested for other reasons. The great importance of enzymes to some food i n d u s t r i e s underlines the need for research into alternate sources or methods of production. There i s a plethora of microbial enzymes on the market, even a s e l e c t i o n to perform any one function. Most, i f not a l l , of 3 these are synthesized by selected s t r a i n s of micro-organisms grown i n large fermentation vessels under c o n t r o l l e d conditions of temperature, aeration, a c i d i t y , n u t r i t i o n and a g i t a t i o n . By contrast, plant enzyme production appears rather p r i m i t i v e , having progressed l i t t l e beyond primary ex t r a c t i o n , drying and improvement of storage s t a b i l i t y (Ortiz et a l . , 1980). Plant tissue and c e l l culture hold great promise with respect to i n d u s t r i a l production of plant products. The a r t of plant tissue culture began about t h i r t y years ago, and has since progressed to a young science, with respect to n u t r i t i o n a l and biochemical aspects. A knowledge of the physiology and c e l l metabolic a c t i v i t y i s necessary for each species and each plant product of i n t e r e s t , s i m i l a r to, but more complex than, harnessing micro-organisms. Useful compounds of plant o r i g i n generally f a l l i n t o one of two cla s s e s , primary or secondary metabolites. Primary metabolites include precursors, intermediates and endproducts of metabolism i n actively-growing c e l l s . These would be more c o r r e c t l y v i s u a l i z e d as i n t r a c e l l u l a r pools. Such materials are l i k e l y to be subject to c e r t a i n steady-state l e v e l s , so that an increase i n recovery of a c e r t a i n compound could only be achieved by means of an increased c e l l harvest. Improvements i n c e l l growth would then lead to increased t o t a l p r o d u c t i v i t y . The pharmaceutical industries have contributed most to our present knowledge of plant secondary metabolism. Many drugs and cosmetics are derived from rare plants, or synthesized ,by common plants i n such small quantities that large harvests and complex extraction schemes are required. Plant tissues and c e l l s i n culture have often been found to synthesize the desired materials, a l b e i t generally i n very small amounts. Because 4 secondary metabolites such as a l k a l o i d s are accumulated, e s p e c i a l l y i n mature and even senescent tissue, t h e i r presence i n plant tissue cultures provides impetus for improvements i n methodology. Aharonowitz and Demain (1980) argued against the d i s t i n c t i o n between primary and secondary metabolites, pointing out the p o s s i b i l i t y of multiple functions and concurrent production. A l l plant metabolism, they state, i s subject to c e r t a i n regulatory mechanisms. An understanding of regulatory points i n metabolic pathways may be used to influence synthesis or catabolism of any compounds. The recent CRC (Chemical Rubber Co.) p u b l i c a t i o n , Plant Tissue Culture as a Source of Biochemicals, edited by J . Staba (1980) provides ample evidence f o r i n t e r e s t and p o t e n t i a l i n t h i s f i e l d . Table I l i s t s many plant products i d e n t i f i e d i n cultures: asterisked items have been produced i n plant cultures i n quantities at l e a s t equal to the parent plants (Zenk, 1978). Table I Products detected i n plant cultures. (*) compounds produced i n quantities at least equal to intact plants (dry weight basis) References: Campbell, et a l . (1965); Heinstein & El-Shagi (1981); Khanna & Staba (1968); Misawa (1977); N i c k e l l (1980); Tabata, et al.(1978); Townsley (1974); Turnbull, et a l . (1980); Zenk (1978).. Class Examples a l k a l o i d s ajmalicine*, atropine, c a f f e i n e * , codeine, glycoalkaloids, indole a l k a l o i d s , morphine, n i c o t i n e , serpentine*, tropane a l k a l o i d s , vindoline antileukemic/antitumor agents campothecin, elephantin, maytansine, harringtonene, lolamarine, v i n c r i s t i n e a n t i m i c r o b i a l agents benzo-compounds carbohydrates cardiac glycosides enzymes ethylene foods; flavours; sweeteners fragrances/perfumes furano-compounds l i p i d s / o i l s miscellaneous medicinals organic acids phenolies pigments st e r o i d s , saponins vitamins plumbagin, and u n i d e n t i f i e d compounds in cultures of poplar, avocado, l e t t u c e , c a u l i f l o w e r and marijuana coumarin, g e n t i s i c acid, tocopherol, ubiquinone*, v a n i l l i c acid agar, sugars, polysaccharides, starch, l i g n i n cultures of D i g i t a l i s amylases, catalase, dehydrogenases, invertase, kinases, myrosinase, phosphatase, proteases, ribonuclease cultures of mung bean, soybean, rose, f l a x , wheat, r i c e cultures of c a r r o t , grape, tomato; aroma/flavours i n cultures of onion, l i c o r i c e , cocoa, coffee; m i r a l i n , stevioside g e r a n i a l , c i t r o n e l l o l v i snagin*, rutamarin mint anthraquinones*, plasmin i n h i b i t o r , ginsengoside*, L-dopa* ascorbic, chlorogenic, cinnamic, c i t r i c , fumaric, o x a l i c , shikimic, v a n i l l i c rosmarinic a c i d * , hydrangenol, putrescine (under study) anthocyanins, betanin, carotenoids, c h l o r o p h y l l , flavonoids, gossypol campestrol, c h o l e s t e r o l , diosgenin*, la n o s t e r o l , s i t o s t e r o l , squalene ascorbic a c i d , thiamine, vitamin K 6 I I . LITERATURE REVIEW 1. Plant proteases: Description and uses P r o t e o l y t i c enzymes f i n d uses i n the following food industry sectors: baking, brewing, protein hydrolysate production, cheese-making and meat processing. There i s also some i n t e r e s t i n t h e i r use i n waste treatment or reduction, such as hydrolysis of scleroproteins (Jones & Mercier, 1974; Yamamoto, 1975). C r i t e r i a for enzyme s e l e c t i o n for a p a r t i c u l a r purpose include s p e c i f i c i t y , h e a t - s t a b i l i t y , pH optimum and the possible presence of i n h i b i t o r s i n the enzyme preparation or the intended substrate. The major plant proteases currently employed are papain, bromelain and f i c i n , estimated to t o t a l over $16 m i l l i o n i n i n t e r n a t i o n a l sales i n 1980 (Wolnak, 1980). Papain i s i n use i n many food and non-food i n d u s t r i e s , described by Jones and Mercier (1974). They pointed out the i n s t a b i l i t y of crude enzyme preparations (dried latex) and the necessity for refinement and low-temperature storage. Appropriate f a c i l i t i e s are generally confined to large enzyme companies located i n North America, Europe or Japan, while the crude papain i s purchased mainly from t r o p i c a l nations. Ortiz et a l . (1980) investigated the storage and drying c h a r a c t e r i s t i c s of papaya latex. They reported an optimum drying temperature of 50-55°C. P r o t e o l y t i c a c t i v i t y declined i n the presence of sodium chloride while addition of EDTA to fresh latex had a preservative e f f e c t on a c t i v i t y . Table II summarizes properties of the three major plant proteases, with most information available on papain. A l l three enzymes are monomers containing at least one cysteine residue which must be i n a reduced form to be enzymatically a c t i v e . B a s i c a l l y , c a t a l y s i s depends on the formation of Table II C h a r a c t e r i s t i c s of the three major plant proteases cu r r e n t l y used i n the food industry. Data f o r t h i s table came from the following references: (1) Bergmeyer, 1974 (2) Murachi, 1970 (3) Liener & Friedensen, 1970 (4) Arnon, 1970 (5) Yamamoto, 1975 (6) Gould, 1975 (7) Englund, et a l . , 1968 (8) Tang, 1974 (9) Kunimitsu & Yasunobu, 1970 (10) S g a r b i e r i , et a l . , 1964 S i m i l a r i t i e s i n primary structure near the reac t i v e cysteine* papain Pro-Val-Lys-Asn-Gln-Gly-Ser-Cys-Gly-Ser-Cys*-Trp f i c i n Pro-Ile-Arg-Gln-Gln-Gly-Gln-Cys-Gly-Ser-Cys*-Trp bromelain Ser-Val-Lys-Asn-Gln-Asn-Pro-Cys-Gly-Ala-Cys*-Trp property papain f i c i n bromelain source molecular weight terminal residues substrates s p e c i f i c i t y temperature pH s t a b i l i t y P i i n h i b i t o r s active s i t e associated proteases notes N C latex of papaya f r u i t 21,000 (5,6) 23,000 (4) isoleucine asparagine proteins, peptides, esters basic carbonyl residues: Arg, Lys, Phe most active @ 50-60 °C, stable 30 min, 70 °C (5) optimum pH 5-7; stable pH 3-11(5), 3-9 (6) 8.75 (4) heavy metals, oxidants, sorbic acid (5) isothiocyanates (8) c l e f t i nvolving Cys„ s, A s p 1 5 g and H i s 1 5 9 IS) chymopapain A & B (4,5,9) lysozyme synthetic a c t i v i t y i n p l a s t e i n reactions (5) latex of f i g f r u i t 24-27,000 (3) 25,500 (7) leucine alanine proteins, peptides, esters Phe or Tyr carbonyl group most active @ 50-60 °C optimum pH 6-8; stable pH 3.5-9 (5) 9.0 (3) heavy metals, oxidants, sorbic acid (5) c l e f t involving cysteine (3) up to 10 active components (3, 10) f r u i t or stem f l u i d of pineapple 33,000 (1,2) v a l i n e glycine proteins, peptides, esters basic or aromatic carbonyl residues: Arg, Phe, Tyr most active @ 50-60 °C optimum pH 6-8, except gela-t i n (optimum pH 5) 9.55 (2) heavy metals, oxidants, sorbic acid (5) c l e f t involving cysteine and h i s t i d i n e (2) 5 isozymes (2) glycoprotein: 1.5-2.5% carb-ohydrate, bonded to Asn 9 an intermediate complex of the substrate a c y l moiety with the active cysteine residue. This has been elaborated upon v i a several avenues of exploration as reviewed by Polgar (1977) with respect to papain. In t h i s case, the enzyme c l e f t i s capable of binding seven amino acid residues. One of these i s the discriminating residue, second i n the N-terminal d i r e c t i o n to the point of cleavage ( F i g . 1). The imidazole group of H i s ^ g i s probably coupled with the t h i o l of Cys 25 forming the active nucleophile fo r substrate attack. The l i n e a r separation of these amino acids i s overcome by peptide f o l d i n g to produce the c a t a l y t i c c l e f t , possibly spanned by the discriminatory substrate residue. Brocklehurst and Kierstan (1973) proposed a zymogen-like mechanism for papain. They gave evidence suggesting that a t h i o l - d i s u l f i d e interchange e f f e c t s the conversion of i n a c t i v e propapain to the active enzyme configuration. The basic scheme for t h i s t r a n s i t i o n i s given i n Figure 2. Less i s known of the p r o t e o l y t i c action of f i c i n , but recent investigations indicate a mechanism very s i m i l a r to that of papain. Two s u l f h y d r y l groups have been demonstrated i n denatured f i c i n , but only one i n active f i c i n (Englund et a l . , 1968). Papain contains seven cysteine residues, only one of which i s a reactive s u l f h y d r y l , the other s i x being involved i n d i s u l f i d e bonds. S i m i l a r l y , f i c i n appears to have three d i s u l f i d e bonds, since the t o t a l h a l f - c y s t i n e value i s eight. Reports i n the l i t e r a t u r e indicate that f i c i n preparations are very heterogeneous (Kramer & Whitaker, 1964; Sgarbieri et a l . , 1964). For this reason, precise a c t i v i t y of commercial preparations i n complex systems cannot r e a d i l y be predicted. Englund and co-workers (1968) demonstrated that the m u l t i p l i c i t y of active f r a c t i o n s could be a t t r i b u t e d to a u t o l y t i c action which had not i n t e r f e r e d with p r o t e o l y t i c capacity of the enzyme. H 0 I II N - C - C I ,R H I ' N - C I r i 0 j H 0 j H H ! I n i l C f N - C - C j - N - C ,R DISCRIMINATING RESIDUE R 0 H II I C - N 0 II C - C I R1 SITE OF CLEAVAGE H O H 0 I II I II N - C - C - N - C - C igure 1. Substrate fragment accommodated by the papain active s i t e . Seven amino acid residues are held in the enzyme c l e f t . The second residue i n the N-terminal d i r e c t i o n from the point of cleavage, the "discrimination residue", i s p r e f e r e n t i a l l y hydrophobic. »-o From L. Polgar, 1977, J . Biochem. 8: 171-176. 11 CyS[63U CVS25, H 'S l59 — V — T V S H R - S or C N e S PROPAPAIN ' (22) Cys 1 2 2 1 Cys ' (63U V J ' : 3 25 cysK ° y s 25^ H ' S 1 5 9 v-r-Cys (63) (22) His 159 R - S e or C N e PAPAIN Figure 2. T r a n s i t i o n of propapain to papain: t h i o l - d i s u l f i d e interchange as the method of zymogen a c t i v a t i o n . From K. Brocklehurst & M.P.J. Kierstan, 1973 Nature, New Biology 242:167-170 12 Most current technical information on the bromelains i s outlined i n Table I I . Although stem and f r u i t bromelains are also s u l f h y d r y l proteinases, they are glycoproteins and d i f f e r from f i c i n and papain i n other respects. Esterase and amidase a c t i v i t i e s of the three enzymes indic a t e a fundamental di f f e r e n c e : both papain and f i c i n show approximately equal a f f i n i t y for corresponding synthetic ester and amide substrates while stem bromelain has a k c a t for BAEE (oC-N-benzoyl-L-arginine e t h y l ester) 140 times as large as the k c a t f o r BAA (oc-N-benzoyl-L-arginine amide), (Murachi,1970). Stem and f r u i t bromelains have h a l f - c y s t i n e values reported between 5 and 11, with only one reactive cysteine residue. Meat processing i s the major a p p l i c a t i o n of plant proteases. Polynesians and Hawaiians have t r a d i t i o n a l l y treated meats with papaya or pineapple, presumably for the improved texture, though flavour improvement i s no doubt also important. Meat was rubbed, or even bo i l e d , with the raw f r u i t or i t was marinated by storing wrapped i n papaya leaves. The meat industry has applied and elaborated on these ancient recipes for meat ten d e r i z a t i o n . Tenderizers are now used immediately pre- or post-slaughter and upon preparation for cooking or canning meat and f i s h products. The North American product r e t a i l e d under the name of ProTen, no longer a v a i l a b l e , was beef tenderized by antemortem i n j e c t i o n of papain. This has been considered a great breakthrough by the meat industry i n t h e i r quest for an inexpensive and thorough means of meeting the consumers' demand for tender, yet lean, meat (Goeser, 1961). Muscle f i b r e proteins and collagen, including c a p i l l a r y walls, are hydrolyzed to some extent i n vivo, and further degraded upon cooking (Kang & Warner, 1974). Organ tissues (kidneys, l i v e r , heart, etc.) receive higher doses of the enzyme unless slaughter r a p i d l y follows i n j e c t i o n . Orsi and Major (1973) developed an assay procedure for routine 1 3 use i n the Hungarian meat industry. By t h i s method, a c t i v i t y of a l l three plant proteases ( f i c i n , papain and bromelain) was found to c o r r e l a t e w e l l with tenderness ratings of a panel of judges. Plant proteases have also been investigated for p o t e n t i a l a p p l i c a t i o n i n the dairy industry, p r i m a r i l y for rennet s u b s t i t u t i o n i n cheese manufacture (Balls & Hoover, 1937; Cooke & C a y g i l l , 1974). Scott (1973) and Sardinas (1976) have both l i s t e d a number of plant sources of coagulants, among them pumpkin, cardoon, sundew and the three already discussed. Scott also pointed out the two d i s t i n c t functions the enzyme must perform: coagulation of the milk, allowing expression of whey but retention of other constituents, and curd hydrolysis for further microbial or enzymic action i n aging, to produce c h a r a c t e r i s t i c texture, flavour and aroma. F i c i n and papain preparations used to make cheese are too highly p r o t e o l y t i c , according to Scott and others (Sardinas, 1972; Cooke & C a y g i l l , 1974; Kosikowski, 1977). This r e s u l t s i n lower y i e l d s due to whey expression by the firm curd as well as losses due to casein h y d r o l y s i s . Furthermore, b i t t e r flavours may develop upon ripening. A c e r t a i n degree of bitterness i s sometimes desirable, though, i n such cheeses as roquefort. Sardinas (1976) explained patented methods for the use of bromelain, f i c i n and papain: the enzymes are added to milk but, when casein has been converted to paracasein, they are inactivated (eg., by peroxide treatment); then b a c t e r i a l cultures are added for ripening. Another solution to b i t t e r peptide formation might be the i n c l u s i o n , i n ripening cultures, of bacteria that w i l l metabolize these products. The extract of cardoon i s reportedly s t i l l i n use i n I b e r i a f o r preparation of an unripened cheese ( V i e i r a de Sa & Barbosa, 1970b; Kosikowski, 1977). V i e i r a de Sa and Barbosa (1972) made Edam, Serra and 14 Roquefort cheeses using an extract from cardoon flowers (Cynara  cardunculus). They found the maximum c l o t t i n g a c t i v i t y at 70°C but most s i m i l a r to the c l o t t i n g time of animal rennet at 32°C. In sheep's milk, only one-third as much extract was required as i n cow's milk for s u i t a b l e curd firmness. They concluded that the cardoon extract was suitable f or manufacture of soft-bodied cheeses l i k e Serra, not as good for Roquefort due to decreased y i e l d s , and unsuitable for Edam production. Ladies' bedstraw (Galium verum) has been extracted for use i n t r a d i t i o n a l cheese-making i n the Middle East. I t was also i n use i n Cheshire, England i n the late 1 8 t h and early 1 9 t h centuries (Scott, 1973). Sardinas (1976) c i t e d a report of cheese manufacture using berries of Withania coagulans, h i s t o r i c a l l y used i n India. In summary, i t appears that there are numerous plants known to have m i l k - c l o t t i n g properties, few of which have been investigated for p r a c t i c a l a p p l i c a t i o n or for i d e n t i f i c a t i o n of the coagulating p r i n c i p l e s . If plant proteases are to f i n d use i n t h i s aspect of the dairy industry, i t i s e s s e n t i a l to develop close controls over enzyme a c t i v i t y . Because of t h i s requirement, many researchers have turned to work with immobilized enzymes and "cold-renneting" techniques. To date, these methods have not produced an e f f e c t i v e means of replacing rennet i n the manufacture of t r a d i t i o n a l cheeses. Microbial rennets and pepsin are commonly used, but do not generally equal the rennet-coagulated cheeses i n sensory properties (Sardinas, 1972; Sternberg, 1976). 2. Plant tissue and c e l l culture Plant tissue cultures are commonly derived from excised lea f , f r u i t , seedling or bud t i s s u e . These tissues are incubated a s e p t i c a l l y on a 15 nutrient agar medium, several of which have been developed for general use or s p e c i a l purposes (Murashige & Skoog, 1962; White, 1943; Gamborg et a l . , 1968). Under appropriate conditions c a l l u s t i s s u e , c o n s i s t i n g of masses of u n d i f f e r e n t i a t e d plant c e l l s , forms. In a developing c a l l u s , c e l l d i v i s i o n i s rapid u n t i l the c e l l mass begins to r e s t r i c t nutrient uptake. To encourage c e l l p r o l i f e r a t i o n , the c a l l u s must be dissected and transferred often (every 10-40 days, depending on the t i s s u e ) . Plant c e l l suspension cultures can sometimes be established from c a l l u s by dispersion of the c a l l u s i n l i q u i d medium with appropriate a g i t a t i o n . Success or f a i l u r e of plant tissue and c e l l suspension cultures depends upon both growth conditions (media, temperature, aeration) and genetics (species, plasmids, nucleic acid r e p l i c a t i o n ) . There i s a large body of l i t e r a t u r e on manipulation of these two factors (see Reinert & Bajaj, 1977, and Thorpe, 1978, for example). Dougall (1980) has presented a concise and very readable synopsis on the current status of n u t r i t i o n research i n plant tissue and c e l l c u l t u r e . Much l i k e the development of knowledge regarding microbial metabolism, plant c e l l cultures are the subject of numerous ongoing i n v e s t i g a t i o n s : carbohydrate and nitrogen metabolism, micronutrient requirements and functions, plant hormone a c t i v i t i e s and the r e l a t i o n s h i p of n u t r i t i o n to d i f f e r e n t i a t i o n and senesence. New cultures are i n i t i a t e d i n media proven successful for other species and, i f adequate, are then modified by supplementation or omission of n u t r i e n t s . Such work requires dedication and the investment of many years of research, the contributions of O.L. Gamborg (1975) and H.B. Street (1977) being outstanding examples. Plant tissue culture has been quite successful i n h o r t i c u l t u r a l endeavours (Boxus & Druart, 1980; Murashige, 1978). As discussed i n the 16 introductory chapter, this has become a popular and expanding f i e l d i n pharmaceutical research as w e l l . Since the advent of axenic culture of plant tissues, there have been a few attempts to grow papaya tissue i n  v i t r o . The f i r s t report of papaya c a l l u s cultures was probably that of Medora et a l . (1973), i n which they gave evidence of p r o t e o l y t i c a c t i v i t y i n these c u l t u r e s . They have subsequently published information about medium composition and substrate s p e c i f i c i t i e s (Bilderback et a l . , 1976; Medora et a l . , 1979; Mell et a l . , 1975, 1979). Enzyme a c t i v i t y was f i r s t assessed using a casein substrate to which buffered extracts of mortar-ground, l y o p h i l i z e d tissue was added (Medora et a l . , 1973). Later work showed that azocasein and casein yellow were better suited for assay of crude extracts, while hemoglobin, a z o c o l l , and hide powder azure were a l l inadequate. Medhi and Hogan (1976, 1979) have also propagated papaya c a l l u s and produced p l a n t l e t s from embryoids. Arora and Singh (1978) found that the most e f f e c t i v e growth hormones for papaya c a l l u s development were NAA (naphthalene acetic a c i d , 1.0 mg/1), k i n e t i n (0.5 mg/1) and g i b b e r e l l i c (1.0 mg/1) a c i d . Propagation of papaya plants v i a tissue c u l t u r e , however, required changing of hormone concentrations during p l a n t l e t development ( L i t z & Conover, 1978). Apte et a l . (1979) reported p r o t e o l y t i c a c t i v i t y i n tiss u e cultures of pineapple. C a l l u s , i n i t i a t e d from l a t e r a l buds, was grown i n the presence of NAA (10 mg/1), casein hydrolysate (0.4 g/1) and 15% coconut milk. This group extracted the enzyme(s) by acetone f r a c t i o n a t i o n of blendor-homogenized t i s s u e . Using a casein hydrolysis assay method, they found great fluctuations i n a c t i v i t y over a 50-day period, but pro t e o l y s i s was lower i n c a l l i and regenerated p l a n t l e t s than i n the mature plant. No one has yet» reported attempts to improve protease yields or to produce 17 ac t i v e c e l l suspension cultures from papaya or pineapple. Other species investigated herein have not previously been studied as undifferentiated t i s s u e . L i t z and Conover (1977) propagated Dieffenbachia from excised l a t e r a l buds, with a minimum of c a l l u s formation. There appear to be no reports i n the l i t e r a t u r e regarding i n v i t r o propagation of f i g . The f o c a l point of this research project was to assess p r o t e o l y t i c enzyme a c t i v i t y i n c e l l suspension cultures derived from plants known to produce proteases. Preliminary work therefore required preparation of ca l l u s cultures from a number of plant sources: papaya, f i g , pineapple, cardoon, Dieffenbachia and bedstraw. From th i s stage, i t was necessary to develop c e l l suspension c u l t u r e s . Successful establishment of these then led to an evaluation of protease a c t i v i t y . A l l subsequent work was aimed at stimulation of p r o t e o l y t i c enzyme production and a c t i v i t y i n c e l l suspension cultures, with concomitant observations on growth and t o t a l protein production under the test conditions. 18 I I I . MATERIALS and METHODS 1. Callus culture of selected species General methods and conditions: The following seven plants were used as tissue sources for c a l l u s culture: papaya, f i g , cardoon, dumbcane, pineapple, bedstraw and t h i s t l e . A s i m i l a r procedure of exc i s i o n , s t e r i l i z a t i o n , trimming and p l a t i n g was followed for a l l tissues, with d e t a i l e d methods given below. Explants and c a l l i were incubated i n p l a s t i c basins covered with aluminum f o i l to maintain humidity and minimize contamination and l i g h t exposure. Incubation was at a temperature of 28 ± 3°C and i n t o t a l dark-ness, so that cultures were exposed to l i g h t and temperature f l u c t u a t i o n s only for br i e f periods, no longer than one hour, during observation and tr a n s f e r . A s t e r i l e transfer cabinet of the hori z o n t a l laminar a i r flow type was used (Envirco, Becton Dickinson Co., USA). Disposable 5 cm p e t r i dishes were used throughout. Table I II gives the composition of B5 medium (Gamborg et a l . , 1968), the medium most commonly used: MS medium (Murashige & Skoog, 1962) was used for some young t i s s u e s . Bacto-agar (Difco) was added at 0.6% (w/v) f o r c a l l u s t i s s u e s . Media were s t e r i l i z e d i n an autoclave a t 15 psi and 121 °C for 15 min. Developing c a l l i were assessed for protease a c t i v i t y by v i s i b l e c l e a r i n g of B5 agar media containing 3% skimmed milk, B5-M. Carica papaya; Papaya was propagated from explants of germinated seeds as outlined i n Figure 3. The seeds were a s e p t i c a l l y removed from a market papaya and the a r i l s peeled off each seed. The seeds were then soaked i n two rinses of s t e r i l e d i s t i l l e d water for about 5 minutes each then blotted dry with s t e r i l e f i l t e r paper. At t h i s point, about half the seeds so pre-Table I I I B5 Medium and Plant Hormones From Gamborg, M i l l e r and Ojima (1968): Exp. C e l l Res. 50: 151-1 Salts Micronutrients Vitamins Carbohydrate Plant Hormones NaH_P0.' 2 4 KN0„ H 20 '3 h MgSO (NH 4) 2S0 4 CaCl, KI 7H20 2H 20 Fe (Sequestrene 330-Iron) stock s o l u t i o n ( i n 100 ml) MnSO^' H3BO3 ZnSO,' H 20 7H20 Na 2Mo0 4* 2H20 CuSO, CoCl, 6H 20 1.0 g 0.3 g 0.3 g 25 mg 25 mg 25 mg stock s o l u t i o n ( i n 100 ml) n i c o t i n i c a c i d 10 mg thiamine 100 mg pyridoxine 10 mg myo-inositol 1 g sucrose 150 mg/1 2500 mg/1 134 mg/1 250 mg/1 150 mg/1 0.75 mg/1 28 mg/1 1.0 ml/1 10 ml/1 20 g/1 IAA (indole-3-acetic acid) 1.0 mg/1 2,4-D (dichlorophenoxyacetic acid) 1.0 mg/1 or p-cpa (parachlorophenoxyacetic acid) 1.0 mg/1 or 2,4,5-T (trichlorophenoxyacetic acid) 1.0 mg/1 k i n e t i n (6-furfurylaminopurine) Adjuncts (optional) agar skimmed milk casein hydrolysate yeast extract 0.1 mg/1 6.0 g/1 30 ml/1 0.5-2.0 g/1 0.5-2.0 g/1 20 papaya fruit seeds seed arils removed germinated on water agar callus from seedling explants I B5 agar) callus dispersed fpZ5\ in small /Ml volume liquid B5 multiple transfers of small fragments multiple transfers with decreasing inoculu gure 3. Preparation of papaya f o r tissue and c e l l c u l t u r e . A l l seeds, c a l l u s and c e l l suspension cultures were maintained at 28 °C i n darkness. 21 pared were mechanically damaged so as to break the tough seed coat: the other h a l f were l e f t i n t a c t . A l l seeds were placed on s t e r i l e d i s t i l l e d water-agar (WA) i n p e t r i dishes with a minimum surface area of 5 cm2 per seed. Plates were incubated as described above u n t i l germination. About the fourth day af t e r germination, the new seedlings were a s e p t i c a l l y explanted and segments no longer than 2 cm transferred to nutrient media. These were based on B5 or MS and generally contained 2,4-D (dichlorophenoxyacetic acid, 1.0 mg/1 ), IAA (indole-3-acetic acid, 1.0 mg/l) and k i n e t i n (6-furfurfyrlaminopurine, 0.1 mg/1). Plates containing explants were incubated under the same conditions and observed for c a l l u s formation i n 2-5 weeks. When ca l l u s tissue was approximately f i v e times the o r i g i n a l explant si z e (estimated tissue volume), i t was dissected away from the parent tissue and transferred to fresh agar medium. Such passages were repeated a minimum of three times, with at l e a s t two weeks' growth each time, p r i o r to further experiments or propagation i n l i q u i d media. The inoculum selected for transfer was always near the fringes of the c a l l u s so as to take only very young, rapidly-growing t i s s u e . Ficus c a r i c a : F i g tissue cultures were i n i t i a t e d from leaves of f i g tre e s . The leaves were prepared for explantation by gently washing large pieces of both laminae and pet i o l e s under running water then soaking 5-10 minutes i n two washes of 10% commercial bleach, followed by r i n s i n g i n three changes of s t e r i l e d i s t i l l e d water, 10 minutes each. Cut edges were a s e p t i c a l l y trimmed, exposing fresh surfaces, and these pieces cut into explant fragments averaging 1 cm i n length for petioles and 1-2 cm2 i n area for laminae. These explants, blotted dry, were transferred to B5 or MS agar plates and incubated under conditions described above. After one week, a l l were transferred to fresh agar then incubated again u n t i l c a l l u s was 22 observed. Callus tissue at l e a s t equal to the o r i g i n a l explant i n siz e was dissected free and transferred to fresh agar. At le a s t three passages of young c a l l u s tissue preceded attempts at l i q u i d c u l t u r e . Cynara cardunculus: Seeds of cardoon were a g i f t of Nichols Garden Nursery, Albany, Oregon, USA. The seeds were s t e r i l i z e d by soaking i n 15% commercial bleach ( 2 x 1 0 min) and r i n s i n g i n s t e r i l e d i s t i l l e d water (3 x 10 min). Some were cracked, others l e f t i n t a c t , then germinated on WA as described for papaya. Explants of seedlings and r e s u l t i n g c a l l u s were handled as described above. Dief fenbachia amoena & D. p i e t a : Leaves of two species of dumbcane were obtained from the UBC Plant Science Department greenhouses. They were pre-pared according to the method given above for f i g . The high contamination rate, however, required much more severe s t e r i l i z a t i o n protocol: 1 x 1 0 min and 1 x 20 min i n 15% bleach, followed by 3 s t e r i l e water rinses t o t a l l i n g 30-40 min. Otherwise, the culture method was the same as given for f i g . The auxin, 2,4-D, was replaced with 2,4,5-T (trichlorophenoxyacetic a c i d ) . Ananas comosus: Pineapple tissue was derived from two sources, the market f r u i t and vegetative plant, both purchased r e t a i l . Basal leaf explants from the tops of pineapples were prepared as described for f i g , using f i n a l explants less than 1 cm^ i n s i z e . Cross-sections of vegetative leaves including base and leaf margins were s t e r i l i z e d as described for dumbcane. The l a t t e r procedure was also applied to explants from the f l e s h of s l i g h t l y under-ripe f r u i t . Standard media (B5 and MS) were used, except for the use of 2,4,5-T. Galium verum; Cultures of la d i e s ' bedstraw originated from f r e s h l y -i harvested seeds. These were s t e r i l i z e d according to the method given f o r cardoon, but with the addition of a wetting agent to the f i r s t bleach soak 23 (2 drops Palmolive detergent per 100 ml bleach s o l u t i o n ) . Bedstraw seeds carry dense surface d i s p e r s a l appendages which would otherwise prevent wetting and e f f e c t i v e s t e r i l i z a t i o n . Explants of the germinated seedlings were 0.5-1 .0 cm i n length. Incubation conditions were as described above. Numerous modifications of B5 medium were made i n attempts to encourage c a l l u s formation, rather than the fin e r o o t - l i k e p r o l i f e r a t i v e structures which developed. These modifications included a l l the hormones l i s t e d i n Table I I I , 0.05-0.5% casein, 1-5% skimmed milk, 10% coconut milk, 0.2% yeast extract and 4 mM thiourea. E f f e c t s of these modifications were assessed by v i s u a l examination for c a l l u s i n i t i a t i o n . Circium arvense: Wild t h i s t l e (var. horridum) was c o l l e c t e d fresh p r i o r to e x c i s i o n . Leaves and stems were surface-wetted with 70% ethanol because of surface h a i r s , then s t e r i l i z e d i n two 5-10 min soakings i n 15% bleach and rinsed i n s t e r i l e water (3 x 10 min). Trimmed explant dimensions were 0.5-2.0 cm. Standard media and incubation conditions were used. 2. Suspension culture of f i g and papaya Only f i g and papaya c a l l i p r o l i f e r a t e d r a p i d l y enough to e s t a b l i s h viable cultures i n l i q i u d media. B5 was the basal medium with the following hormones: k i n e t i n (0.1 mg/1), IAA (1 mg/1) and p-cpa (p-chlorophenoxyacetic ac i d , 2 mg/1). C e l l suspension cultures began with d i s p e r s a l , i n l i q u i d medium, of large c a l l u s tissues representing at l e a s t 10% of the f i n a l volume. Flasks of more than double the r e q u i s i t e capacity were used so as to provide a large surface area for aeration of the medium. They were stoppered with cheesecloth-wrapped cotton and further protected with two layers of paper towelling secured about the flask necks with e l a s t i c bands. Incubation conditions were constant: 28 ± 3°C, dark, 110 rpm on rotary New 24 Brunswick shakers. After 1 -4 weeks' growth, depending on how r e a d i l y the c a l l u s sloughed c e l l s and the suspension thickened, these primary c e l l suspension cultures were transferred to fresh media using a large-bore pipette and an inoculum si z e of 10-15% of the f i n a l volume. Herein, these w i l l be referred to as " c e l l suspension cultures" or " c e l l cultures", according to t r a d i t i o n a l terminology i n the l i t e r a t u r e . A minimum of three such consecutive transfers preceded further experimentation i n c e l l l i n e s e l e c t i o n , protease assay or medium composition as described below. 3. Assay methods Biomass: C e l l dry weight obtained from i n i t i a l c e l l suspension volumes ( i e . , medium + inoculum)were converted to y i e l d s based on one l i t r e . Dry weights were determined by three methods: to constant weight i n a convection oven at 60 °C, i n a vacuum oven at 60°C, or i n a V i r t i s freeze-dryer (low heat, condenser temperature -60°C, less than 1 mm Hg). C e l l s were harvested by f i l t r a t i o n through Miracloth (Chicopee M i l l s , Inc., NJ, USA) then transferred to pre-dried, pre-weighed aluminum dishes. A f t e r establishment of the r e l a t i o n s h i p between r e s u l t s of these methods, a l l harvest weights were derived from l y o p h i l i z e d samples since these were then used for enzyme assays. A rapid method of assessing c e l l growth i s based on " s e t t l e d c e l l volume" ( N i c k e l l & Maretzki, 1969), also employed by Behrend and Mateles (1975). This method was compared to c e l l dry weights to evaluate i t s v a l i d i t y because of the advantage that r e s u l t s could be obtained immediately upon harvest. C e l l suspension cultures were allowed to s e t t l e about 30 min. The medium was decanted through Miracloth to trap f l o a t i n g c e l l s and the thick c e l l suspensions poured into large graduated centrifuge tubes. 25 C e l l s c o l l e c t e d on the f i l t e r were also added, by scraping of the f i l t e r on a f l a t surface. Tubes were centrifuged 3-5 min a t 200 xg and the s e t t l e d c e l l volume was read d i r e c t l y . The c e l l s were then l y o p h i l i z e d to obtain dry weights. C e l l e x t r a c t i o n : Three methods were compared for extraction of i n t r a c e l l u l a r material to determine the most e f f e c t i v e one for release of proteins and, i n p a r t i c u l a r , active proteases. C e l l disruption was accomplished by sonication (80 W, 30 & 120 sec; Braunsonic 1510), by homogenizing (1-4 min, Polytron Kinematica PCU-1, Brinkmann Instruments) and by grinding i n a mortar with or without washed sand as an abrasive (45-90 sec at an average rate of 60-80 strokes/min). The l a s t of these became the standard method for further studies. In a l l cases, c e l l s were suspended i n i c e - c h i l l e d extraction buffer, 0.1 M phosphate at pH 6.0, and kept on i c e throughout. These crude extracts were f i l t e r e d through Miracloth to remove c e l l debris then assayed for protein content and p r o t e o l y t i c a c t i v i t y . P r o t e i n content: Crude c e l l extracts were kept on ice and assayed for p r o t e i n content within three hours. The dye-binding method of Bradford (1976) was used r o u t i n e l y due to the s i m p l i c i t y (one reagent, Coomassie B r i l l i a n t Blue-G, Sigma) and r a p i d i t y (10 min). This method was compared to protein determinations by three other methods. U l t r a v i o l e t l i g h t absorbance at 280 nm (Beckman DB spectrophotometer) could not be determined accurately without f i l t r a t i o n and high-speed c e n t r i f u g a t i o n of the extracts due to interference caused by cloudiness. Since this step would also p e l l e t some of the protein, i t was considered unsuitable. Nitrogen content of c e l l s was determined by the micro-Kjeldahl method (AOAC, 1975), and quantitated by a Technicon Autoanalyzer I I nitrogen analyzer. F i n a l l y , the Lowry procedure (Lowry et a l . , 1951) was tested. A d i l u t i o n series of bovine serum albumin 26 (Fraction V, Sigma) was included as the standard protein for Bradford's and Lowry's me thods. Protease a c t i v i t y : The method of the U.S. National Research Council's Food Chemicals Codex (1966), hereafter referred to as the FCC method, was selected. This procedure was modified as follows: Hammarsten casein (BDH) substrate concentration was 0.2 g/100 ml; e x t r a c t i o n buffer (as previously described) was used i n place of a c t i v a t i o n buffer; the incubation period was extended to 2 hr, and d i t h i o t h r e i t o l (Dtt, Sigma, 2 mM f i n a l concentration) replaced cysteine as the a c t i v a t o r , added with EDTA (G. Frederick Smith, Ohio, USA, at 0.4 mM f i n a l concentration) immediately p r i o r to incubation. Samples were both centrifuged (8,000 xg, 10 min, S o r v a l l RC-2) and f i l t e r e d p r i o r to reading the absorbance at 280 nm. Each sample evaluation consisted of four tubes; two incubated sample/substrate/activator reaction mixtures and two incubated substrate/activator mixtures to which sample was added post-incubation, along with TCA ( t r i c h l o r o a c e t i c acid, F i s h e r ) . Results of this method were compared to t r i a l s of seven others: (a) d i g e s t i o n of Hide Powder Azure (a dye-labelled collagen, Sigma) according to Savage and Thompson (1970), using 0.5 ml of extract d i l u t e d to 5.0 ml i n place of beer, and extending the incubation time to 1 hr; (b) esterase a c t i v i t y by pH-dependent i n d i c a t i o n of r a d i a l d i f f u s i o n according to Araki and Abe (1980), using BAEE (N-benzoyl-L-arginine e t h y l ester, Sigma) as the substrate; (c) c a s e i n o l y t i c a c t i v i t y by the a g a r - d i f f u s i o n method of Holmes and Ernstrom (1973); (d) c a s e i n o l y t i c a c t i v i t y using the Bio-Rad "Protease detection k i t " (1978); 27 (e) g e l a t i n digestion from f i l m , by the method of G l e n i s t e r and Becker (1961), using 0.5 ml extract d i l u t e d to 5.0 ml instead of beer, and Kodak Panatomic-X f i l m ; (f) fluorescence loss of ANS (1-anilino-8-naphthalenesulfonate, Mg s a l t , Sigma) according to Spencer and Spencer (1974), using the casein substrate and activated (Dtt and EDTA) crude c e l l extracts (Aminco-Bowman spectrophoto-fluorometer; e x c i t a t i o n and emission wavelengths, 370 and 460nm, r e s p e c t i v e l y ) ; (g) development of fluorescence by reaction of fluorescamine (Sigma) with TCA-soluble casein digestion products, according to the method of Chism e t a l . (1979), using 0.2% Hammarsten casein without sodium azide incubated with 0.025-0.5 ml crude c e l l extract for 1 hr p r i o r to p r e c i p i t a t i o n and reading ( e x c i t a t i o n and emission wavelengths', 390 and 475nm, r e s p e c t i v e l y ) . In a l l cases, the samples tested were f i l t e r e d extracts of l y o p h i l i z e d c e l l s ground i n a cold mortar with i c e - c h i l l e d buffer as described above. Appropriate concentrations of p u r i f i e d papain (African, Calbiochem) or p u r i f i e d f i c i n (Sigma) enzyme standards were used, and are referred to as "standard" throughout. The FCC method was selected for routine protease determinations. Supernatant absorbance at 280 nm i s dependent on the quantity of 10% TCA-soluble aromatic amino acids released from the casein substrate during the incubation period. Hence, a series of standards containing L-tyrosine (Merck) i n quantities of 0-300 ug per reaction volume (2.1 ml already containing 3.6 mg casein) was included i n each assay. This was representative of digestion products as well as accounting for any spontaneous casein hydrolysis, and thus was used to quantitate p r o t e o l y t i c a c t i v i t y . One u n i t of p r o t e o l y t i c a c t i v i t y was defined as that amount of 28 a c t i v e enzyme(s) extractable from one l i t r e of c e l l suspension culture that w i l l release, from casein, TCA-soluble material equivalent to 100 ug tyrosine under reaction conditions defined above. Conditions i n f l u e n c i n g protease a c t i v i t y : The modified FCC protease detection method was evaluated f o r v a r i a t i o n with reaction pH and temperature of incubation. One extract from a large c e l l suspension culture of f i g was used for a l l t e s t conditions, stored frozen 2 days" between the pH series and the temperature s e r i e s . The influence of pH was tested using buffers a t pH 5.5, 6.0, 7.0 and 8.4. At pH 6.0, the following temperatures were tested: 27, 38, 47, 54 and 67 °C. Appropriate series of standard f i c i n and tyrosine were included with each set of conditions. A number of reagents which could p o t e n t i a l l y activate the extracted enzyme(s) were tested under standard conditions, 40 °C and pH 6.0, with three c o n t r o l s . These controls were t r y p s i n i n h i b i t o r (Sigma) a t 0.4 mg/ml reaction mixture, sodium tetrathionate (ICN) a t 6 mM f i n a l concentration, and no adjunct. The p o t e n t i a l a c t i v a t o r s tested were as follows: L-cysteine-HCl (MCB) a t 1.4 mM, d i t h i o t h r e i t o l a t 2.0 mM, EDTA at 2 mM, glutathione (MCB) at 6 mM, SDDC (sodium diethyldithiocarbamide, MCB) at 6 mM, calcium thiocyanate (Anachemia Chemicals, Ltd.) a t 2.5 ul/ml reaction mixture, and thiourea (Mallinkrodt) a t 6 mM f i n a l concentration. Both papain and f i c i n were tested i n the presence of these reagents, and compared to the behaviour of the f i g c e l l e xtract. Milk c l o t t i n g a c t i v i t y : Papaya and f i g c e l l suspension cultures were harvested by f i l t r a t i o n through Miracloth. Collected c e l l s were l y o p h i l i z e d , weighed and extracted according to the methods described above. Extracts were kept on ice and used within 2 hr. The milk c l o t t i n g assay of Ba l l s and Hoover (1937) was used, with no r e s u l t s , so the following 29 method was devised. Skimmed milk was d i l u t e d to a s o l i d s content of 5% and the pH adjusted to 5.6 with 2 M H3PO4 then autoclaved i n 25-ml Erlenmeyer f l a s k s containing 3 ml each. C e l l extract, 0.3 ml, was added to each of three flasks and these were incubated i n a water bath shaker at 35°C up to 24 hr. C l o t t i n g times were compared to highly d i l u t e d standard papain, at a maximum concentration of 0.3 mg/ml. Electrophoresis: Electrophoresis of f i c i n - l i k e proteins i n crude c e l l e xtracts i n acrylamide gel was attempted. The method of Melachouris (1968), as modified by Mr. T. Kuwata (unpublished), was applied to a v e r t i c a l slab gel apparatus. The separation gels t r i e d were 9-10% acrylamide (Bio-Rad) polymerized with 0.24-0.28% bis-acrylamide; the concentration gels were 3-3.6% acrylamide; the electrophoretic buffer was Tris-TEMED-glycine at pH 8.9. After electrophoresis (4.5-5 hr 100 V), gels were fixed i n TCA-isopropanol-water (15% TCA and 25% isopropanol i n d i s t i l l e d water) f o r 40 min, stained with amido black 10B (0.025% w/v i n 4:5:1 H2O:methanol:acetic acid) 30-50 min, and destained 1 .5-2 days i n 3 changes of destaining s o l u t i o n (9.71:3.57:1 H2O: methanol:acetic a c i d ) . The disc gel method of Weber et a l . (1972) was also employed, using 7.5-9.3% acrylamide. Electrophoresis was conducted i n 0.1 M phosphate buffer (pH 7.2) with 0.1% SDS (sodium dodecylsulfate, F i s h e r ) . Gels were subjected to 2 mA per gel 20-30 min then 5 mA per gel u n t i l the bromophenol blue marker dye approached the d i s t a l end. Gels dislodged from glass tubes were stained up to 2 hr with Coomassie B r i l l i a n t Blue-G (0.25%, w/v, i n 1:1:0.2 H20:methanol:acetic acid) and destained 1.5-2 days i n 35:3:2 H20:acetic acid:methanol. Samples were prepared for electrophoresis i n a variety of ways i n search of one that would e f f e c t i v e l y demonstrate proteins present i n c e l l 30 e x t r a c t s . For electrophoresis without SDS, sodium tetrathionate, SDDC and T r i t o n X-100 were added, alone or i n combination, to the extraction buffer i n some t r i a l s . Many samples were concentrated i n d i a l y s i s tubing (Fisher), packed i n Carbowax 20M (Applied Science) 2-6 hr. For electrophoresis with SDS, samples were mixed with SDS, 8 M urea, and p-mercaptoethanol and boiled 5 min i n stoppered tubes according to the method of Deutch (1976). Standards used were f i c i n and t r y p s i n (Sigma). Samples and standards for a l l e lectrophoretic t r i a l s were mixed 4:1 with g l y c e r o l p r i o r to a p p l i c a t i o n to g e l s . 4. Medium supplementation Five groups of nutrient supplements were investigated with respect to enhancement of protease production of a c t i v i t y i n c e l l extracts: inorganic nitrogen, amino acids, proteins, milk and i t s various components, and miscellaneous organic compounds, including two a n t i b i o t i c s , two t h i o l reagents and SDDC, an oxidase i n h i b i t o r . Inorganic nitrogen and amino acids: Nitrogen i s present i n the standard B5 medium as KN03 (2.5 g/1, or 25 mM) and (NH 4) 2S0 4 (0.134 g/1, or 1 mM). Media were prepared omitting e i t h e r of these, but s u b s t i t u t i n g KC1 or Na 2S0 4 as appropriate to maintain the non-nitrogenous ions (Behrend & Mateles, 1975). To each of these was added one of the following: alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine or pro l i n e ( a l l L-isomers from Sigma) a t 5 mM f i n a l concentration. For comparative purposes, a medium containing one-half the standard n i t r a t e (12.5 mM) was prepared, and 3% (v/v) skimmed milk added to a portion of each nitrate-ammonium combination. Small f l a s k s of these media were inoculated simultaneously from one suspension culture of Ficus c a r i c a grown i n a 31 low-nitrogen medium, 20% of the normal B5 l e v e l . Following 9 days' incubation these conditioned cultures were used to inoculate another set of the same media for 14 days' incubation p r i o r to assessment. C e l l s were harvested and both c e l l s and media l y o p h i l i z e d and analyzed for protein content and protease a c t i v i t y , as described above. Proteins and peptides: B5 medium was supplemented with a s e l e c t i o n of proteins and related materials to investigate t h e i r possible stimulation of protease a c t i v i t y . These were as follows: casein powder (Fisher) at 0.3% (w/v), soy protein i s o l a t e ("Farefax", Nu-Life N u t r i t i o n ) a t 0.3% (w/v), wheat gluten ("WhetPro", I n d u s t r i a l Grain Products) a t 0.3% (w/v), beef extract (Difco) a t 0.3% (w/v), g e l a t i n (MCB) a t 0.3% (w/v), fresh egg albumen at 1% (v/v), enzymatic casein hydrolysate (ICN) at 0.3% (w/v), and skimmed milk at 3% (v/v). Two controls included were soluble starch (Difco) at 0.3% (w/v), and no supplements. A l l f l a s k s were harvested a f t e r 18 days. Both media and c e l l s were analyzed for protease a c t i v i t y and these values summed for t o t a l protease. Milk and i t s components: Skimmed milk was used to supplement B5 medium f o r suspension-cultured c e l l s of papaya and f i g . This enriched medium was compared to standard B5 with respect to biomass, protein and protease a c t i v i t y over a growing period of 23 days under standard conditions of incubation. Assay procedures are described i n preceding sections. The e f f e c t of several components of skimmed milk on protease a c t i v i t y of f i g c e l l s was assessed by simple or complex supplementation of the medium. Single supplements, a l l reagent grade, were added at levels approximating that which 3% skimmed milk would provide for B5 medium: c i t r i c acid (49.8 mg/1), r i b o f l a v i n (53 ug/1), n i a c i n (24 ug/1) and calcium, as CaCl3*2H 20, (1.336 g/1 i n addition to that already present i n B5). 32 Lactose replaced 20% of sucrose i n one t e s t medium, that i s 4 g/1. Commercial casein powder (Fisher) was rehydrated, p r e c i p i t a t e d and c o l l e c t e d by c e n t r i f u g a t i o n . Added at 0.3% (w/v), i t was compared to fresh casein a t the same l e v e l . These caseins were prepared by coagulation of agitated casein s o l u t i o n or fresh l i q u i d skimmed milk with slow addition of phosphoric acid at room temperature u n t i l the pH reached 5.2. A f t e r 20 min continued s t i r r i n g , the casein was c o l l e c t e d by ce n t r i f u g a t i o n at 10,000 xg for 15 min and the whey (supernatant) decanted o f f . After adjustment of the pH to 5.6 with NaOH, most of the whey was f i l t e r e d through Whatman No. 5 paper and sintered glass (10-15 um), then subjected to u l t r a f i l t r a t i o n ( P e l l i c o n , M i l l i p o r e ) with a f i l t e r f o r 10,000 daltons. Both f i l t r a t e and retentate were used as B5 supplements, i n d i v i d u a l l y and i n a 1:1 combination to a t o t a l of 2.7% (v/v). A l l media containing casein or whey prepared i n this manner began with a basal medium containing 15% less i n i t i a l phosphate, to accommodate the a d d i t i o n a l phosphate from the casein p r e c i p i t a t i o n step. Flasks were inoculated from pre-conditioned c e l l suspensions grown 7 days i n 20 ml of each respective t e s t medium. Aft e r 14 days' incubation, c e l l s were harvested and both c e l l s and medium fr a c t i o n s analyzed for protein and protease a c t i v i t y . S t a t i s t i c a l a n a l y s i s : Results of major experiments i n medium supplementation were processed by analysis of variance using two programs prepared for the University of B r i t i s h Columbia computing centre, MFAV (Le, 1980) and Genlin (Grieg & B j e r r i n g , 1980). To determine differences among treatments, Duncan's multiple range t e s t was applied at p r o b a b i l i t y l e v e l s of 1 and 5%. Data were grouped according to these ranges. 33 IV. RESULTS 1. Culture of plant tissues Papaya tissues, C. papaya, were s u c c e s s f u l l y cultured from excised segments of r a d i c l e s and hypocotyls of axenically-germinated seeds. Early t r i a l s indicated that seeds germinated very poorly unless a r i l s were f i r s t removed and seeds rinsed. The a r i l s contain substances i n h i b i t o r y to seed germination (Gherardi & V a l i o , 1976). Table IV includes s u r v i v a l l e v e l s of papaya cultures to both c a l l u s and c e l l suspension stages (Figure 4a & b). C a l l i were pale, s o f t and r e a d i l y dispersable i n l i q u i d medium. Fi g tissues, F. c a r i c a , were s u c c e s s f u l l y propagated from leaf segments to c a l l u s and suspension stages at the remarkably high s u r v i v a l l e v e l of 95% (Table IV). Fig suspension cultures were r e a d i l y established and maintained, and were thus used for experimental studies i n c e l l n u t r i t i o n and enzyme production. The appearance of f i g c a l l u s i s i l l u s t r a t e d i n Figure 5 (a&b); c e l l suspension cultures were i d e n t i c a l to those of papaya, c o n s i s t i n g of a pale yellow s l u r r y of c e l l s . Seeds of cardoon, C. cardunculus, showed a 14% germination rate, on average, and tissues were excised from hypocotyls and r a d i c l e s f o r further propagation. S t e r i l i z a t i o n procedures used were not s u f f i c i e n t l y e f f e c t i v e for these hard seedcoats and a high rate of endogenous contamination re s u l t e d . Galium, l a d i e s ' bedstraw, did not form c a l l u s from germinated seeds, but rather developed as a mass of r o o t - l i k e structures (Figure 6a). In attempts to bypass the stage of c a l l u s formation, germinated tissue was transferred to l i q u i d medium. Under these conditions, the roots continued 34 Table IV Survival of Plant Tissue Cultures plant source explants type & number % s u r v i v a l (2 mos.) form i n 6 mos. Ananas comosus  Carica papaya Circium arvense f r u i t , l e a f , stem 223 100 seeds l e a f , p e t i o l e , stem 110 Cynara cardunculus l e a f , p e t i o l e , stem 222 72 seeds 40 seedlings Dieffenbachia  p i c t a & amoena Ficus c a r i c a Galium verum 254 leaf & p e t i o l e l e a f , p e t i o l e , stem 133 150 seeds 77 l e a f & p e t i o l e 81 stem 19 seeds 0 22 11 0 22 23 16 ( a l l D.amoena) 95 0 11 63 0 c a l l u s & 11 suspensions c a l l u s & 2 suspensions c a l l u s c a l l u s l i t t l e c a l l u s c a l l u s & suspensions roots? roots? 35 Figure 4. (a) (b) Dark-grown papaya c a l l u s and c e l l suspension c u l t u r e s . Papaya c a l l u s on B5 agar, 22 days a f t e r transfer. Papaya c e l l suspension culture, 18 days a f t e r t r a n s f e r . Figure 5. F i g c a l l u s development. (a) F i g c a l l u s formation on primary e x p l a n t s , 12 days a f t e r e x c i s i o n from l e a f laminae and p e t i o l e s . (b) Dark-grown f i g c a l l u s a f t e r 4 generations ( t r a n s f e r s ) . 37 Figure 6. Dark-grown bedstraw t i s s u e on agar and i n l i q u i d B5-M. (a) Bedstraw growth on B5 agar medium, 35 days a f t e r t r a n s f e r , (b) Growth of non-callus bedstraw t i s s u e i n l i q u i d B5 medium co n t a i n i n g 3% skimmed m i l k . F l a s k on r i g h t i s uninoculated B5-M. 38 to develop, forming large tangled masses as a r e s u l t of rotary shaker action (Figure 6b). Milk-containing medium i l l u s t r a t e d p r o t e o l y t i c a c t i v i t y . The medium was checked microscopically for sloughed c e l l s which may have been used for sing l e c e l l suspension, but these were very few i n number. They did not p r o l i f e r a t e when transferred to fresh medium, suggesting that they were a c t u a l l y dead c e l l s , possibly sloughed from the rootcap area of developing roots. The s u r v i v a l rate of cultures i s given i n Table IV. Tissues of dumbcane (Dieffenbachia), pineapple (Ananas) and t h i s t l e (Circium) a l l suffered high rates of microbial contamination i n several t r i a l s (Table IV). T h i s t l e explants formed some c a l l u s which was propagated for many months but grew slowly and would not grow as single c e l l suspensions. Dumbcane and pineapple explants callused very poorly and neither formed s u f f i c i e n t tissue to attempt c e l l suspension c u l t u r e s . V a r i a b i l i t y among c e l l suspension c u l t u r e s : F i g and papaya c e l l s grown i n c e l l suspension exhibited differences not ne c e s s a r i l y related to environmental conditions. I t i s l i k e l y that genetic factors influenced growth and production of protein and protease (Mandels, 1972; S k i r v i n , 1978). Figure 7 (a&b) shows yields of protein and protease i n c e l l extracts from four papaya and f i g batch cultures grown two weeks i n B5-M medium p r i o r to harvest. There was approximately a 3-fold difference i n protein content and a 4-fold difference i n protease a c t i v i t y among these four papaya c e l l c u l t u res: the corresponding differences were 4-fold and ' .5-fold, r e s p e c t i v e l y , i n the four f i g c e l l c u l t u r e s . Although protease a c t i v i t y may have been correlated with protein production of papaya and f i g c e l l s , c u l t u r e #1 of each, for example, this was not necessa r i l y true (e.g., papaya culture #2). This v a r i a b i l i t y existed despite s i m i l a r propagation h i s t o r i e s and use of the same medium and incubation conditions. Unfortunately, such Figure 7. Protein-and protease var i a b i l i t y among c e l l extracts from four papaya and four f i g c e l l suspension cultures. A l l samples were grown 13-17 days in B5 medium with 3% skimmed milk. 40 differences did not appear to be p e r s i s t e n t . Figure 8 i s a compilation of protein and enzyme a c t i v i t y data from some papaya and f i g c e l l suspension cultures, but does not represent a s p e c i f i c experiment i n i t s e l f . Protein and enzyme p r o d u c t i v i t y did not appear to be c o r r e l a t e d with number of tra n s f e r s , since some consecutive c e l l suspensions showed an improvement while others declined. Again, protein synthesis and enzyme a c t i v i t y did not ne c e s s a r i l y change together. With t h i s degree of v a r i a b i l i t y i n culture p r o d u c t i v i t y , i t was e s s e n t i a l to use one inoculum of a standard volume f o r each experimental set, as a co n t r o l against changes with number of transfers and inoculum age. Judging from the variations apparent from one generation to the next, heredity alone was probably a minor f a c t o r . Other genetic factors such as nucleic acid synthesis, repair, regulation and foreign inclusions l i k e plasmids or viruses were probably responsible for much of the v a r i a b i l i t y noted. An i n v e s t i g a t i o n of genetic controls i n f l u e n c i n g c e l l growth and production of protein and protease i n c e l l suspension was beyond the scope of the present work. 2. Assessment of assay methods C e l l growth and biomass production: Growth, as indicated by weight of harvested c e l l s , i s the simplest i n d i c a t o r of appropriate n u t r i t i o n . Since there are several methods of assessing growth, some of these were compared. Table V gives harvest weight data from both papaya and f i g suspensions as determined by freeze-drying, oven-drying and vacuum-oven drying. Results were reproducible f o r each method and indicated, further, that the two oven methods were the same (Students's t-test,oc=0.05), with l y o p h i l i z e d papaya samples weighing 10% more and l y o p h i l i z e d f i g c e l l s weighing 6% more ( i e . , containing that much more water af t e r reaching a constant weight). These 41 C- papaya I C- papaya II F- carica c a l l u s c a l l u s c a l l u s Figure 8. The role of heredity i n v a r i a b i l i t y of productive capa-c i t y : a compilation of data from one f i g and two papaya c e l l c u l t ures. Git = generation number i n suspension culture; pn = protein content ( c e l l s + medium), (mg/1); ps = t o t a l protease (units / 1 ) . Table V A comparison of drying methods f o r papaya and f i g c e l l s from suspension cultures. Each was i n i t i a l l y 1.800 g b l o t t e d wet weight, n = number of samples; s = standard deviation of the mean. drying method papaya mean dry weight (mg) f i g mean dry weight (mg) s convection oven 60 °C (n=8) 17.7 0.46 14.9 0.58 vacuum oven 60 °C (n=8) 17.1 2.07 15.3 0.46 freeze dryer (n=6) 19.2 0.75 16.0 0.45 43 differences must be r e c a l l e d i n considering protein and enzyme data, as tissues were always l y o p h i l i z e d upon harvest to avoid problems of enzyme denaturation or hydrolysis of endogenous p r o t e i n . Settled c e l l volumes cor r e l a t e d well with c e l l dry weights up to about 30 ml c e l l s , obtained from 50-ml c e l l suspension cultures (Figure 9). Above t h i s , the curve reached a plateau i n d i c a t i n g that large differences i n s e t t l e d c e l l volumes were not necessarily weight-related. This may have been due to d i f f i c u l t y of c e l l packing i n the r e s t r i c t i v e vessels used, 50-ml centrifuge tubes. Typ i c a l growth curves, on the basis of l y o p h i l i z e d weights, for papaya and f i g c e l l cultures grown i n B5 medium are presented i n Figure 10. The logarithmic growth phase began within four days, with biomass reaching a maximum i n 17-21 days. There was an i n i t i a l drop i n medium pH, which then began to climb a f t e r the fourth day, reaching values more basic than the i n i t i a l medium. With f i g and papaya cultures, approximately equivalent biomass i n B5 medium, about 3 g/1, were produced. C e l l e x t r a c t i o n : Table VI gives protein y i e l d s and enzyme a c t i v i t y of extracts made using a Polytron homogenizer, a Braun sonicator or a mortar and p e s t l e . No d i s t i n c t differences were evident i n protein yields or enzyme a c t i v i t y of homogenized or mortar-ground samples. Sonication did not appear to be as e f f e c t i v e i n protein extraction, and thus also showed lower protease a c t i v i t y . Only a single sample was prepared by each method, though assayed i n duplicate, since a large volume of c e l l s was required i n order to do the comparison on a single c u l t u r e . I t i s therefore possible only to speculate on the differences seen: 150 sec homogenizing resulted i n lower enzyme a c t i v i t y than the shorter time, sonication showed lower protein extraction and protease a c t i v i t i e s than other methods. Where c e l l y i e l d s , 600 400 CT) • 200 10 20 30, settled cell volume (nrA) AO 50 Figure 9. Settled c e l l volume as an indicat o r of harvest weight. Harvested f i g c e l l s were used to determine the r e l a t i o n s h i p of s e t t l e d c e l l volume to c e l l dry weight ( l y o p h i l i z e d ) . Figure 10. Growth of papaya and f i g c e l l suspension cultures i n B5 medium: biomass and pH changes, open symbols, f i g ; closed symbols, papaya; dry weight from l y o p h i l i z e d c e l l s . 46 Table VI Li b e r a t i o n of protein and act i v e protease from f i g c e l l s by d i f f e r e n t e xtraction methods. Each sample consisted of 0.545 g c e l l s (dry weight), and a l l were ground with 0.1 M phosphate buffer, pH 6.0. extraction extraction protein protease a c t i v i t y method time (sec) (mg/g dry c e l l s ) (mg/g dry cells)(mg/g protein) mortar & pestle 90 15.5 6.3 0.50 mortar & pestle with sand 45 90 11.4 14.2 7.1 6.7 0.62 0.47 Polytron homogenizer 120 240 10.4 10.7 7.1 5.7 0.68 0.54 Braun sonicator 30 120 9.5 11.1 4.8 4.7 0.51 0.42 means of duplicate determinations using Bradford's protein assay. means of duplicate determinations using the modified FCC method f or protease. TCA-soluble di g e s t i o n products were equated with quantities of L-tyrosine prepared i n the same manner. 47 and thus extraction volumes, were small, the Polytron homogenizer and Braun sonicator both r e s u l t i n rapid heating of the solution, even with the use of an ice bath. This could activate enzymes e a r l i e r than desired, or cause enzyme denaturation. Because i t i s easier to grind c e l l s while maintaining a low temperature using a mortar, this was the preferred method. A l l further c e l l extractions were conducted with i g n i t e d sand i n a mortar kept on i c e , using c h i l l e d buffer (4-8 °C). P r o t e i n quantitation: Bradford's method of protein quantitation i s rapid and simple. Figure 11 shows a standard curve of absorbance at 595 nm pl o t t e d against protein content, using bovine serum albumen (BSA). A standard curve was produced for each protein assay and r e s u l t s determined by l i n e a r regression. This precaution circumvented possible v a r i a b i l i t y introduced by any change i n the reagent or experimental conditions. Fresh reagent was prepared f o r t n i g h t l y as longer storage resulted i n a loss of s e n s i t i v i t y . The protein content of c e l l extracts was found to be i n the range of 2.5 to 7% of dry weight. Other methods of protein assay were less e f f e c t i v e and/or more cumbersome. The Lowry procedure required higher concentrations of standard protein (BSA) and thus, was not s e n s i t i v e enough for detection of protein i n extracts from small c e l l harvests. Because of t u r b i d i t y and the spectrum of compounds present i n crude c e l l extracts, simple absorbance readings with 280 nm l i g h t cannot be assumed to r e f l e c t protein content alone, so t h i s method was rejected. Nitrogen content has often been used as a measure of p r o t e i n . Kjeldahl digestion gave re s u l t s ranging from 27.4 to 46.5 mg nitrogen per g weight of dried f i g c e l l s , averaging 3.58% for c e l l s grown i n B5 medium. This would indicate an average t o t a l protein content of 22.4% (n=20, s=2.98) Figure 11: Bradford's protein assay: t y p i c a l standard curve using bovine serum albumin (BSA). This p l o t f i t s the regression equation y = O.Olx + 0.02 ( r 2 = 0.99). co 49 i f the average protein nitrogen factor of 6.25 was applied. However, these nitrogen determinations were based on digestion of e n t i r e c e l l s , whereas protease assays were conducted on c e l l e x t r a cts. This method was deemed inappropriate for routine use i n extract digestion because of the time involved and the p o s s i b i l i t y of v a r i a t i o n i n the r a t i o of nucleic acids to i n t r a c e l l u l a r proteins or amino acid pools during development of i n d i v i d u a l c e l l s . As determined by Bradford's method, extracts of f i g c e l l s contained an average of 5.56% protein (n=28, s=1 .65) i n rapidly-growing c e l l suspension cultures two weeks after t r a n s f e r . At the same time, i n B5 medium without milk, the average protein content of f i g c e l l extracts was 3.53% (n=14, s=1.33). These two figures are much lower than that determined by K j e l d a h l digestions since t h i s method omits insoluble proteins i n disrupted c e l l s , and does not encompass nucleic acids and othernon-protein nitrogen. Protease quantitation: The c l e a r i n g of milk-containing agar was the e a r l i e s t i n d i c a t i o n of p r o t e o l y t i c a c t i v i t y i n plant tissue c u l t u r e s . This was evident i n explants and c a l l u s tissues of papaya, f i g , cardoon, t h i s t l e and bedstraw. C e l l suspension cultures of papaya and f i g , and liquid-grown bedstraw also demonstrated c l e a r i n g of milk-containing medium. Casein was evidently a suitable substrate, so this phenomenon was used as an i n d i c a t o r i n the s e l e c t i o n of assay methods. This decision tended to lead i n t o p o t e n t i a l dairy applications, whereas a meat-digestion assay method such as that devised by Orsi and Major (1973) would have led to an i n v e s t i g a t i o n of meat-industry a p p l i c a t i o n s . Gel d i f f u s i o n methods of enzyme detection proved i n e f f e c t i v e f o r c e l l e x t r acts. Araki and Abe's method (1980) r e l i e s on a l o c a l i z e d zone of 50 colour change i n the agar medium containing BAEE substrate. No zones i n d i c a t i n g e s t e r o l y t i c a c t i v i t y could be detected surrounding wells containing c e l l extract, though papain was detectable down to 0.1 mg/ml. The BioRad gel d i f f u s i o n t e s t i s also performed i n agar, but contains casein as the substrate. The d i f f u s i o n zone i s seen as a c l e a r area surrounding protease-containing wells. Again, no zones around c e l l extracts were evident under the s p e c i f i e d assay conditions, although standard f i c i n was detected as low as 10 ug/ml i n the presence of d i t h i o t h r e i t o l . The agar d i f f u s i o n method of Holmes and Ernstrom (1973) i s also based on casein, but i s performed i n tubes ( v e r t i c a l d i f f u s i o n ) , and r e s u l t s were s i m i l a r to the BioRad method. I t would appear that, i f protease was present, conditions of these tests were not optimal for t h e i r d i f f u s i o n through or a c t i v i t y i n an agar environment. Another protein substrate, collagen, was investigated by two methods. The azure-bound hide powder (Savage & Thompson, 1970) was s l i g h t l y digested by an extract of f i g c e l l s , equivalent to 0.03-0.04 ug f i c i n per mg c e l l dry weight extracted. Film g e l a t i n (Glenister & Becker, 1961) was also digested by enzymes i n the crude f i g c e l l extract, equivalent to 0.059 ug f i c i n per mg extracted c e l l s . These are both at the low end of the range of f i c i n concentrations with detectable a c t i v i t y . None of the above methods permits comparison with other proteases without preparation of a standard curve for each enzyme of i n t e r e s t . That i s , each assay of papaya c e l l s would require a d i l u t i o n series of standard papain, and f i g c e l l s would require a s i m i l a r series of the f i c i n standards. Fluorometric methods of protease detection tested gave unsatisfactory r e s u l t s . The method of Spencer and Spencer (1974) appeared to work well with chymotrypsin, but was subject to complications when used 51 with s u l f h y d r y l proteases. ANS fluorescence increased sharply upon addition of the c e l l extracts, possibly due to i n t e r a c t i o n of -SH reactive groups with the casein substrate. Fluorescence did not subsequently decrease. This complication, and the lack of any evidence i n d i c a t i n g p r o t e o l y t i c a c t i v i t y i n crude c e l l extracts, discouraged further use of d i r e c t f l u o r i m e t r i c methods. The procedure given by Chism et a l . (1979) c a l l s for pre-incubation of the sample with the casein substrate, p r i o r to TCA p r e c i p i t a t i o n and fluorescamine a d d i t i o n . There was a great deal of v a r i a b i l i t y among samples containing less than 1.0 unit/ml of papain. The FCC method for papain was adopted for routine quantitation of protease a c t i v i t y , modified as described i n section III-3. This method proved to be the most r e l i a b l e and se n s i t i v e one that could conveniently be applied on a routine b a s i s . One advantage of the FCC method i s that f i n a l data are derived from absorbance readings a t 280 nm and can r e a d i l y be related to quantities of a single aromatic amino a c i d . Tyrosine i s most commonly reported i n the l i t e r a t u r e , so i t was used to prepare a standard curve for each assay, as i l l u s t r a t e d i n Figure 12. C e l l extracts often contained NPN soluble i n 10% TCA which produced absorbance readings equivalent to as much tyrosine as 1 mg/ml. Thus assessment of true p r o t e o l y t i c action on the casein substrate was based on subtraction of absorbances of controls from absorbances of incubated samples. By l i n e a r regression these differences were related to the standards to give equivalent amounts of tyrosine which would have been released from casein by p r o t e o l y t i c a c t i v i t y . If aromatic amino acids, tryptophan, tyrosine and phenylalanine comprise approximately 15% of casein, then r e s u l t s of 80 ug of tyrosine released would i n d i c a t e 1.2 mg of amino acids released from casein, assuming a l l amino acids are released equally. 040 Figure 12. Ty p i c a l protease assay standard curve using L-tyrosine. This plot f i t s the regression equation y = 0.0018x - 0.01 ( r 2 = 0.99). 53 Factors i n f l u e n c i n g protease a c t i v i t y : The e f f e c t of pH on protease a c t i v i t y at 38 °C of f i c i n and f i g c e l l extract i s i l l u s t r a t e d i n Figure 13a. Although f i c i n a c t i v i t y at th i s temperature remained r e l a t i v e l y constant over the pH range 5.5 to 8.4, the f i g c e l l extract demonstrated a c l e a r pH optimum about 6.0. Temperature also had l i t t l e e f f e c t on the standard f i c i n preparation over the range of 28-67 °C, whereas the f i g c e l l extract showed a temperature optimum around 47 °C ( F i g . 13b). The influence of several compounds on f i c i n a c t i v i t y at 38 °C i s shown i n Figure 14. Two concentrations of f i c i n were tested with each compound. Only cysteine and d i t h i o t h r e i t o l (with or without EDTA) had pronounced stimulatory e f f e c t s , while thiocyanate, sodium tetrathionate and try p s i n i n h i b i t o r were i n h i b i t o r y . Glutathione, EDTA alone and SDDC resulted i n a s l i g h t increase i n absorbance over the base l e v e l s , which contained no f i c i n . Protease a c t i v i t y of the f i g c e l l extract was not affected greatly by any of the test compounds, but was stimulated s l i g h t l y by Dtt, with or without EDTA. SDDC also appeared to be stimulatory, but there was interference by colour development i n those reaction tubes. Thiocyanate, sodium tetrathionate and tr y p s i n i n h i b i t o r had e s s e n t i a l l y no e f f e c t on assay r e s u l t s . 3. Milk c l o t t i n g a c t i v i t y C l o t t i n g times of c e l l extracts were determined, according to the procedure i n section III-3, for c e l l extracts from f i v e cultures of papaya and one of f i g . Results, given i n Table VII, show the f a s t e s t c l o t t i n g time was 5.5 hr (papaya #4). The only f i g c e l l extract tested required 12 hr to c l o t milk casein. Also included i n Table VII are c l o t t i n g times required 54 £ c 8 0 1 5 0 CM II 0, 0100 o c o _Q ° 0O50l X I a 5.5 6-5 pH 7-5 ft5 E c CM 0-240 II 0> 0-180t u c o I 0-120 o &060T '0 V>-+-26 — 34 42 50 58 66 temperature (°C) Figure 13. E f f e c t of pH and temperature on a c t i v i t y of f i c i n and f i g c e l l extract. Protease a c t i v i t y was determined by the modi-f i e d FCC method (see t e x t ) . F i c i n was used at 0.42 mg/ml ( X ) . F i g c e l l s were extracted by grinding with 0.1 M phosphate buffer, (a) Casein substrate prepared i n buffers of d i f f e r e n t pH, a l l samples incubated at 40 °C. (b) Casein substrate, pH 6.0, samples incubated at 27-57 °C. 55 DITHIOTHREITOL (Dtt) CYSTEINE Dtt • EDTA EDTA GLUTATHIONE SDDC TRYPSIN INHIBITOR Dtt • TRYPSIN INHIBITOR Ca-THIOCYANATE Na-TETRATHIONATE THIOUREA CONTROL - x -o • XO-. 1 2 4 % 40 % activity 80 Figure 14. Influence of some act i v a t o r s and i n h i b i t o r s on p r o t e o l y t i c a c t i v i t y . Papain (100 ug) , cysteine designated as 100% of a c t i v i t y (—o-); f i c i n (21 ug) , Dtt designated as 100% of a c t i v i t y (-¥r); f i g c e l l extract, Dtt + EDTA set as 100% of a c t i v i t y (-*-). SDDC i s sodium d i e t h y l d i t h i o -carbamate. 56 Table VII Milk c l o t t i n g a c t i v i t y of plant c e l l extracts i n comparison to standard papain. Five d i f f e r e n t papaya c e l l cultures and one f i g c e l l culture (numbers corresp-onding to data i n Figure 7) were extracted and tested for c l o t t i n g a c t i v i t y as described i n the text. test material c l o t t i n g time (hr) (a) c e l l extracts papaya 1 13.0 papaya 2 12.5 papaya 3 15.0 papaya 4 5.5 papaya 5 18.0 f i g 4 12.0 papain standards (ug/ml) 16.3 3.5 8.2 5.0 5.4 10.8 4.1 14.0 0 >24 means of duplicate determinations 57 f o r a d i l u t i o n s e r i e s of crude papain. These standards ind i c a t e that the extract with the shortest c l o t t i n g time possesses c l o t t i n g a c t i v i t y equivalent to about 8 ug/ml papain, by l i n e a r i n t e r p o l a t i o n . Others were even less a c t i v e . Milk c l o t t i n g a c t i v i t y was not assessed routinely so no data are a v a i l a b l e regarding other c e l l cultures such as l a t e r generations. 4. Electrophoresis The greatest d i f f i c u l t y encountered i n attempts at separation of proteins i n c e l l extracts was concentration. Extracts normally used for protein and protease determinations were obtained by grinding l y o p h i l i z e d c e l l s ( i n the form of a spongy mat) i n a mortar with s u f f i c i e n t phosphate buffer to wet the t i s s u e , and the thick s l u r r i e s were then f i l t e r e d . Extracts were generally found to contain 0.2-1.2 mg protein/ml. No protein bands were detectable by electrophoresis of these c e l l e x t r a c t s . Passage of one sample through a small Sephadex G-25 column, e q u i l i b r a t e d with extraction buffer, to remove compounds possibly i n t e r f e r i n g with protein mobility did not appear to help. SDS-electrophoresis of samples prepared by b o i l i n g with urea and p-mercaptoethanol was no more succe s s f u l . The standard sample was a Sigma f i c i n preparation, twice r e c r y s t a l l i z e d , containing 0.5 mg protein/ml. Over a period of 4-5 hr electrophoresis, there was s u f f i c i e n t movement to in d i c a t e three, possibly four, poorly-separated protein bands i n the p u r i f i e d f i c i n . Trypsin was applied to the gel as a standard and was found to migrate more r e a d i l y though i t , too, showed three bands. Only one attempt was made to locate p r o t e o l y t i c bands by contact of the f i n i s h e d , unfixed gel with Bio-Rad casein protease detection agar. No c l e a r i n g was v i s i b l e i n 8 hr at 28 °C. Concentration of samples by 58 long-term contact with Carbowax 20 M (Applied Science, Pa.), 5-8 hr at 6 °C, samples being contained i n d i a l y s i s tubing, was also inadequate. In many cases, f l o c c u l a t i o n of the concentrate occurred, and none showed improved mo b i l i t y . I t i s postulated that proteins i n c e l l extracts were p h y s i c a l l y bound to other extract components, or to the polyacrylamide gel i t s e l f , thus preventing t h e i r mobility i n a gel environment while causing no interference with protein or protease assays, conducted i n f l u i d environments. This suggestion would be supported by the negative r e s u l t s obtained with agar-based protease assays. 5. Medium supplementation Four groups of nutrient supplements were investigated with respect to enhancement of protease production and a c t i v i t y i n c e l l e xtracts: low molecular weight nitrogen sources, proteins, skimmed milk and i t s components, and miscellaneous organic compounds including two a n t i b i o t i c s . Each group of materials comprised-a separate series of c e l l suspension cultures handled uniformly and each inoculated from a single c e l l suspension. Only f i g c e l l cultures were used for these studies. Nitrogen n u t r i t i o n i n f i g c e l l c u l t u r e s : The r e l a t i v e importance of the two nitrogen sources i n B5 medium was determined using complete B5, B5 with half the normal n i t r a t e concentration, B5 without n i t r a t e (ammonium only) and B5 without ammonium (n i t r a t e o nly). Each of these was also prepared with skimmed milk (3%, v/v). The r e s u l t s , presented i n Figure 15, i n d i c a t e that n i t r a t e was the most important of the nitrogen sources. Biomass, t o t a l protein and t o t a l protease a c t i v i t y were a l l lowest i n the absence of n i t r a t e . Although milk went a long way toward correcting t h i s d e f i c i e n c y i n terms of biomass, i t did not provide a s i g n i f i c a n t improvement i n protein or protease y i e l d over the non-milk media where n i t r a t e was absent. 12 T 10t 6f o E . 2 4 - O [ [N03+MILKl-t N » H NH t] MILK 5 b, II ex. in ai o z < cr INH4 •MILKkJ t 800 600 E 400 c a> .*-» o u. Q . 200 [N%*,ti<H4 [NOyMILKl 16-0 'c 3 14-0 tr I N t y N H ^ I N0 3 ]—-INH^MILKK^ CO? *> LU . II <°-o a 30 o a» O I— a. 10 Figure 15. Inorganic nitrogen supply i n f i g c e l l cultures, and int e r a c t i o n with milk. (a) E f f e c t on biomass. Shaded bars give c e l l weights from the same medium with the addition of 3% skimmed milk. £ (b) E f f e c t on pr o t e i n and protease a c t i v i t y . Range spans encompass groups of s i g n i f i c a n t l y d i f f e r e n t media, at the 1% l e v e l , using Duncan's multiple range test. 60 Several amino acids were added to incomplete B5 medium (only n i t r a t e or ammonium) i n search of keys to nitrogen metabolism that may be u t i l i z e d to stimulate production of protein and active protease. Results of c e l l harvests (Figure 16) underlined the importance of n i t r a t e as the base of nitrogen metabolism. In i t s presence, several amino acids stimulated growth while i t s absence produced generally poor growth i r r e s p e c t i v e of other supplements with the sole exception of milk. Glutamate a c t u a l l y stimulated protein synthesis i n non-nitrate medium to a l e v e l equivalent to unsupplemented n i t r a t e medium, an adequate replacement for n i t r a t e (Figure 17a). Stimulation of protease a c t i v i t y was most obvious with the addition of aspartate to n i t r a t e - c o n t a i n i n g medium, with glutamate, cysteine and possibly arginine also causing a s i g n i f i c a n t improvement i n protease a c t i v i t y , r e l a t i v e to complete B5 (Figure 17b). With the ammonium-based medium, there were no outstanding s i g n i f i c a n t differences among supplements with respect to protease a c t i v i t y . Duncan's multiple range t e s t at the 5% p r o b a b i l i t y l e v e l gave overlapping ranges over the e n t i r e data base. Only milk and cysteine were s i g n i f i c a n t l y better than arginine and alanine, a l l others f a l l i n g between and overlapping these extremes. These data indicate that the amino acids capable of stimulating p r o t e i n synthesis were also apt to increase protease a c t i v i t y . With the a d d i t i o n a l case of cysteine, the inverse was also true, high protease l e v e l s i n d i c a t i n g high protein l e v e l s . Cysteine at 5mM may have caused a c t i v a t i o n of enzymes normally produced by the c e l l s , or a c t u a l l y stimulated enzyme synthesis. I t i s doubtful that glutamate and aspartate serve only enzyme-related functions, since they caused stimulation of t o t a l protein synthesis as well as an improvement i n t o t a l p r o t e o l y t i c a c t i v i t y of 61 ALANINE ARGININE ASPARTIC ACID C Y S T E I N E GLUTAMIC ACID GLYCINE PROLINE MILK Cr rzr I •V/ H / / — ( -2 cell 4 biomass (g/l)11 1 2 Figure 16. Amino acid supplements and skimmed milk: e f f e c t on biomass in f i g c e l l suspension c u l t u r e s . Shaded bars give c e l l weights from NO^-based media, open bars from NH^ media. 150 NO • MILK 900 850 250 • G L U -• ASP 150 • A R G -• A L A -• CYS-Control-• PRO-50 cr NH, cn E O f—* MILK y G L U / • A S P A L A ARG ^Control - • C Y S PRO '145 •MILK 4 . 0 ASP-30 • GLU ^ 1 • CYS — • ARG — • A L A - ' Control -r t t • PRO—^1 'c 3 UJ o O II z a. < — NH o a* to a a> .•MILK • CYS Figure 17. Amino acid supplements and milk: e f f e c t on protein and protease a c t i v i t y i n f i g c e l l cultures deprived of e i t h e r n i t r a t e or ammonia. Nitrate-based media are on the l e f t and ammonia-based media on the r i g h t side of both (a) protein, and (b) protease. The control contained only NO^ or NH^ but no amino acids. Range spans encompass groups of a l l media d i f f e r i n g from each other at the 5% l e v e l , using Duncan's multiple range t e s t . 63 suspension-cultured f i g c e l l s . E f f e c t of proteins and peptides on f i g c e l l s : Protease a c t i v i t y of f i g c e l l cultures was not stimulated by most of the proteinaceous materials tested. Results are given i n Figure 18, in c l u d i n g two control media, one with no supplementation and one containing soluble starch, a polymer of very d i f f e r e n t chemistry. Only milk and egg albumen resulted i n protease a c t i v i t y l e v e l s s i g n i f i c a n t l y higher than B5 medium alone. On the contrary, beef extract, casein, wheat gluten, g e l a t i n and casein hydrolysate produced s i g n i f i c a n t l y lower protease a c t i v i t y i n f i g c e l l cultures (p=0.01). Starch was also i n h i b i t o r y . Causes of enzyme i n h i b i t i o n were not examined, though i t i s speculated that adsorption of p r o t e o l y t i c enzymes or associated ions by polymers could lead to interference with a c t i v i t y . Skimmed milk, being easier to work with i n media than egg albumen, was selected for further study with respect to i d e n t i f i c a t i o n of stimulatory f a c t o r s . E f f e c t of skimmed milk and i t s components on f i g c e l l s : The addition of 3% skimmed milk to B5 medium had a profound stimulatory e f f e c t on growth of papaya and f i g c e l l s . T y p i c a l protein and protease p r o d u c t i v i t i e s are shown i n Figure 19. Some of the supplements added to B5 medium i n t h i s study were derived d i r e c t l y from skimmed milk as described i n s e c t i o n III-4. Others were pure compounds added at le v e l s approximately equivalent to quantities found i n milk. The r e s u l t s , Figure 20, showed a 10-fold spread i n biomass y i e l d s from t h i s range of supplements. Whey and the retained portion of u l t r a f i l t e r e d whey both produced growth which was at least equal to that obtained with milk. Casein and e s p e c i a l l y u l t r a f i l t e r e d whey were i n e f f e c t i v e growth promoters. Of the simple supplements, only c i t r i c a c i d produced a good growth response, approaching that of milk i t s e l f . 64 SKIMMED MILK EGG ALBUMEN SOY PROTEIN I S O L A T E C O N T R O L CASEIN HYDROLYSATE B E E F E X T R A C T W H E A T G L U T E N G E L A T I N C A S E I N POWDER STARCH 5 d> n ,UJ lo z < p r o t e a s e a c f i v i t y ( u n i t s / 1 ) 2 0 Figure 18. Proteins and peptides: effect on protease activity in f i g c e l l cultures. Two controls were included, starch and unsupplemented B5 medium. Range spans encompass groups of differing at the 1% probability level (Duncan's multiple range test). Figure 19. Protein and protease a c t i v i t y i n f i g and papaya c e l l suspension cultures over 3 weeks, open symbols, f i g ; closed symbols, papaya 66 WHEY CASEIN • WHEY RETENTATE MILK CITRIC ACID UF RECOMBINED CASEIN CASEIN POWDER CONTROL LACTOSE NIACIN UF FILTRATE CALCIUM RIBOFLAVIN ID O 2 4 6 8 cell dry weight ( g / l ) 10 12 Figure 20. Skimmed milk and i t s components as supplements: e f f e c t on biomass. F i g c e l l cultures were grown i n B5 containing the above supplements. UF refe r s to u l t r a f i l t r a t i o n prod-ucts, retentate, f i l t r a t e or these recombined (1:1). Range spans encompass groups d i f f e r i n g from each other, using Duncan's multiple range test at the 5% l e v e l . 67 Pr o t e i n y i e l d was highest with skimmed milk, whey and casein+whey (recombined), followed by u l t r a f i l t r a t i o n retentate, casein (fresh and powdered) and c i t r i c acid ( F i g . 21a). C i t r i c acid led to only 57% as much protein synthesis as whole milk, and the other supplements f e l l far short of t h i s . P r o t e o l y t i c a c t i v i t y , as shown i n Figure 21b, was greatest when the milk u l t r a f i l t r a t i o n retentate was added to the medium. Milk, whey-containing media, fresh casein and c i t r a t e a l l produced s i g n i f i c a n t l y greater enzyme a c t i v i t y than other supplements. The unusually high protease a c t i v i t y achieved using u l t r a f i l t r a t i o n retentate was due to i t s addition at the same l e v e l as whole whey, although i t a c t u a l l y comprised about two-thirds of the t o t a l whey volume. In e f f e c t , t h i s resulted i n the addition of 50% more of these components than are normally added i n the skimmed milk supplement. The active component of whey was retained by the 10,000-dalton f i l t e r , so the u l t r a f i l t r a t e had no stimulatory e f f e c t on y i e l d s of biomass, protein or protease. Recombined with retentate, intermediate r e s u l t s were obtained. The effectiveness of the retained f r a c t i o n of whey was most notable i n t o t a l protease a c t i v i t y , where i t yielded 38% higher a c t i v i t y than skimmed milk. Although fresh casein and reconstituted casein powder produced s i m i l a r c e l l harvests, 6.57 and 5.26 g/1 respectively, f i g c e l l s grown with the l a t t e r demonstrated only about hal f the protease a c t i v i t y of those grown with fresh casein. C i t r i c acid produced a s i g n i f i c a n t improvement i n protease a c t i v i t y r e l a t i v e to unsupplemented B5 medium (p=0.05). Enzyme a c t i v i t y i n the citrate-supplemented f i g c e l l culture released the equivalent of approximately 400 mg of tyrosine per l i t r e of 14-day c e l l s , a l e v e l not s i g n i f i c a n t l y d i f f e r e n t from that obtained with skimmed milk, casein or whey. 68 MILK CASEIN •WHEY WHEY UF RETENTATE, UF RECOMBINED CASEIN. CASEIN POWDER' CITRIC ACID-LACTOSE CONTROL UF-FILTRATE f NIACIN —X J-700 i n cn O §3 CALCIUM RIBOFLAVI / I N ^ l E 500 c o CL + 300 100 UF RETENTATE MILK UF RECOMBINED, CASEIN CASEIN •WHEY O O ~ + 6 0 WHEY CITRIC ACID. CASEIN POWDER LACTOSE CONTROL NIACIN UF-FILTRATE CALCIUM RIBOFLAVIN 8-0 E k-o +2 0 >» •4—• O OJ in s e Q . Figure 21. E f f e c t of skimmed milk components on (a) protei n , and (b) protease a c t i v i t y , i n f i g c e l l suspension c u l t u r e s . UF r e f e r s to u l t r a f i l t r a t i o n products, retentate, f i l t r a t e , or these recombined (1:1). Range spans encompass groups of media producing s i g n i f i c a n t l y d i f f e r e n t r e s u l t s (Duncan's multiple range t e s t , p=0.05). 69 Influencing synthesis and release of p r o t e i n and protease: Table VIII includes data from two experiments to influence protein and protease p r o d u c t i v i t y by materials not d i r e c t l y r e l a t e d to c e l l n u t r i t i o n . The sulfur-containing compounds, glutathione and thiourea, were compared to B5 medium alone. Glutathione produced a 40% higher biomass, but protein and protease le v e l s were not improved. Thiourea resulted i n a decrease i n a l l three factors (biomass, protein and protease), not a growth stimulant as suggested by Erez (1978). Chloramphenicol and cycloheximide both had negative e f f e c t s on biomass, protein content and protease a c t i v i t y i n f i g c e l l c u l t u r e s . Chloramphenicol resulted i n 83% of the c e l l harvest weight obtained i n the c o n t r o l , while cycloheximide produced only 32% as much biomass. The d r a s t i c e f f e c t of cycloheximide i s a t t r i b u t e d to i t s mechanism of acti o n , interference with the role of t-RNA i n peptide bond formation at the 80S ribosomal subunit. Chloramphenicol also i n t e r f e r e s with protein synthesis, but only on mitochondrial ribosomes. There was no s i g n i f i c a n t difference i n medium protease le v e l s among samples. The presence of the detergent Span-80, di d not stimulate production of proteins i n general, and protease a c t i v i t y was somewhat lower i n t h i s sample than i n the c o n t r o l , 3.5 and 3.9 units/1 r e s p e c t i v e l y . Within each t e s t set, biomass was p a r a l l e l l e d by both protein synthesis and p r o t e o l y t i c a c t i v i t y . I t did not appear that p r o t e o l y t i c a c t i v i t y was due to rupture of dying c e l l s , r eleasing i n t r a c e l l u l a r (eg., vacuolar) proteases (B o i l e r & Kende, 1979). C e l l s exposed to cycloheximide would have been expected to s u f f e r damage, yet did not indicate higher enzyme l e v e l s than the c o n t r o l . Table VIII E f f e c t of miscellaneous organic materials on growth of f i g c e l l s i n suspension cul t u r e , and production of pr o t e i n and protease. Data from two d i f f e r e n t experiments are presented: (a) based on B5 medium, and (b) based on B5 with 3% skimmed milk. See text f o r concentrations of test materials. Relative values are based on the c o n t r o l . n= number of determinations of biomass, protein, protease. s= standard deviat ion of the mean medium (a) B5 B5 + thiourea B5 + glutathione biomass (g/D r e l a t i v e protein (mg/1) 0.86 1.00 39.3 n=4, s=0.07 n=2 0.75 0.87 22.2 n=4, s=0.10 n=2 1.20 1.39 27.0 n=2 n=2 r e l a t i v e 1.00 0.57 0.69 protease a c t i v i t y (units/1) r e l a t i v e 1.12 n=2 0.54 n-2. 0.94 n=2 1.00 0.48 0.83 (b) B5-M 6.47 1.00 n=3, s=0.41 B5-M + 4.37 0.68 chloramphenicol n=3, s=1.02 B5-M + 1.80 0.28 cycloheximide n=3, s=0.31 B5-M + 7.33 1.13 Span-80 n=3, s=1.85 407.9 1.00 n=3, s=6.57 185.4 0.45 n=2 162.2 0.40 n=3, s=3.63 425.8 1.04 n=3, s=5.35 3.91 1.00 n=3, s=0.12 3.24 0.83 n=2 1.60 0.41 n=3, s=0.30 3.507 0.90 n=3, s=0.18 71 Furthermore, f i g c e l l s grown i n B5 plus skimmed milk generally gave evidence of casein h y d r o l y s i s , by c l e a r i n g of the medium, within a week of subculture. Occasionally, c l e a r i n g could be detected i n four days. This a c t i v i t y , then, occurred well before mid-log phase and was not l i k e l y to be associated with mass c e l l death. As shown i n Figure 19, the l e v e l of p r o t e o l y t i c a c t i v i t y detected decreased a f t e r the t h i r d week, during the stationary phase, when c e l l death became an important factor i n the population. A comparison of r e s u l t s of four experiments in v o l v i n g assessment of protease a c t i v i t y i n f i g c e l l s i s given i n Figure 22 (a&b). The enzyme a c t i v i t i e s are presented i n t h e i r two component f r a c t i o n s , i n the medium ( e x t r a c e l l u l a r ) and i n the c e l l extracts ( i n t r a c e l l u l a r ) . With or without the addition of milk to B5 medium, a much larger proportion of t o t a l p r o t e i n was contained i n the c e l l s than i n the medium: average proportions of 0.95 and 0.80 i n c e l l s grown i n B5 and B5-M, r e s p e c t i v e l y . Protease a c t i v i t y was divided almost equally between c e l l s and medium, average proportions i n c e l l s being 0.44 and 0.49 i n B5 and B5-M media, r e s p e c t i v e l y . However, there was a wide scatter i n the d i s t r i b u t i o n of enzyme a c t i v i t y , the proportion i n c e l l s ranging from about 0.3 to 0.7, so i t was not possible to assay only one of these two f r a c t i o n s then multiply by a p r o p o r t i o n a l i t y constant. The i n c l u s i o n of the above t e s t compounds i n media did not appear to influence the proportion of e x t r a c e l l u l a r enzyme a c t i v i t y . 8 A B B5 A B C B5-M ~ 6 'c 3 o O 0» <U 2 8 C L A B B5 A B C B5-M re 22. In t r a - or e x t r a c e l l u l a r l o c a t i o n of protein and protease a c t i v i t y i n f i g c e l l suspension c u l t u r e s . Both media and c e l l extracts were assayed. Data from three B5-grown cultures (A,B,C) and four B5-M-grown cultures (A,B,C,D) are presented, where the l e t t e r codes r e f e r only to d i f f e r e n t experiments. S o l i d l i n e , c e l l extract; dotted l i n e , medium. 73 V. DISCUSSION 1 . Tissue d e d i f f e r e n t i a t i o n Successful u t i l i z a t i o n of plant tissue and c e l l cultures i s dependent upon means of in f l u e n c i n g c e l l growth and development. Callus cultures c o n s i s t of immature and r e l a t i v e l y unspecialized c e l l s derived l a r g e l y from cambial and parenchymatous c e l l s . Propagation of such tissues i s , of necessity, very labour i n t e n s i v e . Furthermore, tissues grown on semi-solid media are subject to varying degrees of morphogenesis due, i n part, to the s p a t i a l association of c e l l s with each other and with the medium. The desirable goal i s a system i n which a l l c e l l s are maintained i n i d e n t i c a l conditions, preferably without f l u c t u a t i o n over time, such as continuous-feed fermentors. This s i t u a t i o n requires a knowledge of c e l l growth, genetic status and metabolism, much of which has been derived, to date, from batch culture systems. Several plant species were selected for the purpose of producing p r o t e o l y t i c a l l y active c e l l suspension c u l t u r e s . These were Carica papaya, Ficus c a r i c a , Ananas comosus, Dieffenbachia amoena, D. p i c t a , Galium verum,' Cynara cardunculus and Circium arvense. Hundreds of plant species have been propagated as c a l l u s for a v a r i e t y of purposes. Those that have not, e i t h e r have not been investigated, or have p a r t i c u l a r environmental or n u t r i t i o n a l requirements that have not yet been i d e n t i f i e d or accommodated. The species selected for t h i s study were handled by the t r a d i t i o n a l methods of explant e x c i s i o n and incubation on two of the most common tissue culture media i n use today, MS (Murashige & Skoog, 1962) and B5 (Gamborg et a l . , 1968). Developing c a l l i were incubated under a single environmental regime: 28°C i n t o t a l darkness. Over the f i r s t four to f i v e months of e s t a b l i s h i n g 74 c u l t u r e s , only a few variables were exercised. These were the plant auxins (IAA, NAA, 2,4-D, 2,4,5-T, and p-cpa), transfer time (at c a l l u s i n i t i a t i o n , a f t e r 2 weeks, a f t e r 5 weeks), inoculum siz e (2-15 mm i n diameter), and medium supplements (yeast extract, casein hydrolysate, and skimmed m i l k ) . V i s u a l examination of growing tissues determined the most suitable conditions f or those species which did c a l l u s and develop i n v i t r o . C a l l u s cultures were obtained from papaya, f i g , dumbcane, cardoon and t h i s t l e . The f i r s t two of these showed the f a s t e s t growth. Explants from pineapple, dumbcane and mature cardoon were a l l subject to heavy endogenous contamination, and no pineapple explants survived more than three weeks. Bedstraw presented an unusual reaction, i n that seeds developed no c a l l u s but stem explants resulted i n a non-callus outgrowth to produce a r o o t - l i k e mass. Fig and papaya tissues responded equally well to MS and B5 media. Of the auxins, IAA alone was less e f f e c t i v e than the remainder, but could be used i n combination with the phenoxyacetic acids, so p-cpa was a r b i t r a r i l y adopted. The best transfer i n t e r v a l varied from one c a l l u s to another, but appeared to be su i t a b l e about one week a f t e r rapid outgrowth of f r i a b l e c a l l u s . This generally resulted i n transfers a f t e r 20-30 days, with the minimum fragment transferred s u c c e s s f u l l y being about 5 mm i n diameter. Both yeast extract and casein hydrolysate i n h i b i t e d c a l l u s development, the l a t t e r r e s u l t i n g i n browning of t i s s u e s . Skimmed milk was an e f f e c t i v e growth stimulant. This supplement had been i n use previously as an in d i c a t o r of p r o t e o l y t i c a c t i v i t y i n papaya c a l l u s cultures (Townsley, unpublished). Thus, skimmed milk i n agar media provided an e f f e c t i v e rapid method for determination of p r o t e o l y t i c enzyme production. 75 C a l l u s i n i t i a t i o n from papaya explants has also been reported from other laboratories (Medora et a l . , 1973; Arora & Singh, 1978; L i t z & Conover, 1978; Medhi & Hogan, 1976). Plant propagation was the goal of L i t z and Conover, while Medora's group reported the presence of proteases i n t h e i r papaya c a l l u s cultures (Medora et a l . , 1973; Bilderback et a l . , 1976; Mell et a l . , 1979). Pineapple has also been used for micro-propagation, but t h i s work bypasses c a l l u s formation by use of a p i c a l and a x i l l a r y buds, thus requiring only a continuation of the natural morphogenetic sequence (Mathews et a l . , 1976). Development of c a l l u s cultures from f i g has not been previously reported. 2. C e l l suspension cultures For economic reasons, the establishment of c e l l suspension cultures i s r e a l l y the f i r s t step towards large-scale production of p l a n t c e l l s or t h e i r metabolites (Dougall, 1980). The advantages of suspension culture systems include: faster growth rate, uniformity of c e l l environment and inoculum, s i m p l i c i t y of microscopic examination, ready a l t e r a t i o n of the medium and the p o s s i b i l i t y of d i r e c t p l a t i n g for cloning purposes (Widholm, 1980). Fragments of the r o o t - l i k e outgrowth from bedstraw explants transferred to l i q u i d medium continued to develop i n the same d i f f e r e n t i a t e d form, r e s u l t i n g i n large tangled masses. Microscopic examination showed no tendency to c a l l u s formation and l i t t l e root-cap c e l l sloughing. Transfer of medium containing only free c e l l s resulted i n no further growth, so i n v e s t i g a t i o n of these cultures was discontinued. Callus cultures of f i g , papaya and t h i s t l e grew well enough to attempt suspension c u l t u r e . C a l l i were dissected and transferred to l i q u i d 76 medium of the same composition and maintained on rotary shakers, again at 28°C i n t o t a l darkness. Because t h i s t l e grew poorly, even i n milk-contain-ing medium," suspension culture of this species was not pursued f u r t h e r . Papaya c a l l u s broke up slowly, forming a fine c e l l suspension by the t h i r d t r a n s f e r , while f i g c e l l s r e a d i l y dispersed within two weeks of introduction to l i q u i d medium. Neither species presented the common problem of c e l l aggregation. Microscopic examination revealed single c e l l s and small clumps up to about 20-30 c e l l s i n suspension cultures beyond the t h i r d t r a n s f e r . When skimmed milk was present i n the medium, cl e a r i n g of the milk t u r b i d i t y could be detected i n 5-10 days and thick c e l l s l u r r i e s were formed i n 14-20 days. I t was apparent that Gamborg's B5 medium, p a r t i c u l a r l y with the addition of skimmed milk (at a rate of 3%,v/v), was n u t r i t i o n a l l y adequate for growth. In terms of dry weight, a 50-65% conversion of sucrose was not unusual over a two-week period. Papaya and f i g c e l l suspension cultures have not previously been reported i n the l i t e r a t u r e , and the rapid, luxuriant growth attained by both was remarkable. 3. Determination of biomass, protein and protease C e l l harvest y i e l d i s the most common method of assessing growth i n plant c e l l cultures (Rose & Martin, 1974). If dry weights are determined, t h i s i s also the most accurate measure a v a i l a b l e . Settled c e l l volumes and fresh weight y i e l d s have also been used as growth i n d i c a t o r s ( N i c k e l l & Maretzki,1969; Byrne & Koch 1962). Because water content of harvested c e l l s i s d i f f i c u l t to standardize, fresh weight values can be highl y v a r i a b l e . Settled c e l l volumes from f i g cultures were found to accurately r e f l e c t dry weight during the e a r l y phases of growth, but the l a t t e r method was selected for i t s r e l i a b i l i t y and g e n e r a l i z a b i l i t y . Papaya 77 and f i g c e l l s were found to be very d i f f e r e n t i n size and structure, so i t was supposed that they would pack d i f f e r e n t l y . Gamborg et a l . (1968) used c e l l s dried 18-20 h i n a vacuum oven at 60°C, as others have since. Kato and Asakura (1981) are among those who have used l y o p h i l i z e d c e l l s . Due to the p o s s i b i l i t y of heat l a b i l e proteases and the p o t e n t i a l f o r endogenous pr o t e o l y s i s , a l l harvested c e l l s were freeze-dried to obtain biomass data p r i o r to e x t r a c t i o n . These weights bore a constant r e l a t i o n s h i p to oven-dried weights from both papaya and f i g c e l l suspensions. Proteins are considered primary metabolites of plant c e l l s i n culture and t h e i r production i s thus more c l o s e l y linked to general n u t r i t i o n a l status and rapid growth than are products of secondary metabolism. Plant c e l l s i n s i t u contain approximately 5% protein (db) i n non-storage organs (Thomas & Davey, 1975). In seeds and tubers, protein content i s more commonly 10-20% of the dry weight. Suspension-cultured f i g and papaya c e l l s grown i n B5 medium with or without milk were generally found to contain 3.5-7.0% protein (x=4.55, n=45) i n crude extracts. This range i s comparable to that reported by Gamborg and Finlayson (1969), a range of 1.4-8.7% protein i n fourteen species cultured i n v i t r o . If K j eldahl nitrogen values obtained for f i g c e l l s were represen-t a t i v e , averaging 3.58% of the dry weight, then i t must be surmised that approximately 80% of the nitrogen i s either non-extractable or non-proteinaceous. It could be bound i n the c e l l wall and membranes, or present as nucleic acids or other non-protein nitrogen (NPN) i n the extracts. High absorbance values of unincubated TCA-soluble reaction mixtures i n protease assays were an i n d i c a t i o n of high NPN content of c e l l e x t r a cts. This p o s s i b i l i t y was not considered i n c a l c u l a t i o n of protein from Kjeldahl nitrogen by Gamborg and Finlayson (1969). The range of nitrogen contents 78 i n f i g c e l l s was not p a r t i c u l a r l y high i n comparison to other l i t e r a t u r e values, lower than the reported 5-7% i n tobacco c e l l s (Kato & Asakura, 1981) and 8.3% i n r i c e c e l l s ( C i f f e r i et a l . , 1980). The l a t t e r authors reported that suspension-cultured r i c e c e l l s contain 46% protein (db), a very high f i g u r e i n comparison to f i g c e l l s and even to r i c e grain, which i s 6-8% p r o t e i n . Several methods of p r o t e i n assay were considered for routine use i n t h i s p r o j e c t . The method of choice was the dye-binding technique of Bradford (1976). I t was found to be s u f f i c i e n t l y reproducible and s e n s i t i v e for t h i s work, but i s e s p e c i a l l y to be praised for i t s s i m p l i c i t y and r a p i d i t y . Most work d e t a i l i n g enzyme synthesis and a c t i v i t y i n c e l l suspension cultures has dealt with the enzymes involved i n normal metabolic functions, and l i t t l e information e x i s t s with respect to enzymes with p o t e n t i a l commercial a p p l i c a t i o n . Reports of proteases from papaya c a l l u s t i s s u e s i n v i t r o are the exception, a r i s i n g from a group of researchers i n the United S t a t e s — D . Bilderback, D.E. Bilderback, R. Medora, G.P. Mell, J . Ong and J.M. Campbell. I t i s mainly t h e i r work which stimulated the present study. They produced papaya c a l l u s cultures, then l y o p h i l i z e d and extracted these to t e s t for protease a c t i v i t y on a v a r i e t y of substrates. The authors used the assay method for papain given i n the Food Chemicals Codex (National Academy of Sciences, 1966). Unfortunately, r e s u l t s were reported i n absorbance units, and thus could not r e a d i l y be compared herein. The present work began with an assessment of a l t e r n a t i v e protease detection methods. Adapting the g e l a t i n digestion method of G l e n i s t e r and Becker (1961) to plant c e l l extracts provided the f i r s t evidence that p r o t e o l y t i c a c t i v i t y v i s u a l i z e d by the c l e a r i n g of milk i n l i q u i d media was 79 extractable and s u f f i c i e n t l y stable f o r exogenous assay. This method, however, i s not precise enough to be useful i n comparison of c e l l cultures grown i n media with only s l i g h t modifications. Three agar d i f f u s i o n methods (Araki & Abe, 1980; Holmes & Ernstrom, 1973; "Protease detection k i t " from Bio-Rad) a l l f a i l e d to detect a c t i v i t y that could be quantitated by the Food Chemicals Codex method. This i s believed to be due to the f a i l u r e of the enzyme(s) to pass through the agar medium, thus no zones of c l e a r i n g , t u r b i d i t y or colour change could be seen at any distance from the o r i g i n . Fluorescence detection methods for proteases have received much attention i n recent years -because of t h e i r high s e n s i t i v i t y (Udenfriend e t a l . , 1972; Schwabe, 1973). The technique reported by Spencer and Spencer (1974) r e l i e s on the loss of fluorescence as the substrate-bound ANS reagent i s released upon p r o t e o l y s i s . Following the rapid i n i t i a l increase i n fluorescence upon addition of the plant c e l l extract, no s i g n i f i c a n t decline could be seen over 30 minutes. I t was postulated that the cysteine a c t i v a t o r , or other substances present i n the extracts caused an increase i n t u r b i d i t y of the reaction mixture due to aggregation of the casein substrate. The c o n t r o l reaction, using chymotrypsin, demonstrated the predicted loss of fluorescence. Another complication which arose was the operating temperature of the assays. For short-time incubation, i t was c l e a r that a temperature higher than ambient would be necessary. Another method, described by Chism et a l . (1979), circumvented t h i s problem by preincubation of the substrate-enzyme mixture, followed by fluorescamine l a b e l l i n g of TCA-soluble peptides. This approach appeared to work i n the range of 50-500 ug tyrosine and with papain concentrations at the higher end of the range previously employed (200 ug per reaction tube). With the 80 amount of time, labour and cost involved, t h i s method held no advantage over the Food Chemicals Codex assay. A few modifications were made to the Food Chemicals Codex method for papain for ease of preparation and q u a n t i t a t i o n . The activator used, d i t h i o t h r e i t o l rather than cysteine, was added at the s t a r t of the incubation period instead of being included i n the extraction buffer. This s i m p l i f i e d advance preparations and assured a minimum of p r o t e o l y t i c a c t i v i t y i n the extracts p r i o r to determination of protein content. Secondly, because a c t i v i t y was very low, i t was convenient to decrease the substrate concentration from 10 to 2 mg/ml, a step which improved the p r e c i s i o n of absorbance readings, p o s s i b l y by improving the e f f i c i e n c y of casein d i g e s t i o n . Proteolysis i n extracts of l y p h i l i z e d c e l l s was low, so the 0.2% casein substrate would r a r e l y have approached complete h y d r o l y s i s . The incubation period was also extended to two hours, from one, increasing the differences between t e s t and c o n t r o l samples. This would have been done more e f f e c t i v e l y by increasing the incubation temperature to 47°C. Unfortunately, at the time that temperature s t a b i l i t y of the protease was determined, a great deal of data had been c o l l e c t e d a t 40°C, as recommended i n the o r i g i n a l method. This would not have been t r u l y comparable to res u l t s obtained at a higher incubation temperature. Despite the lengthy and complicated procedure, t h i s method was adopted for routine assay. I t was, i n part, compensated for by the s i m p l i c i t y of protein determinations done concurrently. A s i m i l a r protease assay method involved addition of a ninhydrin reagent to the TCA supernatants for absorbance readings a t 570 nm (Reimerdes & Klostermeyer, 1976). Besides being more cumbersome i n that yet another reagent would be required and a l l supernatants would have to be pH-adjusted, there was interference by the enzyme activators such as cysteine or d i t h i o t h r e i t o l . 81 Milk c l o t t i n g a c t i v i t y has sometimes been mistakenly understood as an i n d i c a t o r of p r o t e o l y t i c a c t i v i t y (Pozsar-Hajnal et a l . 1974; Pozsar-Hajnal & Hegedus, 1975). Although commonly present i n protease preparations, milk c l o t t i n g a c t i v i t y i s not necessarily d i r e c t l y c o r r e l a t e d with p r o t e o l y t i c a c t i v i t y , as demonstrated by Skelton (1971) using papain. This d i s t i n c t i o n may occur due to a difference i n optimum conditions for the two a c t i v i t i e s , and i t could be possible to favour one or the other by manipulation of incubation conditions. Whitaker (1959) reported on the e f f e c t s on pH, temperature, substrate concentration and i n h i b i t o r s on the milk c l o t t i n g a c t i v i t y of f i c i n , following s i m i l a r methodology to that devised by Balls and Hoover (1937) f o r papain. To date, however, few plant species have provided s u f f i c i e n t milk c l o t t i n g a c t i v i t y for the manufacture of cheese without also having too much p r o t e o l y t i c a c t i v i t y . Extracts of papaya and f i g c e l l cultures yielded c l o t t i n g times far i n excess of a reasonable range for cheese manufacture. An i n v e s t i g a t i o n of conditions conducive to milk c l o t t i n g may yet improve the p o t e n t i a l for f i g c e l l cultures i n t h i s f i e l d . A long c l o t t i n g time may be desirable i f conditions preventing formation of b i t t e r peptides are defined. I t was unfortunate that cardoon tissues could not be r e a d i l y propagated i n suspension culture, since t h i s i s one of two plants that have been used for t r a d i t i o n a l cheese-making ( V i e i r a de Sa & Barbosa, 1970a&b). I n s u f f i c i e n t young material was a v a i l a b l e for extraction to t e s t milk c l o t t i n g a c t i v i t y . P r o t e o l y t i c a c t i v i t y i n f i g and papaya suspension cultures became evident i n 5 days at the e a r l i e s t , by a loss of opacity i n milk-containing media. Protease determinations over time established 14 days as the optimum time for pr o d u c t i v i t y , hence, for harvest. In media without milk, t h i s was 82 s l i g h t l y delayed i n papaya cultures, to 16-18 days, but unchanged i n f i g c u l t u r e s . Location of enzyme a c t i v i t y , assessed only with f i g c e l l c ultures, showed no s i g n i f i c a n t difference between B5 and B5-M with respect to the proportions of i n t r a - and e x t r a c e l l u l a r a c t i v i t y . I t was c l e a r that the e n t i r e culture ( c e l l s and medium) would have to be l y o p h i l i z e d , or otherwise concentrated, i n order to obtain the f u l l p r o t e o l y t i c a c t i v i t y . One s o l u t i o n might be achieved by a l t e r i n g the c e l l permeability, as suggested by Brodelius and Mosbach (1982) and Reese and Maguire (1969). Only one material, the detergent Span-80, was tested i n t h i s regard. No s i g n i f i c a n t difference i n medium protease a c t i v i t y was detected between Span-grown and milk-grown f i g c e l l s . The p o s s i b i l i t y s t i l l e x i s t s that a r e v e r s a l of t h i s p r i n c i p l e might be applied, i e . , addition to media of substances which prevent c e l l leakage of enzymes, thereby necessitating only the harvest of c e l l s and requiring much less e f f o r t to concentrate. There appear to be no reports i n the l i t e r a t u r e t esting t h i s hypothesis. Assay d i f f i c u l t i e s : Several p o t e n t i a l problem areas may be encountered i n dealing with crude extracts of dried plant c e l l s and i n transforming y i e l d s of small volume cultures to large volumes. In t h i s study, harvest weights were determined by c o l l e c t i o n of c e l l s on Miracloth then l y o p h i l i z i n g the mass removed by scraping of the f i l t e r . Depending upon the t o t a l harvest weight obtained, the c e l l s adsorbed into the f i l t e r may sometimes have constituted a s i g n i f i c a n t proportion of the t o t a l weight. By estimation from pre-weighed f i l t e r s , t h i s loss was u n l i k e l y to be more than 5%. Also with respect to weights, l y o p h i l i z a t i o n was chosen i n order to better preserve endogenous protein and protease a c t i v i t y , although other 83 methods of drying yielded harvest weights about 10% lower (papaya) and 6% lower ( f i g ) . Where calcu l a t i o n s based on biomass were made, the l y o p h i l i z e d weight was used. However, most data are reported on the basis of culture volume. This was done p a r t l y to avoid estimation of true dry weight from l y o p h i l i z e d weight, but mainly to provide a c l e a r e r picture of o v e r a l l productive capacity with a view to fermentor-scale production. E x t r a c t i o n of dried material was performed as r a p i d l y as possible i n the cold to prevent loss of e i t h e r proteins or proteases due to p r o t e o l y s i s . The thoroughness of the adopted extraction procedure was not investigated, however, and i t i s possible that t h i s may have varied from one sample to another. In compensation, a grinding time of 60-75 seconds using a buffer:dry weight r a t i o of about 15:1 became standard procedure. The presence of endogenous protease i n h i b i t o r s i n c e l l cultures was a r e a l p o s s i b i l i t y . Such compounds could have been active during protease assays. Materials shown to i n h i b i t a c t i v i t y of standard f i c i n and/or papain preparations included t r y p s i n i n h i b i t o r , calcium thiocyanate, sodium tetrathionate and thiourea. Sodium tetrathionate i s a known i n h i b i t o r of s u l f h y d r y l enzymes, and was included as a c o n t r o l : both papain and f i c i n were strongly i n h i b i t e d i n i t s presence, as expected. The i n h i b i t o r y e f f e c t of t r y p s i n i n h i b i t o r was somewhat s u r p r i s i n g since the a c t i v i t y of plant proteases i s not generally decreased by t h i s material (Fossum & Whitaker, 1968). I t i s believed that most plants probably contain protease i n h i b i t o r s of some sort as a mechanism for regulation of metabolic enzymes associated with p a r t i c u l a r developmental periods, such as seed germination (Ryan, 1973). In fact, papaya contains endogenous isothiocyanates, shown herein to be i n h i b i t o r y to a c t i v i t y of both papain and f i c i n . These have 84 not yet reported been i n f i g (Tang, 1974; Tang & Tang, 1976). The same mechanism may be responsible for the strong i n h i b i t i o n of papain and f i c i n reported here. Cyanide i s known to be an a c t i v a t o r of both enzymes (Arnon, 1970; Liener & Friedenson, 1970), but the thiocyanate form i s i n h i b i t o r y to t h e i r a c t i o n . Ascorbic acid i s present i n papaya at the 1% l e v e l , and may also be produced by c e l l s i n v i t r o . Skelton (1968) demonstrated the i n h i b i -t i o n of papain a c t i v i t y i n the presence of 2.3 mM ascorbic acid, but without an enzyme a c t i v a t o r . Because of t h i s report and one by Whitaker (1959) ascorbic acid was not tested for possible stimulatory e f f e c t s on standard enzymes or e x t r a c t s . L a s t l y , thiourea was included both because i t i s a sulfur-containing compound, and because of a report by Erez (1978) that i t stimulates growth of plant tissue i n c u l t u r e . Thiourea was included i n l i q u i d media i n e a r l y attempts to e s t a b l i s h c e l l suspension cultures f o r t h i s reason, but because i t appeared to produce no improvement i n growth, i t was subsequently omitted. If i t could stimulate enzyme production, however, i t may have become a useful addition despite having no e f f e c t on growth per  se. This t e s t showed i n h i b i t i o n of papain by thiourea ( f i c i n was not tested), and thus dispensed with such a theory. In conclusion, the most e f f e c t i v e enzyme activators were those used i n other reports, cysteine and d i t h i o t h r e i t o l . F i g c e l l extracts indicated l i t t l e e f f e c t of e i t h e r sodium tetrathionate, trypsin i n h i b i t o r or calcium thiocyanate, with thiourea being s l i g h t l y i n h i b i t o r y . This leaves room for the p o s s i b i l i t y that proteases are already functioning under i n h i b i t o r y conditions. D i t h i o t h r e i t o l was a stronger a c t i v a t o r of proteases i n the f i g c e l l extract than was cysteine. If natural protease i n h i b i t o r s were present i n the cultures and extracts, o t h e i r e f f e c t s were at l e a s t p a r t i a l l y overcome by these two a c t i v a t o r s . 85 Sodium d i e t h y l dithiocarbamate showed an apparent stimulatory e f f e c t , but t h i s can be a t t r i b u t e d to an a r t e f a c t u a l colour change achieved upon heating with the c e l l e x t r a c t . The f a c t that these reactions do not e n t i r e l y agree with those of standard f i c i n suggests that p r o t e o l y t i c a c t i v i t y of f i g c e l l extracts i s due, at l e a s t i n part, to proteases other than f i c i n . Protease assays were performed using Hammarsten casein as the substrate. No other substrates were used on a routine basis, though g e l a t i n d i g e s t i o n was demonstrated. P r o t e o l y t i c enzymes i n plants often appear to be associated with protein turnover (Ryan, 1973). For t h i s reason, they may be s p e c i f i c to the type of storage protein present i n c e l l s , such as the g l o b u l i n - s p e c i f i c protease i n pumpkin seeds (Spencer & Spencer, 1974). Although t h i s s p e c i f i c i t y does not seem to apply to the f i c i n and papain extracted from f r u i t latex for commercial use, i t may play a role i n p r o t e o l y t i c a c t i v i t y detected i n plant c e l l suspension c u l t u r e s . The proteases present may have shown higher a c t i v i t y f o r more suitable substrates. However, i t can be argued that only protein substrates of commercial importance should be used for assay i f the eventual a p p l i c a t i o n i s intended for t h i s purpose. No progress was made i n i d e n t i f i c a t i o n of proteases from c e l l c u l t u r e s . Electrophoretic separation of proteins i n crude plant c e l l extracts should have made an important contribution toward answering questions of i d e n t i t y and m u l t i p l i c i t y of p r o t e o l y t i c enzymes. I t was assumed that proteases synthesized i n c e l l culture would be present i n larger quantity than the s t r u c t u r a l proteins and normal metabolic enzymes i n c e l l s . Although i t was possible to v i s u a l i z e protein bands i n a "pure" f i c i n preparation, almost no protein mobility was evident i n crude f i g c e l l e xtracts, even when concentrated as much as four times. Mereaptoethanol and 86 SDS treatments did not s i g n i f i c a n t l y improve mobility. There are at l e a s t two possible explanations for the lack of success. There may have been material present i n the extracts which bound proteins, preventing t h e i r migration into the acrylamide gel, possibly the same forces p r o h i b i t i n g use of gel d i f f u s i o n methods of protease detection. A l t e r n a t i v e l y , extracts containing 0.3-1.2 mg/ml t o t a l protein, as determined by Bradford's method, may not contain s u f f i c i e n t quantities of any p a r t i c u l a r protein to be v i s i b l e as a banding pattern using the same s t a i n . 4. Nitrogen n u t r i t i o n i n f i g c e l l cultures I t was postulated, on the basis of dramatically improved growth i n the presence of 3% skimmed milk, that proteins or other nitrogenous compounds were responsible for increased p r o d u c t i v i t y of suspension-cultured f i g and papaya c e l l s . The approach taken to medium nitrogen supplementation was the addition of amino acids or other nitrogenous materials to the basal B5 medium. Growth was the primary i n d i c a t o r of s u i t a b i l i t y of the supplements. Production of protein and protease were also monitored, since growth alone could not ensure enzyme synthesis. There i s a large body of information i n the l i t e r a t u r e on the subject of nitrogen n u t r i t i o n and metabolism i n plant c e l l cultures, as well as i n i n t a c t plants (Dougall, 1977, 1980; Hewitt & Cutting, 1977). I t has been estimated that 50-90% of t o t a l plant c e l l nitrogen i s assimilated from environmental sources, the actual proportion being affected by a v a i l a b i l i t y and ease of metabolism (Hewitt et a l . , 1977). P r e f e r e n t i a l sources of inorganic nitrogen vary among species, and nitrogen metabolism r e f l e c t s the a v a i l a b l e sources (Tischner & Lorenzen, 1980). Reports indicate that the majority of plant c e l l cultures u t i l i z e 87 ammonium nitrogen before reduction of n i t r a t e (Gamborg & Shyluk, 1970; Bayley et a l . , 1972; Dougall, 1980). This appeared to be the case i n both papaya and f i g c e l l cultures as well, evident i n the i n i t i a l decline and subsequent r i s e i n pH of the growth medium (Gamborg et a l . , 1968; Bayley e t a l . , 1972). Gamborg pointed out, i n the same pu b l i c a t i o n , that ammonium ions at l e v e l s greater than 2mM depressed growth of soybean c e l l s i n suspension cul t u r e , the same maximum experienced i n i n t a c t plants ( M i f l i n & Lea, 1976). Only one paper encountered recommended a higher ammonium l e v e l , 1OmM, possibly related to his goal of inducing embryogenesis i n carrot c e l l c ultures (Wetherell & Dougall, 1976). Ammonia i s assimilated i n i n t a c t plants v i a two pathways, depending upon i t s concentration i n the environment: ei t h e r by glutamine synthetase a c t i v i t y , forming glutamine from glutamate, and/or by glutamate synthase a c t i v i t y , forming glutamate from oc-ketoglutarate (Rhodes et a l . , 1976; Koiwai & Noguchi, 1972). The r a t i o of th e i r a c t i v i t i e s i s dependent on ammonium a v a i l a b i l i t y , the former being most important at low ammonia concentrations, but the l a t t e r becoming in c r e a s i n g l y active at higher le v e l s of ammonia, possibly to prevent ammonia t o x i c i t y . Some plant c e l l s i n suspension culture, such as wheat, appear to have no s p e c i f i c requirement for ammonia, while soybean c e l l s are an example of a species which grows very poorly without i t (Bayley et a l . , 1972). Gamborg and Shyluk (1970) reported that soybean c e l l s would grow quite well on ammonium s a l t s as the sole nitrogen source i f Krebs cycle acids were also supplied. The medium used herein, B5, contained 25 mM n i t r a t e i n addition to 2 mM ammonium ions [1 mM ( N ^ ^ S O g ) ] . Ojima and Ohira (1978) stated that r e s i d u a l n i t r a t e i s usually s u f f i c i e n t for growth even a f t e r a l l the carbohydrate has been metabolized, although they also recommend 40 mM 88 n i t r a t e to enhance growth of suspension-cultured r i c e c e l l s . U t i l i z a t i o n of n i t r a t e requires a c t i v a t i o n of n i t r a t e reductase, which i n turn requires adequate medium molybdenum and calcium, and quite possibly the induction of a n i t r a t e - s p e c i f i c permease (Oaks, 1977). Nitrate may be reduced to n i t r i t e i n the cytosol and reduced further, to ammonia, most l i k e l y i n p l a s t i d s such as chloroplasts ( M i f l i n & Lea, 1976). The potassium n i t r a t e supplied i n B5 medium i s probably used to produce both n i t r i t e and potassium malate (DeKock et a l . , 1977). If amino acids are to be synthesized from n i t r a t e , both ATP and NADPH are required. This energy and reducing power. Fowler has suggested (1978), could be derived from the pentose phosphate pathway of carbohydrate metabolism and would therefore be unavailable under conditions carbohydrate d e f i c i e n c y . In the i n t a c t plant, n i t r a t e need not be processed immediately, but can be transported through the xylem or stored i n vacuoles without p r i o r reduction (Oaks, 1977). Storage i n i n t r a c e l l u l a r pools may occur i n single c e l l culture as w e l l . The reduction products, ammonia and amino acids, and the cytoplasmic n i t r a t e pool a l l serve to i n h i b i t further n i t r a t e uptake. Inadequate carbohydrate supplies accomplish the same e f f e c t (Bidwell et a l . , 1964). E f f e c t on biomass: I t was evident, from the growth of f i g c e l l s i n B5 medium lacking ammonium, that n i t r a t e was an adequate nitrogen source. .On the contrary, ammonia was i n s u f f i c i e n t as the sole nitrogen supply, not a s u r p r i s i n g r e v e l a t i o n due to the low concentration i n comparison to n i t r a t e . What was unexpected, though, was the i n a b i l i t y of i n d i v i d u a l amino ac i d supplements, and even milk, to compensate for the nitrogen shortage. Apparently, the sum of nitrogen present i n the medium i s c l o s e l y linked to growth, but not i n a l i n e a r manner. Fig c e l l growth i n the t o t a l of 7 mM nitrogen supplied by ammonium su l f a t e plus glutamate, for example, exceeded 89 h a l f the y i e l d from medium containing 25 mM nitrogen i n the form of n i t r a t e alone. S i m i l a r l y , the combination of ammonia and n i t r a t e nitrogen i n B5 medium produced a higher biomass y i e l d than the sum of the i n d i v i d u a l y i e l d s . The n i t r a t e concentration i n B5 i s not excessive for f i g c e l l suspension cultures, since a one-half reduction i n t h i s nutrient resulted i n a concomitant decline i n y i e l d . E f f i c i e n t n i t r a t e u t i l i z a t i o n , however, seemed to have required the presence of a reduced source of nitrogen, e i t h e r ammonia or some of the amino acids. Amino acid metabolism i n suspension-cultured plant c e l l s has been reviewed by Dougall (1980) and Ojima and Ohira (1978). Amino acids are a reduced form of nitrogen but cannot e n t i r e l y replace ammonium s a l t s . Glutamine-grown soybean c e l l s were found to produce only about two-thirds the y i e l d obtained i n the presence of ammonia, for example (Gamborg et a l . , 1968). The i n c l u s i o n of amino acids i n plant tissue culture media has wide-ranging e f f e c t s on growth from strongly i n h i b i t o r y to strongly stimulatory, depending on the plant species, inorganic nitrogen n u t r i t i o n , n u t r i t i o n a l h i s t o r y of the culture and the p a r t i c u l a r amino acids tested (Maretzki & Thorn, 1978; M i f l i n et a l . , 1977). Following are a few examples: (a) r i c e c a l l u s grew best when supplied with any of alanine, arginine, asparagine, glutamic acid or p r o l i n e (Furuhashi & Yatazawa, 1970); (b) soybean root c e l l s required the combination of l y s i n e , arginine, h i s t i d i n e , aspartic and glutamic acids or protein hydrolysate for best y i e l d s (Gamborg et a l . , 1968); (c) the most useful amino acids for growth of sugarcane c e l l s were found to be arginine, h i s t i d i n e , aspartic and glutamic acids ( N i c k e l l S Maretzki, 1969); (d) alanine and aspartic acids stimulated growth of Datura  innoxia c e l l s while most other amino acids fed i n d i v i d u a l l y were i n h i b i t o r y to growth (Fukunaga & King, 1982). 90 There are also at l e a s t two reports of i n h i b i t o r y e f f e c t s of some amino acids being reversed by others. Cattoir-Reynaerts et a l . (1981) found that lysine and threonine i n h i b i t i o n of growth i n barley cultures was reversed by the addition of arginine. Growth of tobacco, tomato, carrot and soybean c e l l s i n n i t r a t e medium was i n h i b i t e d by a common spectrum of amino acids, with strongest e f f e c t s on each species being seen with d i f f e r e n t p a r t i c u l a r amino acid supplements. The same authors (Behrend & Mateles, 1975) reported the a b o l i t i o n of such i n h i b i t o r y e f f e c t s , most notably by the addition of arginine or i s o l e u c i n e . A s e l e c t i o n of amino acids, based on these reports i n the l i t e r a t u r e , were added i n d i v i d u a l l y to B5 medium d e f i c i e n t i n e i t h e r n i t r a t e or ammonium. In the presence of n i t r a t e , s i x amino acids stimulated growth above the unsupplemented l e v e l , with only glycine producing a decrease i n biomass. Aspartic and glutamic acids resulted i n yields more than double that achieved with ammonium, a l l i n the presence of n i t r a t e . This indicated a near complete u t i l i z a t i o n of the amino acid nitrogen, presented a t 5 mM i n place of ammonium s u l f a t e nitrogen at 2 mM. By the same reasoning, cysteine was u t i l i z e d at one-sixth, and alanine at about one-third the e f f i c i e n c y of ammonium a s s i m i l a t i o n . Because no combinations of amino acids were tested, antagonism to growth i n h i b i t i o n could not be i n v e s t i g a t e d . In n i t r a t e - d e f i c i e n t media, f i g c e l l growth was poor with or without supplementation by the t e s t amino acids. However, the stimulatory e f f e c t s of glutamic and aspartic acids were again noted. Biomass y i e l d s i n ammonium media were a l l less than 1 g/1 so i t would not be r e a l i s t i c to c a l c u l a t e percentages of growth i n h i b i t i o n below the unsupplemented ammonium medium. Fig c e l l s appear to require both n i t r a t e and a source of reduced nitrogen for best growth. The l a t t e r was adequately supplied by ammonium or 91 the a c i d i c amino acids. While skimmed milk was stimulatory to growth i n both n i t r a t e and ammonium media, i t produced a greater biomass y i e l d i n the former. No other supplement to B5 medium tested resulted i n equivalent stimulation of growth. Improved nitrogen n u t r i t i o n may, however, induce protein synthesis, p o t e n t i a l l y enhancing production of p r o t e o l y t i c enzymes. C e l l yields alone could not provide t h i s information for f i g c e l l c u l t u r e s . E f f e c t on p r o t e i n and protease: The protein contents and protease a c t i v i t i e s of c e l l s and media were determined and compared with respect to the nitrogen sources provided. Two series of supplements were s t u d i e d — t h e amino acids discussed above, and a few plant and animal proteins. Skimmed milk was included i n both series for comparison. Of the amino acids used to supplement B5, aspartic and glutamic acids induced the greatest protein production i n both n i t r a t e and ammonium media, though the l a t t e r , as a group, gave much lower protein y i e l d s . In n i t r a t e media, arginine, alanine and cysteine were also stimulatory of p r o t e i n production, r e l a t i v e to the basal l e v e l s . The same f i v e amino acids also stimulated protease a c t i v i t y i n n i t r a t e media, while the other amino acids were i n h i b i t o r y . Protease a c t i v i t y i n ammonium-based media, regardless of supplement, was c o n s i s t e n t l y lower than i n the unsupplemented medium. No amino acids induced s i g n i f i c a n t l y higher p r o t e o l y t i c a c t i v i t y than unsupplemented ammonium medium. These r e s u l t s r e i t e r a t e d the i n e f f e c t i v e n e s s of ammonium-based media when enhancement of protease production was the goal. The stimulatory e f f e c t of aspartic and glutamic acids was most l i k e l y related to t h e i r ease of a s s i m i l a t i o n and metabolism i n protein synthesis. These compounds supply reduced nitrogen, carbon skeletons for carbohydrate and hydrocarbon 92 metabolism, and a v a i l a b l e f u e l for ATP production. Some of the proteins added to B5 medium i n t e r f e r e d with determinations of harvest weights due to i n s o l u b i l i t y , undigested protein becoming trapped with c e l l s upon f i l t r a t i o n . S i m i l a r l y , t h i s r e s i d u a l protein resulted i n a r t e f a c t u a l c e l l protein l e v e l s . For t h i s reason, only data for protease a c t i v i t y was evaluated for the purpose of medium improvement. This data indicated that many proteins, including reagent grade casein powder and enzymatic casein hydrolysate were i n h i b i t o r y to p r o t e o l y t i c a c t i v i t y of c u l t u r e s . The causes of th e i r i n h i b i t o r y action are not known, although casein hydrolysate had been shown, e a r l i e r , to be i n h i b i t o r y to growth of c a l l u s t i s s u e s . Soluble starch, added only as a c o n t r o l for the e f f e c t of the presence of high molecular weight material, also proved to be i n h i b i t o r y . Only fresh skimmed milk and egg albumen showed s i g n i f i c a n t stimulation of p r o t e o l y s i s , to approximately the same l e v e l . Note that egg albumen i s not heat-stable, and thus coagulated upon autoclaving the medium. Much undigested albumen remained af t e r the incubation period, yet i t s presence had stimulated p r o t e o l y t i c a c t i v i t y . A comparison of the e f f e c t s of skimmed milk i n complete, n i t r a t e - d e f i c i e n t and ammonium-deficient media exposed a very broad range of protein l e v e l s . A l l milk-containing media yielded highest protein y i e l d s , but ammonium medium supplemented with milk was not s i g n i f i c a n t l y better than a l l the media without milk. This r e s u l t was supported by the protease determinations, which c l e a r l y placed the N^-milk combination at the same l e v e l as n i t r a t e alone. Without skimmed milk, complete B5 produced highest p r o t e o l y t i c a c t i v i t y , and B5 with ammonium alone yielded the lowest a c t i v i t y . 93 5. Stimulatory e f f e c t s of skimmed milk and milk components Inclusion of skimmed milk i n B5 medium resulted i n higher y i e l d s of biomass, protein and p r o t e o l y t i c a c t i v i t y than could be obtained with any other nitrogen-containing supplements. I t was therefore postulated that the stimulatory e f f e c t s observed were due to non-nitrogenous materials. The p a r t i a l f r a c t i o n a t i o n of fresh skimmed milk permitted an evaluation of the possible r e l a t i o n s h i p of the active p r i n c i p l e ( s ) to milk casein and whey. Both of these components enhanced growth, r e l a t i v e to unsupplemented medium, although whey appeared to be more e f f e c t i v e than casein or i n t a c t milk. Lactose, calcium, n i a c i n or r i b o f l a v i n did not s i g n i f i c a n t l y a l t e r biomass y i e l d s . The f i l t r a t e from u l t r a f i l t e r e d whey was also i n e f f e c t i v e , while the retained f r a c t i o n s i g n i f i c a n t l y stimulated growth of f i g c e l l s . The stimulatory e f f e c t of c i t r i c acid on growth was s t r i k i n g . C i t r a t e provides a v a i l a b l e carbon for metabolism via the t r i c a r b o x y l i c a c i d c y c l e , contributing to an improved energy pool for the synthetic pathways associated with growth. Its use i n plant c e l l culture media i s not w e l l documented. Gamborg and Shyluk (1970) found that soybean c e l l s could grow with ammonia as the sole nitrogen source i f Kreb's ( t r i c a r b o x y l i c acid) cycle intermediates were provided. The function to these acids i n c e l l cultures was not known, but several options were proposed: r e l i e f of ammonium t o x i c i t y , enhancement of ammonium transport or s a t i s f a c t i o n of a carbon requirement for amino acid synthesis. Ojima and Ohira (1978) also found that some of the carboxylic acids, i n supplement to ammonia, promoted r i c e c e l l growth. One a p p l i c a t i o n of c i t r i c acid to plant c e l l culture medium which i s of p a r t i c u l a r i n t e r e s t was reported by Erner et a l . (1975). C i t r u s species, e s p e c i a l l y orange, were known to require approximately 10% orange j u i c e i n the growth medium. This group discovered that the 94 stimulatory e f f e c t of orange j u i c e was completely reproducible with c i t r i c acid (2.5 g/1). Figs, the fresh f r u i t , contain 6 meq c i t r i c acid per 100 g fresh weight, but no information on c i t r a t e content of the vegetative plant was a v a i l a b l e . I t i s possible, then, that a high endogenous c i t r a t e l e v e l i s c r i t i c a l to normal metabolic a c t i v i t y i n f i g c e l l . The capacity to synthesize the required l e v e l s may be l o s t over the course of successive c e l l transfers (Erner et a l . , 1975). Protein p r o d u c t i v i t y of f i g c e l l s i n media containing c i t r i c a c i d or the milk f r a c t i o n s , whey or casein, was s i g n i f i c a n t l y higher than that i n unsupplemented B5. As usual, t o t a l protein content c l o s e l y followed biomass production, with calcium, lactose, and the vitamins r e s u l t i n g i n p r o t e i n le v e l s not s i g n i f i c a n t l y d i f f e r e n t from B5 alone. A s i m i l a r pattern was observed i n assessment of p r o t e o l y t i c a c t i v i t i e s of f i g c e l l suspension cultures grown i n basal medium containing milk or i t s components. Whey, whey retentate from u l t r a f i l t r a t i o n , f resh casein, milk and c i t r i c a c i d a l l induced p r o t e o l y t i c a c t i v i t i e s s i g n i f i c a n t l y higher than other media. Casein powder was not s i g n i g i c a n t l y stimulatory, whereas fresh casein was, suggesting that the active p r i n c i p l e ( s ) was/were u n l i k e l y to be the casein i t s e l f . A complication a r i s e s with the hypothesis that c i t r a t e was responsible for stimulation of growth and of protease a c t i v i t y . The u l t r a f i l t r a t e from whey, which should have contained low molecular weight materials (less than 10,000 daltons), including c i t r a t e , a c t u a l l y showed no growth or protease enhancing e f f e c t s . The most e f f e c t i v e milk f r a c t i o n i n these respects was the whey u l t r a f i l t r a t i o n retentate. This discrepancy may have ar i s e n as a r e s u l t of c o - p r e c i p i t a t i o n with calcium and phosphate at neutral pH. C i t r a t e , soluble i n whey during the process of casein coagulation, may have become insoluble 95 i n neutralized whey and could not pass through the u l t r a f i l t r a t i o n membrane. 6. Applications, problems and p o t e n t i a l of plant c e l l cultures A b r i e f look at both p o s i t i v e and negative aspects of plant c e l l c u l t u r e can help to put the present work into perspective i n terms of a p p l i c a b i l i t y . The introductory chapter presented an overview of the multiple research d i r e c t i o n s taken by those propagating plant tissues and c e l l s . Yet much of th i s work i s s t i l l i n the research stages, or i s intended s o l e l y for research purposes. Growth of f i g c e l l s could be improved by the addition of whey to basal B5 medium. Under these conditions, i t may be possible to omit or decrease the plant hormone l e v e l s . Technically, the r e s u l t i n g c e l l s l u r r y could be considered the equivalent of the same species i n d i f f e r e n t i a t e d form. As such, d i f f i c u l t i e s i n meeting regulatory guidelines f o r FDA approval would be minimal. The spectrum of requirements and tests f o r GRAS status described by Nelson (1980) and Whitaker (1980) might be avoided altogether for papaya c e l l cultures, i f the presence of papain can be proven, since papain has already been declared GRAS. Appl i c a t i o n of c e l l suspension cultures would require development, but i t i s not d i f f i c u l t to envision meat marinades or yogurt containing such a puree. Re f r i g e r a t i o n would prevent a c t i v i t y before use of a marinade, or a f t e r s e t t i n g of a milk-based product. In the l a t t e r case, the slow milk c l o t t i n g a c t i v i t i e s encountered i n papaya c e l l suspension cultures may a c t u a l l y be favourable i n terms of shelf l i f e . Recent advances i n techniques of whole c e l l immobilization (Brodelius & Mosbach 1982), though p o t e n t i a l l y useful for plant c e l l s producing flavour constituents, would not l i k e l y be applicable to these protease producers. 96 A p p l i c a t i o n of plant tissue and c e l l c ulture techniques has met with a few major hurdles. Underlying many d i f f i c u l t i e s l i e s the influence of genetics, both of the parent plant and of progeny c e l l s . Much information i s a v a i l a b l e on cytogenetic studies but few generalizations have been substantiated. Plant c e l l s are commonly held to be totipotent, to contain the genetic information required to regenerate new, f u l l y - d i f f e r e n t i a t e d p lants. Within the mature plant, expression of genetic information i s li m i t e d by surrounding c e l l s and regulated, i n part, by hormones. Such l i m i t a t i o n s to genetic expression are believed to be f u l l y r e v e r s i b l e (Street, 1977). Undifferentiated c e l l s growing i n suspension culture should, therefore, respond only to environmental conditions, which are under external c o n t r o l . This hypothesis has been supported by the large numbers of plant species that have been propagated i n v i t r o then responded to deliberate induction of morphogenesis (Reinert & Bajaj, 1977; Murashige, 1978; Winton, 1978). The f a c t that some species have not yet been s u c c e s s f u l l y subjected to d e d i f f e r e n t i a t i o n followed by morphogenesis could be explained e i t h e r by a lack of totipotency or by a lack of knowledge with respect to n u t r i t i o n a l and environmental requirements for these transformations. A c o r o l l a r y genetic phenomenon concerns metabolic v a r i a t i o n among c a l l i (Townsley, 1977) and c e l l suspensions (Street, 1977). Rather than the temporal c y t o l o g i c a l changes accompanying c e l l d i f f e r e n t i a t i o n , metabolic differences may be manifest concurrently. That i s , c e l l s a t the same age and under the same culture conditions do not necessarily e x h i b i t i d e n t i c a l metabolic behaviour. Street suggested that one cause may be the continuous production of genetic v a r i a t i o n . This could very l i k e l y be the case i n c a l l u s and batch suspension cultures, which are i n a continual state of 97 f l u x , providing a s e l e c t i v e advantage f o r d i f f e r e n t f r a c t i o n s of the c e l l populations over time. K i b l e r and Neumann (1980) found a wide range i n ploi d y l e v e l s i n Datura and barley c u l t u r e s . They described c e l l s as f a l l i n g into one of two genetic c l a s s e s , meristematic or parenchymatous. Their appearance was distinguishable microscopically though t h i s d i f f e r e n c e has probably been mistakenly a t t r i b u t e d to c e l l age i n other work. Differences i n nucleic acid content may then be manifest metabolically, noted as quantitative differences i n p r o d u c t i v i t y . To avoid c e l l heterogeneity, c e l l s e l e c t i o n has often been recommended. The simplest approach involves repeated transfer of a small inoculum of rapidly-growing c e l l s , those best suited to the culture conditions (Noguchi et l a . , 1977). This method was applied, i n t h i s study, to papaya and f i g c a l l u s and suspension c u l t u r e s . Although p r o t e o l y t i c a c t i v i t y varied from one generation to the next, the range of v a r i a t i o n d i d not appear to exceed three- or f o u r - f o l d . These c e l l populations were not e n t i r e l y homogeneous so p r o d u c t i v i t y represented the mean of a l l c e l l s . I t i s quite l i k e l y , though not investigated herein, that the differences between i n d i v i d u a l c e l l s were much greater, p a r t i c u l a r l y i n the e a r l i e s t c a l l u s c u l t u r e s . Related to c e l l s e l e c t i o n i s another f i e l d r e quiring further i n v e s t i g a t i o n — r a p i d methods of c e l l i d e n t i f i c a t i o n . Tabata e t a l . (1978) selected for high-nicotine producers on the basis of examination of small c a l l i . They had a rapid method to i d e n t i f y the compounds of i n t e r e s t , the " c e l l squash method" and paper chromatography for a l k a l o i d s (Ogino e t a l . , 1978). They found the selected cultures to be quite stable i n the l e v e l of nicotine production over successive t r a n s f e r s . No such method was av a i l a b l e f o r detection of high protease producers. Timing the cl e a r i n g of 98 milk agar showed no differences among c a l l i . Noting pH changes by i n c l u s i o n of i n d i c a t o r s i n agar was also unsuccessful i n detecting d i f f e r e n c e s . The Bio-Rad protease detection method was applied i n the preliminary search for a rapid detection method, again without r e s u l t s . For lack of such a s e l e c t i o n method, the approach chosen was that dependent on endogenous s e l e c t i o n pressures, using only rapidly-growing t i s s u e . A more refined technique for c e l l s e l e c t i o n , described by Bergmann (1977), i s based on the m i c r o b i o l o g i c a l method of p l a t i n g i n agar. C e l l d e n s i t i e s of 10^ to 10^ c e l l s / m l are generally required to induce growth. For most plant species, there appears to be a minimum c e l l density, below which no growth occurs. Successful p l a t i n g r e s u l t s i n a dense c e l l population i n agar, upwards of two clones per mm2 not conducive to i n c l u s i o n of i n d i c a t o r s i n the medium. C e l l p l a t i n g i s t e c h n i c a l l y a d i f f i c u l t procedure and has met with l i m i t e d success. This was not attempted with papaya or f i g c e l l suspersion c u l t u r e s . Zenk (1978) c l a s s i f i e d plant c e l l s i n two groups i n terms of p r o d u c t i v i t y i n v i t r o : (a) a l l c e l l s having s i m i l a r productive capacity, usually less than the parent plant, requiring that the e n t i r e c e l l population be influenced to increase p r o d u c t i v i t y , or (b) productive c a p a c i t i e s d i f f e r i n g among c e l l s i n a population, with a few variants being much more productive, sometimes more than evident i n s i t u . The v a r i a b i l i t y noted among papaya and f i g cultures suggested that e i t h e r the v a r i a b i l i t y within c e l l populations was r e l a t i v e l y small, or that c e l l s with high growth rates included variants with high p r o t e o l y t i c a c t i v i t y . Cytogenetic i n s t a b i l i t y i s one of the major d i f f i c u l t i e s enountered by plant tissue c u l t u r i s t s . I t appears now that there are several solutions to t h i s . C e l l s e l e c t i o n , as discussed above, i s only one 99 answer. Others include methods of haploid c e l l production from ovules, anthers or microspores, or by chromosome el i m i n a t i o n . Details of these techniques are provided i n several chapters i n Plant C e l l , Tissue and Organ  Culture, edited by Reinert and Bajaj (1977), and i n Plant C e l l Cultures:  Results and Perspectives, edited by Sala and others (1980). Furthermore, desirable c e l l s can sometimes be r e a d i l y i d e n t i f i e d by c h a r a c t e r i s t i c s such as pigment production, a l k a l o i d content and nucleic acid content. Recent advances have also been made i n the a p p l i c a t i o n of radio-immunoassays (Weiller, 1977), and i n i d e n t i f i c a t i o n of g e n e t i c a l l y linked c h a r a c t e r i s t i c s , "genetic markers" (see Sala et a l . , 1980). With the aid of s e n s i t i v e detection methods, c e l l s e l e c t i o n i s s i m p l i f i e d , and genetic v a r i a b i l i t y i s minimized. Plant c e l l clones could, i n theory, be t a i l o r e d to s p e c i f i c purposes: production of c e r t a i n metabolites, biotransformation of precursor materials (Alferman & Reinhard, 1980), resistance to disease or environmental stress ( V a s i l et a l . , 1980), autotrophy, or catabolism of xenobiotics. Plant species and purposes may be matched and modified, but each s i t u a t i o n requires s p e c i a l consideration. The use of papaya or f i g c e l l suspension cultures for the production of p r o t e o l y t i c enzymes i s a r e a l p o s s i b i l i t y . This work has provided only an introduction to t h i s plant-.purpose r e l a t i o n s h i p . Improvement of p r o d u c t i v i t y , and a c l e a r d e f i n i t i o n of n u t r i t i o n a l e s s e n t i a l s , must precede attempts at bioreactor-scale production. Plant c e l l culture i s an expensive pr o p o s i t i o n , worthwhile only when the cost of harvesting the o r i g i n a l plant becomes p r o h i b i t i v e , or the plants become e x t i n c t . H o r t i c u l t u r i s t s , plant breeders and plant pathologists are interested i n plant tissue culture as a propagative method and model study system, as indicated by the August/82 s p e c i a l issue of " C a l i f o r n i a Agriculture", an applications-oriented 100 p u b l i c a t i o n of the University of C a l f o r n i a , Department of A g r i c u l t u r e . With regard to plant product synthesis, however, only i n d u s t r i e s related to pharmaceuticals have looked s e r i o u s l y at plant c e l l c ulture as a means of production, despite the costs. A lack of knowledge and expertise i n the industry have so far stymied developmental work. There i s a requirement for long-term research and development programs due to the slow, labour-intensive establishment of cultures and cloning procedures. I t would appear that the North American food industry does not yet have the need or incentive for such a committment. 101 VI. SUMMARY Tissue cultures were established from explants of cardoon (Cynara  cardunculus), t h i s t l e (Circium arvense), dumbcane (Dieffenbachia amoena), papaya (Carica papaya), and f i g (Ficus c a r i c a ) . Two species f a i l e d to form c a l l u s ; pineapple (Ananas comosus) and ladies *bedstraw (Galium verum). Cultures were a l l maintained at 28 °C i n darkness, preventing normal photosynthetic a c t i v i t y and d i f f e r e n t i a t i o n . Papaya and f i g were s u c c e s s f u l l y maintained under these conditions as und i f f e r e n t i a t e d c e l l suspensions. Growth i n l i q u i d media produced greatest c e l l dry weight i n 14-21 days, the faster-growing cultures being selected for sub-culture. P r o t e o l y t i c a c t i v i t y was apparent i n tissue and c e l l cultures by the c l e a r i n g of milk i n media within a week of sub-culture. Casein was selected as the substrate for quantitation of extractable protease a c t i v i t y . Assay r e s u l t s were used to compare stimulatory e f f e c t s of medium supplements, and determine temporal p r o d u c t i v i t y . The FCC method, i n modified form, was used r o u t i n e l y with tyrosine content of the TCA-soluble components as the u n i t of measurement. Extractable protein was rou t i n e l y determined using the dye-binding method of Bradford. Biomass, c e l l dry weight produced over the te s t period, was determined by freeze-drying samples. A l l data were converted to a p e r - l i t r e basis for consistency and ease of comparison. B5 medium, which contains 2% sucrose, was an adequate nutrient supply for growth. No modifications were made to the basal ingredients except where s p e c i f i e d . Supplements to B5 medium were investigated for t h e i r influence on growth and production of protein and protease. Skimmed 102 milk, at 3% v/v, was one of many supplements. Both papaya and f i g c e l l cultures produced p r o t e o l y t i c enzymes that would hydrolyse milk casein i n the medium within one week. Other medium supplements included amino acids, t h i o l s , proteins, starch, lactose and other milk components. Results showed that protein synthesis and p r o t e o l y t i c a c t i v i t y i n extracts of l y o p h i l i z e d f i g c e l l cultures followed growth (biomass production) c l o s e l y . Those medium supplements which stimulated c e l l growth, such as milk, led to higher le v e l s of t o t a l protein and protease a c t i v i t y . Thus, n i t r a t e i n B5 medium was shown to be more important, i n terms of o v e r a l l growth, than ammonia as a source of inorganic nitrogen for f i g c e l l s . The most e f f e c t i v e amino acid supplements for stimulation of growth and protease production i n f i g c e l l cultures were glutamic and aspartic acids, with cysteine also improving protease a c t i v i t y , but not growth. Of the proteins and peptides added to B5 medium, only milk and egg albumen produced s i g n i f i c a n t l y higher protease a c t i v i t y than the c o n t r o l . Dried casein and casein hydrolysate were both i n e f f e c t i v e medium supplements. The cause of the stimulatory e f f e c t of egg albumen i s not known. Data obtained from f i g c e l l suspension cultures grown i n B5 supplemented with milk or various components thereof, showed that growth and protease a c t i v i t y were highest with whey, milk, fresh casein and c i t r i c a c i d . Unsupplemented medium resulted i n s i g n i f i c n a t l y lower biomass, protein and protease l e v e l s . The b e n e f i c i a l e f f e c t of milk may be l a r g e l y due to i t s c i t r i c acid content, which would have supplied growing f i g c e l l s with and a d d i t i o n a l energy source and carbon source. It could also contribute to i n t r a c e l l u l a r levels of c i t r a t e , possibly d r i v i n g c e l l metabolism at a rate greater than normal. Whether c e l l s grown i n t h i s 103 medium were normal i n size or ploidy was not determined. Since whey produced such a dramatic increase i n biomass, although not quite as high as i n t a c t milk, t h i s unrefined material could be used i n l i q u i d form as an inexpensive supplement for growth of f i g c e l l s . I t would also be worthwhile i n v e s t i g a t i n g f o r propagation of other plant species i n tissue or c e l l c u l t u r e . There was much v a r i a b i l i t y among papaya and f i g c e l l suspension cultures, often not stable from one generation to the next. Each experiment required a standard inoculum f o r each t e s t condition, and c e l l s grown i n the same medium did not n e c e s s a r i l y produce comparable r e s u l t s i f the inoculum d i f f e r e d . I t would have been desirable to s e l e c t clones on the basis of high protease p r o d u c t i v i t y , but no method f or rapid and non-destructive i d e n t i f i c a t i o n was a v a i l a b l e . 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