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Studies on the protease of Pseudomonas aeruginosa Lacko, Andras G. 1963

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STUDIES ON THE PROTEASE OF PSEUDOMONAS AERUGINOSA by ANDRAS G. LACKO B.S.A. U n i v e r s i t y of B r i t i s h Columbia, 1961. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF , THE REQUIREMENTS FOR THE DEGREE OF M.Sc. i n the Department of Animal Science We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requ irements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re f erence and s t u d y . I f u r t h e r agree that p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted' by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s unders tood that copying, or p u b l i -c a t i o n o f t h i s t h e s i s for f i n a n c i a l g a i n s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h C o l u m b i a , Vancouver 8, Canada, Date - I X -ABSTRACT Contrary to some reports i n the l i t e r a t u r e Pseudomonas  aeruginosa ATCC 9027 as w e l l as other s t r a i n s of Pseudomonads produced d e f i n i t e l y l a r g e r amounts of protease when supp l i e d w i t h proteinaceous n u t r i e n t s than i n a glucose mineral s a l t s medium. The enzyme appeared to be e x t r a c e l l u l a r i n character and l i b e r a t e d under conditions of at l e a s t p a r t i a l s t a r v a t i o n i n the presence of an inducer. Of the pr o p e r t i e s of the enzyme temperature and pH optima were found to be 60°C and pH 8.0 r e s p e c t i v e l y . C h e l a t i n g agents were found to i n h i b i t enzyme a c t i v i t y . - i v -ACKNOWLEDGEMENT o I wish to express my sincere appreciation to Dr. J.J.B. Campbell for his direction and c r i t i c i s m s throughout th i s investigation. - i i i -TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF THE LITERATURE 3 1.. C u l t u r a l Requirements f o r the production of b a c t e r i a l proteinases 3 2. The mode of l i b e r a t i o n of b a c t e r i a l enzymes 8 3. Pro p e r t i e s of the proteases of Pseudomonads 12 MATERIALS AND METHODS 14 1. The organisms and t h e i r c u l t i v a t i o n 14 2. Enzyme Assays 14 3. Preparation of the supernatant l6 4. Preparation of c e l l s f o r r e s t i n g experiments 16 5. Preparation of c e l l r f r e e e x t r a c t s 17 RESULTS AND DISCUSSION 18 I . N u t r i t i o n a l requirements f o r production of the protease I I . Studies on mechanism of l i b e r a t i o n of protease by c e l l s of P. aeruginosa 25 I I I . P r o p e r t i e s of the protease 36 GENERAL DISCUSSION 46 I . N u t r i t i o n a l requirements f o r production of the protease I I . The mechanism of l i b e r a t i o n of the protease 48 I I I . P r o p e r t i e s of the enzyme 52 SUMMARY 53 BIBLIOGRAPHY 55 INTRODUCTION The widespread occurence of p r o t e o l y t i c enzymes i n Nature i s explained by the vast d i v e r s i t y of l i v i n g systems c o n t a i n i n g p r o t e i n s d i f f e r i n g i n : composition and s t r u c t u r e . According to Schoenheimer (19^6), the dynamic s t a t e of animal p r o t e i n s i s f a c i l i t a t e d by t h e i r s p e c i f i c p r o t e o l y t i c enzymes which break down body p r o t e i n s to smaller u n i t s . These smaller u n i t s can be r e a s s i m i l a t e d i n t o p r o t e i n molecules or other-wise u t i l i z e d . Although i n simpler systems, such as b a c t e r i a , there i s very l i t t l e turnover of p r o t e i n s , p r o t e o l y t i c enzymes are produced to f i l l other fundamental r o l e s . The breakdown of prot e i n s to smaller molecules i s of paramount importance to mankind f o r n u t r i t i o n a l reasons as w e l l as f o r other p r a c t i c a l considerations. D i s s i m i l a t i o n of large molecules i n the s o i l and i n sewage by b a c t e r i a l proteases provides n u t r i e n t s f o r plant l i f e and at the same time helps to perpetuate the nit r o g e n c y c l e by doing away with organic waste m a t e r i a l s . Although p r o t e o l y t i c enzymes of b a c t e r i a l o r i g i n can cause food s p o i l a g e , the food i n d u s t r y makes good use of b a c t e r i a l proteases i n cheese making and i n the production of some fermented beverages. -2 -Besides p r o v i d i n g n u t r i e n t s f o r the c e l l s by breaking down i n g r e d i e n t s of the c u l t u r e medium, the scavenger a c t i v i t y of b a c t e r i a l proteases i s s i g n i f i c a n t . They may f a c i l i t a t e the l y s i s and degradation of o l d c e l l s a l l o w i n g the younger ones to use the breakdown products as n u t r i e n t s . The phenomenon of b a c t e r i a l p r o t e o l y s i s a t t r a c t e d the mounting i n t e r e s t of b a c t e r i o l o g i s t s and biochemists a l i k e , i n the past f i f t e e n years. Due to r a p i d advances i n the f i e l d of p r o t e i n chemistry, new p r o t e o l y t i c enzymes are being searched f o r continuously to e l u c i d a t e the primary s t r u c t u r e of p r o t e i n s by p a r t i a l degradation. Meanwhile, i n the f i e l d of b a c t e r i a l physiology the discovery of new methods paved the way f o r the study of e x t r a c e l l u l a r proteases. To date considerable'data have been, accumulated- • <•»•-; . *. . : :c l concerning the carbohydrate metabolism and endogenous r e s p i r a t i o n of Pseudomonas aeruginosa ATCC 9027 yet very l i t t l e i s known about the r o l e of i t s potent protease i n c e l l u l a r metabolism. This study was assigned to i n v e s t i g a t e a) - the c u l t u r a l -c o n d i t i o n s necessary f o r the production of the protease, b) i t s metabolic r o l e , c) the mode of i t s l i b e r a t i o n and d) i t s p r o p e r t i e s . -3-Review of L i t e r a t u r e I. C u l t u r a l Requirements f o r the Production of B a c t e r i a l Proteinases The f i r s t demonstration of an a c t i v e p r o t e o l y t i c enzyme i n a c u l t u r e supernatant was reported by Fermi (1890). Since that time a d i f f e r e n c e of opinion has e x i s t e d as to whether the presence of p r o t e i n s or p r o t e i n d e r i v a t i v e s i s e s s e n t i a l f o r the e l a b o r a t i o n of such enzymes. Some authors have reported the presence of enzymes a f t e r the growth of b a c t e r i a i n s y n t h e t i c media containing amino a c i d s or ammonium s a l t s as the source of n i t r o g e n . Others, on the other hand, have maintained that organic nitrogen,,in the form of peptone or compounds of s i m i l a r s i z e , are r e q u i r e d f o r protease formation. Much of the work before 1930 seems u n r e l i a b l e when viewed from the modern standpoint, s i n c e (a) the concept of pH, i t s a p p l i c a t i o n to b a c t e r i o l o g y and i t s fundamental importance i n enzymic a c t i v i t y had not been elaborated. (b) the s i g n i f i c a n c e of vitamins i n n u t r i t i o n was unknown and discordant r e s u l t s with s y n t h e t i c media may have been due to t h e i r presence i n the amino acids or other substances used. (c) the organisms were not washed and consequently organic matter was probably c a r r i e d over from the stock c u l t u r e . M e r r i l l and Clark (1928) reported the presence of p r o t e o l y t i c enzymes a f t e r the growth of b a c t e r i a i n s y n t h e t i c media c o n t a i n i n g amino acids or only ammonium s a l t s when calcium and magnesium ions were present. Wilson (1930) c r i t i c i z e d M e r r i l l and C l a r k ' s (1928) r e s u l t s on the ground that only poor growth was obtained i n the absence of calcium and magnesium s a l t s and concluded that these s a l t s are necessary f o r the e l a b o r a t i o n of proteases only i n so f a r as they sti m u l a t e growth. Haines (1931) working with B. mesentericus and a Pseudomonas sp found that mixtures of calcium and magnesium s a l t s or magnesium s a l t s alone exert a s t i m u l a t i n g i n f l u e n c e on the growth of micro organisms i n s y n t h e t i c media, while calcium s a l t s alone have but l i t t l e a c t i o n i n t h i s respect. In the same paper Haines a l s o demonstrated that proteases were formed i n simple s y n t h e t i c media with ammonium c h l o r i d e as the source of nitrogen provided that s a l t s of magnesium and calcium were present. The s t i m u l a t o r y e f f e c t of calcium s a l t s on protease production was sub s t a n t i a t e d by the observation that l i t t l e or no enzyme appeared i n the c u l t u r e c e n t r i f u g a t e when calcium s a l t s were absent from the medium. A f t e r improving h i s assay method Haines (1933) set out to study q u a n t i t a t i v e l y the g e l a t i n a s e production of d i f f e r e n t b a c t e r i a . He found that i n the absence of calcium and magnesium s a l t s only very minimal amounts of gelatinase appeared i n the c u l t u r e supernatant regardless of whether a mixture of amino acids or ammonium c h l o r i d e served as the source of nitrogen. -5-Berman (1918) invest igated the effect of the presence of glucose on the production of p r o t e o l y t i c enzymes by b a c t e r i a . He found that the production of these enzymes was i n h i b i t e d i n those organisms which carr i ed out vigorous fermentation with the formation of a c i d , but that glucose had l i t t l e effect on the p r o t e o l y t i c a c t i v i t i e s of organisms with l i t t l e or no fermentative power towards t h i s sugar. Furthermore, i f steps were taken to neutra l i ze the a c i d i t y by s trongly buffer ing the medium, the i n h i b i t o r y effect was checked. The above f indings ind ica te that the i n h i b i t o r y ef fect of glucose on protease production i s due to the a c i d i t y evolved during fermentation. G o r i n i (1950) showed that reagents such as c i t r a t e , oxalate and EDTA, which bind calcium, i n h i b i t e d the act ion of proteases. The i n a c t i v a t i o n of the enzyme i n t h i s case could be completely prevented by addi t ion of excess calc ium, provided that t h i s was done immediately after the addi t ion of the reagent. G o r i n i also demonstrated that calcium protected the protease against heat i n a c t i v a t i o n and suggested that calcium played a fundamental ro le i n these b a c t e r i a l proteinases , not only i n t h e i r protect ion against i n a c t i v a t i o n , but also i n t h e i r s t a b i l i t y . Calcium was thus considered to be a constituent of these proteases which must therefore be considered a meta l loprote in , i n which, according to t h e i r o r i g i n , the bond between the prote in and the metal i s more or l e s s s tab le . -6-Calcium as the a c t i v e substance i n p r o t e o l y s i s can be replaced only by strontium, and then only to a s l i g h t extent. As a c o n s t i t u e n t of the proteinase molecule, calcium can be p a r t i a l l y s u b s t i t u t e d by magnesium, but the protein-magnesium complex, however, has no p r o t e o l y t i c a c t i v i t y . Morihara (1959) presented evidence, using r a d i o a c t i v e calcium, to the e f f e c t that the metal was an i n t e g r a l part of the protease of Pseudomonas myxogenes. The a c t i v i t y of t h i s enzyme was not a l t e r e d by EDTA u n t i l the temperature reached 50° C. This might suggest that calcium i s f i r m l y combined with the enzyme p r o t e i n . By supplying CaCO^ as the source of calcium Morihara ( 1956 ) also overcame the i n h i b i t i n g a c t i o n of glucose metabolites, since the n e u t r a l i s i n g e f f e c t of CaCO^ prevented the considerable decrease i n pH that would otherwise take place. F i s h e r (i960) was unable to i s o l a t e any protease from the supernatants of Pseudomonas aeruginosa when the organism was grown on mineral s a l t s medium c o n t a i n i n g carbohydrate as the carbon source. The enzyme was found to be elaborated upon the a d d i t i o n of c e r t a i n peptides or p r o t e i n s . Morihara (1962), however, reported.that a n a t u r a l medium c o n t a i n i n g meat extract and peptone, i n h i b i t e d protease production by d i f f e r e n t s t r a i n s of P. aerugihosa and by s t r a i n s of other pseudomonads as w e l l . The greatest y i e l d s of enzyme were obtained w i t h a medium containing 7% glucose, mineral s a l t s i n c l u d i n g calcium and 0.125% corn steep l i q u o r . Most of the enzyme was released i n t o -7-the medium when the c e l l s were w e l l i n t o t h e i r s t a t i o n a r y phase of growth and when a l l the glucose had been metabolized. The f i n d i n g s of Morihara might be substantiated by the theory of Castaneda-Agullo (1956), who demonstrated with S e r r a t i a  marcescens, that i n p r o t e i n free media no p r o t e o l y t i c a c t i v i t y was found unless a c e r t a i n l e v e l of b a c t e r i a l growth had been reached and calcium was present. The i n d u c t i o n of the protease thus might be brought about by the proteins of dead b a c t e r i a or by proteinaceous d e r i v a t i v e s of metabolism, formed only when the c u l t u r e has at t a i n e d a high concentration of b a c t e r i a l mass. Castaneda-Agullo's suggestion i s supported by h i s f i n d i n g s that c e l l suspensions placed i n a low concentration of g e l a t i n gave r i s e to the r a p i d appearance of i n c r e a s i n g amounts of protease i n the medium before even one c e l l u l a r d i v i s i o n had taken place. In the s y n t h e t i c medium, on the other hand, the same suspension produced only n e g l i g i b l e amounts of the proteinase even a f t e r undergoing two d i v i s i o n s . -8-H• The Mode of L i b e r a t i o n of B a c t e r i a l Exoenzymes For the determination of the p h y s i o l o g i c a l f u n c t i o n of a b a c t e r i a l enzyme, i t i s very important to e s t a b l i s h whether the enzyme i s e x t r a c e l l u l a r or not. Po l l o c k (1962) i n h i s recent review a r t i c l e o u t l i n e d i n d e t a i l the c r i t e r i a determining whether an enzyme i s to be considered e x t r a c e l l u l a r or c e l l bound to some extent. He attempted to c l a s s i f y the b a c t e r i a l exoenzymes as f o l l o w s . I . C e l l bound enzymes I I . E x t r a c e l l u l a r enzymes The c e l l bound enzymes can be f u r t h e r s p l i t i n t o two sub-classes a) I n t r a c e l l u l a r enzymes cannot be l i b e r a t e d from the c e l l bound s t a t e by d e s t r u c t i o n of the c e l l w a l l but they can be separated from the i n t a c t cytoplasmic membrane a f t e r l y s i s of the pr o t o p l a s t by d i f f e r e n t i a l c e n t r i f u g a t i o n . b) Surface bound enzymes 1. can be extracted from the c e l l under c o n d i t i o n s where the p r o t o p l a s t i s protected (Lysozyme) 2. Might be n e u t r a l i z e d by a s p e c i f i c anti-serum or by any type of large molecule that i s incapable of penetrating the c e l l membrane. -9-The term " e x t r a c e l l u l a r enzymes" must be r e s t r i c t e d to enzymes which e x i s t i n the medium around the c e l l , having o r i g i n a t e d from the c e l l , without any a l t e r a t i o n to the c e l l s t r u c t u r e , that would i n t e r f e r e with the normal processes of growth and reproduction. From the p r a c t i c a l point of view the c l a s s i f i c a t i o n of an enzyme as e x t r a c e l l u l a r depends upon the demonstration that 1) The enzyme occurs i n the medium separated from the c e l l s . 2) The appearance of the enzyme i n the medium does not depend on i r r e v e r s i b l e damage to c e l l s t r u c t u r e . In most of the reported wofcks on exoenzymes l i t t l e or no e f f o r t i s made to c o n t r o l or estimate c e l l a u t o l y s i s . There are, however, r e l a t i v e l y simple, although i n d i r e c t ways of measuring the degree of l y s i s i n a b a c t e r i a l .culture?, e P o l l o c k (1956). 1. Enzyme synthesis should be followed by young c u l t u r e s pre-f e r a b l y i n the l o g a r i t h m i c phase of growth. I t i s not s u f f i c i e n t to demonstrate that there i s no decrease i n c e l l mass (measured by t u r b i d i t y or by t o t a l or v i a b l e counts) i n order to conclude that l y s i s has not occurred. In a c t i v e l y growing c u l t u r e s l y s i s of some c e l l s may be masked by growth of others. I t i s i n t e r e s t i n g to note that many enzymes, that are considered to be e x t r a c e l l u l a r appear to be l i b e r a t e d toward the end of the l o g a r i t h m i c phase, a peri o d not f a r from when the c e l l s might begin to undergo a u t o l y s i s Morihara (195&). -10-I f an enzyme i s found i n the c u l t u r e supernatant the c e l l s themse lves s h o u l d be t e s t e d f o r a c t i v i t y as i t was done by P o l l o c k (1956) w i t h B. s u b t i l i s P e n i c i l l i n a s e . C e l l a u t o l y s i s can be l o o k e d f o r more d i r e c t l y by t e s t i n g the c u l t u r e s u p e r n a t a n t f o r the p r e s e n c e of subs tances n o r m a l l y l o c a t e d i n s i d e the c e l l . The s i m p l e s t method i s to use the appearance o f i n t r a c e l l u l a r enzymes as markers o f c e l l damage. Other p o s s i b l e markers might be DNA which i s not n o r m a l l y p r e s e n t i n the medium o r on the s u r f a c e o f the . c e l l . Most o f the work on so c a l l e d e x t r a c e l l u l a r p r o t e a s e s do not meet the above s t a n d a r d s e s t a b l i s h e d by P o l l o c k (I962) as f a r as p r o d u c i n g ev idence to show the e x t r a c e l l u l a r n a t u r e o f the enzymes i s c o n c e r n e d . There a r e , however, a number o f papers i n which the a u t h o r s were t r y i n g to e s t a b l i s h the mode o f l i b e r a t i o n and the l o c a t i o n of the enzymes they worked o n . Gorbach (1936) showed w i t h P. f l u o r e s c e n s and w i t h P . p,yo:cyanea that the p r o t e a s e s l i b e r a t e d by these s p e c i e s appeared i n the medium p r o p o r t i o n a l l y to the number of dead c e l l s . They b e l i e v e d that these enzymes were l i b e r a t e d due to a u t o l y s i s . P o l l o c k (1962) e x p l a i n s the above on the b a s i s o f ana logy w i t h c a t h e p s i n l i b e r a t e d by mammalian c e l l s upon t h e i r d e a t h . In o t h e r words, most b a c t e r i a a r e l i k e l y to posse s s i n t r a c e l l u l a r p r o t e a s e s which become a c t i v a t e d a f t e r the death o f the c e l l s and p r o b a b l y have d i f f e r e n t s p e c i f i c t y and -11-f u n c t i o n from those l i b e r a t e d under normal p h y s i o l o g i c a l c o n d i t i o n s . Castaneda-Agullo (1956) also suggested that p r o t e i n s of dead b a c t e r i a might induce l i b e r a t i o n of proteases. Some workers such as Hartmann (1957) presented evidence that the proteases they worked on were e x t r a c e l l u l a r since l i t t l e or no enzyme was bound to the c e l l s . P o l l o c k (1956) studied the p e n i c i l l i n a s e of B a c i l l u s s u b t i l i s i n d e t a i l employing novel methods to e s t a b l i s h the character of t h i s enzyme. L o c a l i z a t i o n of p r o t e o l y t i c enzymes has a t t r a c t e d considerable i n t e r e s t i n the past few years. Amongst others Chaloupka (1961) reported the l o c a l i z a t i o n of i n t r a c e l l u l a r proteases, i n 1 and i n B. megaterium while McDonald (1962) reported the l o c a l -i z a t i o n of a protease i n a species of Micrococcus. The l o c a l i z a t i o n techniques b a s i c a l l y follow, thesame path: 1) S e l e c t the appropriate phase of growth when the enzyme i s d e f i n i t e l y c e l l bound. 2) Subject the properly washed c e l l s to an appropriate procedure y i e l d i n g w e l l defined s u b - c e l l u l a r f r a c t i o n s which are analysed f o r a c t i v i t y . Damodaran (1955), Guntelberg (195*0Campbell (1957) and McDonald (1961) showed that the proteinases of the b a c t e r i a they? used i n t h e i r experiment, were l i b e r a t e d i n d i r e c t r e l a t i o n to growth. - 1 2 -Zimmermarin (1956) reported that the production of the protease of Streptococus l i q u e f a c i e n s took place i n the absence of c e l l m u l t i p l i c a t i o n i n a r e s t i n g c e l l suspension. Castaneda-Agullo (1958) suggested that the production of a b a c t e r i a l protease might be the r e s u l t of b a c t e r i a l s t a r v a t i o n . I l l . P r o p e r t i e s of the Proteases of Pseudomonads A number of workers reported p r o p e r t i e s of proteases of d i f f e r e n t p u r i t y obtained from Pseudomonads Morihara (1956) found that the pH and temperature optima f o r the c r y s t a l l i n e protease of P. myxogenes were 7«0 - 8.5 and 4-5°C r e s p e c t i v e l y . In a l a t e r paper Morihara ( i 9 6 0 ) reported that the proteases of P. aeruginosa and P. myxogenes were immunologically i d e n t i c a l , yet Morihara (1963) claims that the c r y s t a l l i n e protease of r O P. aeruginosa has an optimum temperature of oO C. McDonald (1963) worked with crude preparations of the enzyme f i n d i n g 6o°C as the optimum temperature and so d i d F i s h e r ( i 9 6 0 ) who used a p a r t i a l l y p u r i f i e d protease of P. aeruginosa i n h i s experiments. F i s h e r ( i960) and Morihara (1963) both reported that metal binding agents i n t e r f e r e with the p r o t e o l y t i c a c t i v i t y of the enzyme. Morihara (1963) a l s o suggested that the metal must be t i g h t l y bound to the enzyme, since mild c h e l a t i n g agents di d not i n h i b i t enzyme a c t i v i t y . -13-Morihara (19^ 3) found the protease of P. aeruginosa to be f a i r l y stable at room temperature. - 1 4 -MATERIALS AND METHODS 1. The organisms and t h e i r c u l t i v a t i o n Excluding one experiment with d i f f e r e n t s t r a i n s of Pseudomonads, the organism used throughout these studies was Pseudomonas aeruginosa ATCC 9 0 2 7 . The c e l l s were c u l t u r e d i n va r i o u s media, as i n d i c a t e d with every experiment, i n Roux f l a s k s at 30°C. Each f l a s k contained 100 ml of medium. Casein hydroly-sate was obtained from N u t r i t i o n a l Biochemicals Corp., Cleveland, Ohio while the other proteinaceous i n g r e d i e n t s were sup p l i e d by Difco L a b o r a t o r i e s , D e t r o i t , Michigan. The basic medium used f o r c u l t i v a t i o n was the ammonium phosphate glucose medium of N o r r i s and Campbell ( 1 9 5 0 ) . The ammonium s a l t i n g r e d i e n t of the above medium was always used with carbon sources such as glucose, o£-keto-glutarate and succinate unless the medium included some organic source of nit r o g e n (peptone e t c . ) . With proteinaceous i n g r e d i e n t s , s e r v i n g as the source of nitroge n f o r the b a c t e r i a , equivalent amounts of KH 2P0^. were used i n s t e a d of (NH i f)H 2PO i f since the presence of inorganic nitrogen was found to repress enzyme production to a small extent. 2 . Enzyme Assays a) Caseinase Casein was purchased from General Biochemicals Inc. Chagrin F a l l s , Ohio. Preparation of the c a s e i n s o l u t i o n proceeded as -15-f o l l o w s . 4 gms of Vitamin Test Casein was suspended i n 0.01M t r i s -(hydroxymethyl)amino methane ( T r i s ) b uffer pH 8.0 with constant s t i r r i n g . The pH was readjusted to 8.0 with base u n t i l i t would not change with standing. P r o t e o l y t i c a c t i v i t y was r o u t i n e l y determined by the s l i g h t l y modified method of Hagihara (1954) as f o l l o w s . 1 ml of the 2% casein s o l u t i o n was pre-incubated at the required temperature. 1 ml of the enzyme s o l u t i o n was added at 0 time with mixing. At the end of the incubation p e r i o d 2 ml of the p r e c i p i t a t i n g reagent c o n s i s t i n g of a) 0.1M T r i c h l o r o a c e t i c a c i d b) 0.2M CH^COONa c) 0.3M CH^COOH was added to p r e c i p i t a t e the unhydrolysed casein. The r e a c t i o n mixture was now allowed to stand f o r 30 minutes and then i t was f i l t e r e d through paper. 1 ml of the f i l t r a t e was added to 5 mis of 0.4M NagCO-j. F i n a l l y 1 ml INphenol reagent was added to the r e a c t i o n mixture with r a p i d s t i r r i n g . Color was allowed to develop f o r 30 minutes and read i n the Beckraan Model B spectrophotometer at 670 m^against a reagent blank. The blank was prepared s i m i l a r l y to the procedure above with the d i f f e r e n c e that the p r e c i p i t a t i n g reagent was added to the casein s o l u t i o n at 0 time, before the enzyme. One u n i t of protease was defined as the amount of enzyme that l i b e r a t e d 1 m i l l i e q u i v a l e n t ( l 8 l mg) of ty r o s i n e per hour under the s p e c i f i e d c o n d i t i o n s . -16-b) G e l a t i n was the product of Difco L a b o r a t o r i e s , D e t r o i t , Michigan. G e l a t i n s o l u t i o n was made up by d i s s o l v i n g 8 gms of g e l a t i n i n 0.01M pH 7.4 T r i s b u f f e r . The gelat inase assay was c a r r i e d out employing the s l i g h t l y modified method of Morihara (1956). 6 mis of k% g e l a t i n was pre-incubated and 1 ml of the enzyme s o l u t i o n was added at 0 time. Incubation was c a r r i e d out at 45°C f o r 30 minutes. At the end of the incubation p e r i o d the sample was promptly t r a n s f e r r e d to the Ostwald viscosimeter and the flow time measured i n seconds (E seconds). The same procedure was followed with a blank s o l u t i o n and i t s time f o r outflow (C seconds) was measured. When E/C was equal to 0.700, protease a c t i v i t y of the d i l u t e d enzyme s o l u t i o n was taken as 1 u n i t / m l , p r o v i s i o n a l l y . 3. Preparation of the supernatant f o r assaying p r o t e o l y t i c  a c t i v i t y Cultures were removed from the incubator at the end of the in c u b a t i o n period and centr i f u g e d at 5000xg f o r 15 minutes. The supernatants were d i l u t e d i n order to give the a c t i v i t y corresponding to the desired range of col o r r e a c t i o n . 4. Preparation of c e l l s f o r r e s t i n g experiments Cultures were incubated f o r 16 hours at 30°C i n the re s p e c t i v e media. At the end of l6 hours the suspensions were ce n t r i f u g e d at 5000xg f o r 15 minutes and the c e l l s washed twice i n M pH 7.4 T r i s b u f f e r with i n t e r m i t t e n t c e n t r i f u g i n g . -17-5. Preparation of c e l l free extracts Cells prepared for crushing i n the above manner were put through the French pressure c e l l at 15-20000lbs/sq. inch pressure and the whole c e l l s removed from the extract by centrifuging at approximately 3000xg for 10 minutes. -18-RESULTS AND DISCUSSION I. . N u t r i t i o n a l Requirements f o r the Production of the Protease Morihara (1962) showed that growth on a glucose mineral s a l t s medium r e s u l t e d i n maximum protease production by various Pseudomonads and e s p e c i a l l y P. aeruginosa. These f i n d i n g s seem contrary to the concept that a peptide or a p r o t e i n must be present as inducer f o r the production of e x t r a c e l l u l a r b a c t e r i a ! proteases. The f i r s t task undertaken was, therefore, to e s t a b l i s h whether P. aeruginosa (ATCC 9027) behaved s i m i l a r l y to the s t r a i n s i n v e s t i g a t e d by Morihara (1962). Morihara's (1962) n u t r i t i o n a l experiments were repeated but i n a d d i t i o n to measurements of protease a c t i v i t y by the speed of g e l a t i n l i q u e f a c t i o n , Horihara's (195*0 assay was c a r r i e d out on the c u l t u r e supernatants. TABLE 1. Protease Production of P. aeruginosa on glucose and peptone media Medium* Gelatinase Caseinase :. (Units x 103) (Units) Glucose 1% (SMI) Glucose T/o (SMIlt) 3 days Glucose 7% (SMIII) 5 days 1% peptone + 2% Beef e x t r . (N.M.) 1% peptone 10 50 18 5 6 3.5 2.5 4.0 17.0 10.0 * A l l c u l t u r e s were incubated i n Roux f l a s k s f o r 48 hours unless otherwise i n d i c a t e d . -19-1% peptone medium was also included in this experiment since in preliminary experiments i t had been shown to stimulate protease production. From Table I i t i s apparent that the nutrit ional requirements for protease production of P. aeruginosa (ATCC 9027) differ markedly from those of the strains used in Morihara's (19&2) experiments. The level of protease activity in the "N.M" medium was five times that found in the supernatant of "SMIII" (Table I ) . The next step was to find the medium which supports the highest enzyme production by our organism. Media, containing various sources of nitrogen and carbon, were prepared and the supernatants checked for act ivity at 1,2 and 3 days. (Table 2). Calcium was found to be essential for protease production in the case of glucose and yeast extract medium, since in i t s absence no enzyme could be detected in the supernatant f l u i d . The addition of calcium ions to 1% casein hydrolysate increased the act ivi ty by about 70%. Casein hydrolysate however is bound to contain some but l imit ing amounts of calcium salts thus even without the addition of Ca ions some enzyme was always produced. The enzyme levels in most cases increase or stay stationary as the culture gets older, but in a few instances (3 and 3% casein hydrolysate)the act ivity decreases .s l ightly between 48 and 72 hours. Despite the fact that these changes might seem significant they have to be ignored, since they resulted in those cases where -20-TABEE 2 Protease production in various media by P. aeruginosa Medium* Protease units x 10 5 1 day 2 days 3 days 0.5% glucose + 0.1% yeast ext. 3.5 4.4 5.7 0.3% succinate + " " " . 3-5 . 3.5 3.5 0.5%*ketoglutarate + 0.1% yeast ext. 2.8 2.8 2.8 0.5% glucose 0 0 0 1% peptone 25 27 28 1.5% peptone 36 34 35 2.0% peptone 48 53 58 1% peptone + 0.2% glucose 27 35 35 1% peptone + 0.2% succinate 29 35 38 1% peptone + 2%e<,-ketoglutarate 33 41 42 1% casein hydrolysate 67 71 72 2% » » 145 220 236 3% » " 131 262 248 5% " 11 69 320 312 1% casein hydrolysate + 0^2% glucose 55 89 97 1% casein hydrolysate + 0.2% succinate 104 124 122 1% casein hydrolysate, + 2%<*-keto-124 glutarate 115 122 1% Tryptose 20 22 26 1% Tryptone 41 42 41 1% peptonized acid 2 2 2 0.2% glucose + 0.1% yeast ext., no C a + + 0 0 0 0.2% succinate " " " " " 0 0 0 0.2% ketoglutarate " " " " 0 0 0 1% casein hydrolysate, no C a + + 40 40 40 1% glucose + CaC03 1 2 2.5 -21-extr.emsQy high d i l u t i o n s were used and even then the O.D. readings of the assay did not d i f f e r by more than 0.01-0.03 which i s i n the range of experimental e r r o r . No enzyme production occured when glucose served as the sole source of carbon unless CaCO^ was added to the medium to neutra l i ze the a c t i v i t y during growth. Even with such a buffer ing agent as CaCOj present only minimal amounts of enzyme appeared i n the supernatant after 72 hours of incubat ion . Reasoning from the above, the protease y i e lds i n the media, where 0.1% yeast extract was added as a supplement, must have resulted from the inducing ef fect of yeast extract and not from glucose, succinate and oC -keto-glutar.ate re spec t ive ly . On the other hand glucose, succinate and d. -keto-glutacate st imulate protease product ion, i f an inducer such as peptone or casein hydrolysate i s present, probably through an increased number of c e l l s i n the c u l t u r e . Only temporary repress ion of the protease could be demonstrated by glucose i n the case of 1% casein hydrolysate + 0.2% glucose medium where the enzyme y i e l d i s lower at 24 hours than that generated by the c e l l s i n 1% casein hydrolysate medium. This repress ion can be explained by the fact that r e a d i l y metabolizable compounds such as glucose or amino acids are present, and the c e l l s w i l l use these compounds instead of producing the protease. The y i e lds from 3% and 5% casein hydrolysate are both lower at 24 but higher at 48 and 72 hours than those of 2% casein hydrolysate . This temporary repress ion i s s i m i l a r to that -22-exhibited by glucose, since in 3% and 3% casein hydrolysate there are more low molecular weight peptides and amino ac ids , which are r e a d i l y avai lable to the c e l l without producing the protease, than i n 2% casein hydrolysate . The above trend i s not apparent i n the case of 2% hydrolysate vs 1% casein hydrolysate since i n t h i s case (2%) the repress ion does not las t long enough to express i t s e l f and by 2k- hours (when the f i r s t sample i s taken) there are more c e l l s producing enzyme i n the r i c h e r medium. Further data presented l a t e r show that when dialysed casein hydrolysate i s used as the growth substrate the y i e lds are lower but the enzyme™appears e a r l i e r i n the supernatant probably due to the absence of low molecular weight substrate . 3% casein hydrolysate grown c e l l s produced the largest amounts of enzyme. If the f lasks are shaken p e r i o d i c a l l y the enzyme production can be speeded up but the l e v e l of a c t i v i t y i n the supernatant does not change. \% Tryptose, Tryptone and peptone media yie lded 26, 4 l and 28 uni t s of enzyme respect ive ly while they have p r a c t i c a l l y the same c o n s t i t u t i o n according to the Difco manual (i960). Peptonized mi lk , despite the fact that i t i s considered a complete medium containing large amounts of proteinaceous nutr ients , y i e lds only minimal enzyme a c t i v i t y i n the growth supernatant. The observations l i s t e d above seem to suggest that an inducer or peptide of cer ta in s ize i s required in f a i r l y high concentrations to st imulate higher protease production. -23-Since the data conf l i c t ed with the f indings of Morihara (1962), further inves t iga t ion was necessary to es tabl i sh how widespread was the effect of proteinaceous substrates on the protease production of various Pseudomonas s t r a i n s . Twelve s tra ins from the genus Pseudomonas were cu l t i va ted i n two types of media a) 1% glucose + 0.1% CaCO^ b) 1% casein hydrolysate + C a + + The culture supernatants were assayed for a c t i v i t y a f ter kS hours of incubat ion. The assays were carr ied out at 45 ° C . ( T a b l e A l l the s tra ins produced considerably more enzyme i n 1% casein hydrolysate than i n the glucose mineral sa l t s medium with the exception of a) P. ch loraps is that showed no enzyme production at a l l and b) P. fluorescens ATCC 9721 that produced n e g l i g i b l e but equal amounts of enzyme i n the two media. -24-TABLE 3. Protease production by d i f ferent s t ra ins of the genus Pseudomonas. S t r a i n Dept. Protease A c t i v i t y Units x Stock No. Glucose case, hydi P. f luorescens ATCC 9721 B 114 1 1 P. f luorescens PS 21 Gunsalus' B l l 6 5 l6o P. putrefaciens (Hammer) L B 148 0 6 P.. SP. 120 NA B 176 0 3 Pseudomonas sp (from1 Greek cheese) B 177 2 150 P. ch lororaps is NRCL B 108 0 0 P. aeruginosa U-21 Gunsalus B 101" 0 0 P. aeruginosa ' ATCC 9027 B 102 1 35 P. aeruginosa , K - 257 B 103 2 9 P. aeruginosa PA B 104 0 9 P. aeruginosa (Pyocyaneus) B 105 0 3 P. aeruginosa (Grossowicz, Israel ) B 106 0 2 -25-I I . Studies on the mechanism of l i b e r a t i o n of the protease by the c e l l s of P. aeruginosa Prel iminary experiments ind icated that a) the enzyme was l i b e r a t e d af ter the c e l l s had passed the logarithmic phase of growth and had entered the s ta t ionary phase and}b)dialysed casein hydrolysate induced enzyme l i b e r a t i o n e a r l i e r than did d ia lysed casein hydrolysate when included i n the medium. To obtain an o v e r - a l l p ic ture of the enzyme l i b e r a t i o n i n r e l a t i o n to growth, the c e l l s of P. aeruginosa were grown on three types of media* 1) 5% casein hydrolysate 2) 5% casein hydrolysate (dialysed overnight) 3) 1% glucose Samples were taken at i n t e r v a l s to carry out v iable c e l l counts, pro teo ly t i c assays and determination of remaining proteinaceous compounds. As i t can be seen from Figure I , there i s a s l i g h t decrease i n c e l l numbers from 16-24 hours i n the 3% casein hydrolysate medium. The time of th i s decrease coincides with the period when the enzyme f i r s t appears i n detectable amounts i n the supernatant. A f t e r the l 6 to 24 hour l a g per iod , there i s a second increas ing port ion of the growth curve between 24 and 36 hours i n d i c a t i n g that a f t e r a per iod of stagnation the c e l l s go through about one more d i v i s i o n . This second upsurge inthe number of v iable c e l l s coincides with an e i g h t - f o l d increase i n the enzyme concentration of the supernatant. Af ter 36 hours there i s no s i g n i f i c a n t change i n enzyme l e v e l yet L 0 G 1 o V A B L E C E L L S L O G 10 C E L L C O U N T Oo ii) <$) o Q b\ b <si b o o 6 b E I I Z Y ' M E A C T I V I T Y U N I T S X I O 3 the v iab le c e l l count s t ead i ly decreases from t h i s po int . The substrate concentration i s measured as the materia l reac t ing with the phenol reagent a f ter a t r i c h l o r o a c e t i c ac id (TCA) p r e c i p i t a t i o n and expressed as Moles of Tyros ine /ml . There i s a s i g n i f i c a n t increase i n th i s value between 0 and 8 hours probably due to the hydro lys i s of smaller peptides to amino acids by peptidases. The a c t i v i t y of these enzymes could not be detected i n the supernatant s ince they do not attack intact:- case in , the substrate employed i n the pro teo ly t i c assay. The greatest decrease i n substrate concentration, on the other hand, c l e a r l y corresponds with the period when the enzyme concen-t r a t i o n shows the greatest increase , between 2k and 36 hours. Even a f ter the number of v iable c e l l s and the enzyme a c t i v i t y of the supernatant had ceased to increase , a considerable amount of substrate , about k0% of the o r i g i n a l quantity, remained i n the supernatant. The large amount of unused substrate would ind ica te that the protease can only degrade peptides of p a r t i c u l a r s i ze or type and a large port ion of casein hydrolysate l i e s outside the range of a c t i v i t y of t h i s enzyme. In the case of dialysed casein hydrolysate only of the enzyme i s produced by the c e l l s compared to the y i e l d i n the supernatant of 3% casein hydrolysate . Although the enzyme appears 12 hours e a r l i e r i n the dialysed casein hydrolysate medium, the l e v e l of pro teo ly t i c a c t i v i t y does not increase i n the supernatant s i g n i f i c a n t l y upon further incubat ion . The amount of substrate used by the c e l l s i n both media f a i r l y wel l corresponds with the number of c e l l s produced but there i s a considerable difference of 0.93 M x 10 of substrate per1..!. ell l(*Table :-4)a-t 36 hours that must have been u t i l i z e d to produce the enzyme. TABLE 4 The U t i l i z a t i o n of Substrate for Growth and Production of the Protease i n J>6 hours by P. aeruginosa Media 3% casein Dialysed casein hydrolysate hydrolysate No. of c e l l s / m l 2.8 x H f 1 0 1.4 x J U T 1 0 M of substrate u t i l i z e d / m l 6.8 2.1 M substrate u t i l i z e d / c e l l 2.43 x 10 0 1.5 x 10 Protease units produced 26l x 10" 3 4 x l o " 3 Protease units . . , per no of c e l l s 93.5 x 10" * 2.86 x 10 ^ There i s no i n i t i a l increase i n the tyrosine value (substrate cone.) i n the case of dialysed casein hydrolysate, s ince the majori ty of the peptidase a c t i v a t i n g compounds are probably removed by d i a l y s i s and (due to the shortage of read i ly metabolizable substrate) the l ibera ted amino acids are r a p i d l y u t i l i z e d by the c e l l s . The amount of enzyme produced per c e l l i n the two media -30-d i f f e r s considerably suggesting that d i a l y s i s removes an inducer compound to some extent. The l i b e r a t i o n of the protease seems to take place a f ter the c e l l s have already metabolized the low molecular weight substrates such as amino acids and shorter peptides. Af ter a period of s tarvat ion the enzyme i s produced i f there i s an inducer present i n the medium. The necess i ty of an inducer i s evident from the fact that only a n e g l i g i b l e amount of enzyme production was noted i n glucose medium supplemented with calcium ions and only a minimal amount of enzyme (3 uni ts x 10 ^ i n 72 hours) was produced when CaCO^ was added to the glucose medium i n quant i t i es s u f f i c i e n t to buffer the system. Since the enzyme i s being l i b e r a t e d mainly i n the s ta t ionary phase one might suggest that the mechanism of l i b e r a t i o n involves l y s i s of c e l l s . Evidence obtained by checking the a c t i v i t y of c e l l free extracts , however, seems to contradict the theory that l y s i s might be involved i n the release of the enzyme from the c e l l s . 1. On tes t ing the c e l l free extracts for a c t i v i t y very weakly pos i t i ve resu l t s were obtained i n only one case and negative re su l t s were obtained with 8, 12, 16 hour c e l l s when grown on'5% casein hydrolysate. -31-2. Twenty-four hour c e l l s would be expected to contain the largest amounts of the enzyme since there i s an eight fo ld increase i n a c t i v i t y i n the supernatant between 2k and 36 hours of growth. With the routine procedure of washing the c e l l s twice before crushing them a t o t a l of 20 x 10~^ uni ts of a c t i v i t y was recovered from a f lask containing 100 ml of medium. The supernatant at the same time contained a t o t a l of 3&00 -x 10~-^  i n other words 180 times as much enzyme as did the c e l l s . 3. When washed three times, the c e l l s y ie lded only h a l f of the enzyme reported above and the t o t a l of 10 un i t s (1 unit /ml) approaches the region of the minimal value that could be detected and could conceivably be looked upon as experimental variat ion;: . Since the large majority of the enzyme was always associated with the supernatant and only a minimal amount could be found loose ly bound to the c e l l s , i t was concluded that the enzyme was not l i b e r a t e d through the l y s i s of c e l l s . There was a considerable amount of l y s i s af ter the c e l l s had reached the peak of t h e i r growth curve (36 hours) i n 5% casein hydrolysate . At the same time, i n dialysed casein hydrolysate , h a l f the number of the c e l l s that were produced i n 5% casein hydrolysate showed very l i t t l e tendency to l y s e . C e l l s grown on glucose, having counts i n the same range as produced i n d ia lysed casein hydrolysate , a lso showed l i t t l e decrease i n number of -32-v iab le c e l l s even 36 hours a f ter the s tar t of the s ta t ionary phase. The only major difference between these cultures was the fact that there was considerably more protease present i n the 3% hydrolysate medium, suggesting that the enzyme might have a ro le i n c e l l l y s i s . Since the protease was produced by c e l l s that had reached t h e i r s tat ionary phase, i t seemed obvious to expect res t ing c e l l s to produce the protease too. P. aeruginosa was grown on glucose and the c e l l s harvested at 16 hours. A washed c e l l suspension at f o r t y times growth concentration (17-l8 mg/ml dry weight) was shaken i n a Warburg bath with a v a r i e t y of substrates which under normal growth condit ions induced production of the protease. The r e s t i n g c e l l s prepared from the glucose grown cu l ture , however, did not produce the enzyme despite the fac t , that judging from the oxygen uptake of the c e l l s , they were s tarv ing for periods as long as four hours. C e l l s grown on casein hydrolysate however produced the enzyme even i n the absence of a substrate or inducer. ( F i g . 3)' The endogenously r e s p i r i n g c e l l s produced the enzyme e a r l i e s t , while the other suspensions followed l a t e r , producing approximately the same amount of enzyme by four hours. ( F i g . 3) • There was no change i n the v iable count or t u r b i d i t y during the four hours of incubat ion . Glucose grown c e l l s did not produce the enzyme endogenously even when incubated with calcium. Thus there had to be induct ion of the enzyme during growth since s tarvat ion alone does not br ing -35-about the l i b e r a t i o n of the enzyme. Calcium ions seem to contro l the synthesis of the enzyme to a great extent. Even casein hydrolysate grown c e l l s (no C a + + i o n s added to the medium) f a i l e d to produce the enzyme endogenously i f they are starved without C a + + ions ( F i g . 4). The explanation for th i s might come from the fact that , as i t has been shown e a r l i e r , casein hydrolysate contains only a l i m i t i n g amount of calc ium. This amount of the cat ion i s used up by the c e l l s for the synthesis of the enzyme which i s excreted into the medium during growth. Af ter harvest ing the c e l l s do not contain any calcium reserves and unless the C a + + ions are supplied to the suspension no enzyme synthesis occurs. Enzyme production i s considerably higher endogenously when calcium i s supplied to the growth medium i n abundance, s ince the c e l l s are probably not depleted of calcium ions during washing and enzyme production gets under way at a faster r a t e . The above data strongly ind ica te that the enzyme i s being synthesized while the c e l l s are s tarv ing during endogenous r e s p i r a t i o n . To supplement the above suggestion the harvested casein hydrolysate grown c e l l s were crushed at zero time I and the extracts examined for enzyme a c t i v i t y . No a c t i v i t y was associated, at any time, with the c e l l extracts supporting the view that the - enzyme was synthesized while the c e l l s were r e s p i r i n g endogenously. -36-I I I . Properties of the protease To es tab l i sh the basic propert ies of the enzyme a r e l a t i v e l y crude preparation was obtained by t rea t ing the cul ture super-natants with (NH^^SO^ (90% sa turat ion) ; the p r e c i p i t a t e was disso lved i n buffer and dialysed for 2k hours against 0.01M sp pH 8.0. a) The optimal pH range was found to be between 7.0 and 9 .0 , the a c t i v i t y being highest at about 8.0 (Figure 5 « ) . b) Up to 40°C the a c t i v i t y increases l ogar i thmica l ly : witht". increas ing temperature, the Q10 being about 2. Above kO°Q there i s some denaturation of the enzyme taking place , although the a c t i v i t y keeps on increas ing up to about 60°C. The curve drops sharply at 70°C where denaturation i s so extensive that i t over-'« takes the k i n e t i c inf luence of temperature increase . (Figure 6.). c) K ine t i c propert ies of the enzyme At 60 C enzyme a c t i v i t y was not a common function of time or enzyme concentration. Thus i t was necessary to e s tab l i sh the temperature range where the enzyme a c t i v i t y did follow a s tra ight l i n e , since reproducible resu l t s would not be poss ible otherwise. Furthermore i f the enzyme did not fol low the course of zero order k i n e t i c s , the re su l t s obtained from di f ferent experiments could not be compared. As i t i s seen from Figures 8 and 9, incubation at 6o°C beyond 15 minutes causes denaturation of the enzyme to the extent that -39-the a c t i v i t y does not increase l i n e a r l y with t ime. S i m i l a r l y there i s no l i n e a r increase with time i f the enzyme i s supplied i n a higher or lower concentration than the optimum l e v e l . These re su l t s might have been obtained due to the fact that the enzyme could only p a r t i a l l y break down casein and would gradual ly run out of substrate after 15 minutes. Time curve, s t u d i e s ^ conducted at lower temperature, however, show that a l i n e a r r e l a t i o n s h i p was maintained between enzyme a c t i v i t y and time of incubation for periods as long as 60 minutes, while s i m i l a r amounts of casein were broken down, as indicated by the O.D. readings , as i n the case of incubat ion at 6o°C where the curve l e v e l s off a f ter 15 minutes and at lower O.D. readings. From the data presented, optimum condit ions for carry ing out the enzyme assay i n a reproducible manner have been es tabl i shed. The incubation period should l a s t no longer than 15 minutes at 6o°C y i e l d i n g an O.D. reading between 0.4-0.6 at 670 Vvyx when read on the Beckman Model B spectrophotometer. d) A c t i v i t y of the protease on d i f ferent proteins TABLE 5-The a c t i v i t y of the protease on d i f f erent substrates Substrate A c t i v i t y (Units x 10"^ ) Casein (2%) 4.2 Serum albumin (2%) I.85 CFX of P. aeruginosa 0.6 lactalbumin 0.9 £ OA-o 6 03-FIGURE 7 © Ul N O.f z TH;E course OF ENZYME ACTIVITY AT 30° C '0 20 30 +° T IME M I N U T E S 70 TIME COURSE OF EhlZYhiB CTlVlTy AT <30 °C T I M E M I N U T E S -42-Casein seems to be the best substrate for the protease of i P. aeruginosa, while serum albumin, lactalbumin and the organisms own prote in are considerably less s u i t a b l e . (Table 5). The enzyme was found to be act ive on ge la t in but quant i tat ive comparison was not poss ib l e , since ge la t in does not contain amino acids that could react with the phenol reagent. e) Metal requirements for the a c t i v i t y of the enzyme Both ethylene diamine tetraacetate (Versene) and extensive d i a l y s i s caused reduction i n enzyme a c t i v i t y , that could not be restored by the r e - a d d i t i o n of metal ions . P r a c t i c a l l y a l l metals (0.002M) i n h i b i t the enzyme act ion to ++ some extent except Ca i n small concentrations. TABLE 6 The effect of metal ions on enzyme a c t i v i t y •3 Metal added (0.002m) A c t i v i t y (units x 10 ) contro l 3«2 F e + + 2 ' 1 M g + + 2.8 C a + + 3 .1 N i + + 0.3 M n + + 2.8 Z n + + 0.4 C o + + 2.75 FIGURE 10 T7-/E EFFECT OF VERSGNB ON SNZyMB ACTIVITY. © 0.01 0.0 2 M O L A R C O N C . OF V E R S E N E 0.03 F I G U R E II Tff£ EFFECT OF IONIC STRENGTH ON ENZYME ACTIVITY. 1 0.10 0.20 M O L A R C O N C . OF NaCI The enzyme did not show appreciable s e n s i t i v i t y to r e l a t i v e l y high ion concentrations; (Figure II) therefore, the i n h i b i t i o n by metal ions at low concentrations must have been due to s p e c i f i c i n h i b i t i o n by the respective ions . " 8 - h y d r o x y quinol ine , cysteine and glutathione were also found to i n t e r f e r e with enzyme a c t i v i t y probably through the binding of calcium i n the s tructure of the enzyme. f) S t a b i l i t y of the enzyme The enzyme was stored i n d i s t i l l e d water, 0.03M T r i s buffer pH 8.0 and i n cone. (NH^)2S04 a t i f ° c a n d a t r o o , n temperature. The enzyme showed reasonable s t a b i l i t y under a l l condit ions inves t iga ted . Most i n t e r e s t i n g , of course, i s the fact that no loss of a c t i v i t y occured af ter four days i n concentrated ammonium su l fa te s o l u t i o n . (Table 7). -45-TABLE 7 The s tabi l i ty of the enzyme under various conditions i o days Hoom temp 4 C Dist . water ENZ. A6T UNITS x 0 4.6 4.4 1 4.4 4.4 2 4.5 ^ 3 4.3 ^.35 4 4.4 4.5 0.03M Tris pH 8.0 0 1 2 4.4 .^6 4.6 k.5 4.5 k A 3 4.4 4 4.5 CONC. ( N H 2 F ) 2 S 0 I + 0 1 2 3 4.2 ^ . 8 4.3 -^7 4.3 ^ 4.4 ^ . 8 4 4*2 '^7 -46-GENERAL DISCUSSION I . N u t r i t i o n a l Requirements for the Production of the Protease The re su l t s (Table 2 and 3) concerning the enzyme production i n various media are in contrast with the f indings of Morihara (1962), who obtained the best protease y ie lds with a glucose mineral sa l t s medium supplemented with corn steep l i q u o r . • Morihara (19&2), however, did not invest igate a number of media, s i m i l a r to peptone and beef ex tract , besides h is "natural medium". Under such circumstances one would f ind i t d i f f i c u l t to draw c lear cut conclusions, since the low enzyme y ie lds i n the "natural medium" (N.M.) might have resul ted from the presence of a s p e c i f i c i n h i b i t o r or repressor, or from the absence of an inducer. A good i l l u s t r a t i o n of such phenomenon can be seen i n Table 2, where the protease production of P. aeruginosa ATCC 9027 var ies widely i n the media ranging from peptonized milk to casein hydrolysate . Since they are derived from natura l sources through p r o t e o l y t i c digest ion a l l these media would come under the heading "Natural media", yet the enzyme production by bacter ia d i f f e r s considerably i n these media. For the production of an e x t r a c e l l u l a r protease, one should f ind i t most reasonable that peptides of cer ta in s ize and composition, contained i n these enzyme digests mentioned above, -47-would serve as inducers for the l i b e r a t i o n of the protease, and the abundance of t h e i r presence might control enzyme l i b e r a t i o n . The minimal amounts of enzyme found i n the glucose + CaCO-, cu l ture supernatants of several Pseudomonas s t ra ins ind ica te that these s t ra ins d e f i n i t e l y require add i t iona l nutr i en t s , most l i k e l y those of proteinaceous nature, for the production of the protease. The fact that any protease production occured at a l l , i n the absence of proteinaceous substrates can be explained by the theory of Castaneda-Agullo (1956). He suggested that induct ion of a b a c t e r i a l proteinase might be brought about by the proteins of dead bacter ia or by proteinaceous der ivat ives of metabolism, formed only when the cul ture has obtained a high concentration of b a c t e r i a l mass. The above theory would f i t i n with the f indings of Morihara (1956) who could detect the presence of enzyme only a f ter the growth curve l e v e l l e d of f and p r a c t i c a l l y a l l the substrate was used up. The effect of calcium on enzyme production was found to be very s i g n i f i c a n t . The enzyme y i e l d s i n a l l media were increased considerably upon supplementation with C a + + ions . Morihara (1959 A, 1959 B) c l e a r l y established the ro l e of X calcium i n the synthesis of the protease produced by P. mycogenes. F i sher (i960) was unable to obtain enzyme from the supernatant of P. aeruginosa with glucose and mineral s a l t s medium. Although he did not t ry to supplement the medium with calcium s a l t s , he -48-concluded that cer ta in peptides and proteins should be added to the medium to obtain the enzyme. I I . The mechanism of l i b e r a t i o n of the protease a) Mechanism of induct ion The most remarkable feature i n the course of l i b e r a t i o n of the protease by P. aeruginosa ATCC 9027 was that the enzyme did not appear i n the medium u n t i l r e a d i l y a v a i l a b l e , smaller molecular weight substrates were used up. Morihara (1956) also showed that only n e g l i g i b l e amounts of enzyme appeared i n the supernatant u n t i l a l l the glucose was metabolized by the b a c t e r i a . Castaneda-Agullo (1958) even went as far as suggesting that the protease of S. marsescens i s produced due to s tarvat ion of the c e l l s . There was a d e f i n i t e lag i n c e l l m u l t i p l i c a t i o n and i n the breakdown of 5% casein hydrolysate (Figure I) between 16 and 24 hours, i n the very period when the enzyme was f i r s t detected i n "I the supernatant. The—fact—that i n dia lysed casein hydrolysate the c e l l s produced the protease much e a r l i e r than i n 5% casein hydrolysate , despite the fact that the former medium was consider-ably less r i c h i n nutr i en t s . On the other hand, dia lysed casein hydrolysate was an espec ia l ly poor source of lower molecular weight substrates that can be r e a d i l y metabolized; i n other words, the c e l l s would reach the s tarv ing stage e a r l i e r than i n 5% casein hydrolysate . - 4 9 -Resting c e l l s r e s p i r i n g endogenously l i b e r a t e the enzyme as ear ly as af ter ha l f an hour incubat ion at 30°C provided they are.; grown on casein hydrolysate (or s imi lar substrate) and there i s an ample supply of Ca ions a v a i l a b l e ) . At the same time, i f the c e l l s are provided with a substrate the enzyme production i s somewhat retarded probably u n t i l the oxidizable substrate i s used up. (Figure 3)• The above discuss ion suggests strongly that s tarvat ion for some period of time i s one of the pre -requ i s i t e s for the l i b e r -a t ion of the protease by P. aeruginosa. Although i t i s an important fac tor , s tarvat ion could not be the only one governing enzyme synthesis and excret ion, s ince c e l l s s tarv ing i n a glucose medium^liberated only a minimum amount of the enzyme probably due to the lack of a proper inducer. The amount of the inducer present seems to play an important part i n enzyme synthes is , s ince i n d ia lysed casein hydrolysate there i s about—— of the enzyme produced by ha l f as many c e l l s as there 80 are i n the 5% casein hydrolysate c u l t u r e . The necessity of an inducer i s apparent from the fact that starved c e l l s only produce the enzyme when previous ly grown on casein hydrolysate, while glucose grown c e l l s f a i l to manufacture the enzyme when r e s p i r i n g endogenuously. Such a contro l mechanism cons i s t ing of repress ion by low molecular weight compounds and induct ion by peptides of c e r t a i n i&i'z'e, would seem very economical. - ' • ~ - 50 -g) The E x t r a c e l l u l a r nature of the enzyme Pol lock (I962) out l ines a number of c r i t e r i a by which one can determine the extent of the c e l l bound character of b a c t e r i a l enzymes. One of these i s the assay of a cytoplasmic enzyme i n the supernatants as the i n d i c a t o r of l y s i s . Working with p r o t e o l y t i c enzymes, however one might f ind i t d i f f i c u l t to look for i n t a c t prote ins i n the supernatant, s ince espec ia l ly at the l a t e r stages of growth when the substrate i s l a r g e l y used up, the b a c t e r i a l protease w i l l probably break down the cel ls 'own prote in i n c l u d i n g the cytoplasmic enzyme one i s looking f o r . In the 5% casein hydrolysate medium f a i r l y extensive l y s i s takes place s t a r t i n g at Zk hours, the time corresponding with the appearance of the protease i n the supernatant. Meanwhile there i s l i t t l e or no l y s i s i n the other media where the enzyme i s present only i n minimal quant i t i e s . These observations combined would strongly suggest a ro l e for the enzyme i n l y s i n g the c e l l s , a f ter the substrate has been broken down, as far as the enzyme's s p e c i f i c i t y b a r r i e r permits . Judging the course of l i b e r a t i o n of the enzyme (Figure 1), one would not expect l y s i s to be the mechanism by which the enzyme i s l i b e r a t e d . Furthermore, only very small portions of the enzyme were found to be very loose ly associated with the c e l l s , a c r i t e r i o n that also character izes e x t r a c e l l u l a r enzymes. -51-The resu l t s with r e s t i n g c e l l s strongly suggest that the enzyme was a c t u a l l y synthesized while the c e l l s were shaken i n the Warburg cup. To back up t h i s c la im, one should consider the fact that even casein hydrolysate grown c e l l s f a i l e d to produce the enzyme unless they were provided with the necessary amounts ++ of Ca ions . The v iable c e l l counts did not change during these experiments i n d i c a t i n g that no l y s i s took place . Presuming that the enzyme was synthesized i n the cup while there was no loss of v iable c e l l s one can safe ly conclude that the enzyme i s l i b e r a t e d i n a t y p i c a l l y e x t r a c e l l u l a r manner. Another question however a r i s e s , regarding the l i b e r a t i o n of the enzyme by s tarv ing c e l l s . Why would these c e l l s keep producing the enzyme when there i s no inducer provided for them and the synthesis of such enzyme prote in re su l t s i n considerable losses of energy to the c e l l ? One might o f fer an acceptable explanation by presuming that the induced c e l l s produce the enzyme u n t i l there are enough precursors ins ide the c e l l perhaps u n t i l the amino-ac id pool i s depleted. In the case of s tarving c e l l s the enzyme production l eve l s of f:.. r a p i d l y while i f amino acids are suppl ied the enzyme production increasesss lowly u n t i l presumably the substrate i s used up and the enzyme l e v e l increases somewhat fas ter from here on. Further experiments should be carr i ed out to c l a r i f y the s ign i f i cance of t h i s somewhat pecu l iar phenomenon. -52-I I I . Propert ies of the Enzyme Amongst the numerous reports i n the l i t e r a t u r e descr ib ing the propert ies of proteases there are several deal ing with the proteases .of Pseudomonas sp e s p e c i a l l y that of P. aeruginosa. Morihara (1956) found that the protease of P. myxogenes that i s immunologically i n d e n t i c a l to the protease of P. aeruginosa (Morihara (1962) has an optimum temperature, for the l i q u e f a c t i o n of g e l a t i n , of about 45°. . Yet F i sher (i960) , Morihara (1963) and McDonald (1963) a l l c laim that the optimum temperature for the protease of P. aeruginosa i s about 60 C. Ge la t in does not seem to be the proper substrate for determining optimum temperature probably due to the fact that i t changes i t s phys ica l propert ies with increas ing temperature. The above authors found the pH optimum to be between pH 7.5 and 8.5. The pH and temperature optimum reported i n t h i s study thus agree with other reports i n the current l i t e r a t u r e . The permanent damage caused to the enzyme by the add i t ion of che la t ing agents deserves some a t t e n t i o n . Enzyme a c t i v i t y cannot be restored to the preparation by adding back divalent ions . On the other hand the enzyme a c t i v i t y was not enhanced but i n h i b i t e d by Ca ion concentrations higher than 0.010N. The above observations s trongly support the claims of G o r i n i (1950) and Morihara (1959 ) that calcium i s e ssent ia l for the synthesis of the enzyme, s ince i t ; par t i c ipa te s i n b u i l d i n g the s tructure of the enzyme, and not i n ca ta lys ing p r o t e o l y s i s . -54-The enzyme was found to be except ional ly stable at both 4°C and at room.temperature. Metal binding agents such as EDTA and 8-hydroxy quinol ine ser ious ly in ter fered with enzyme a c t i v i t y . It was suggested that ++ Ca ions were required for the synthesis of the enzyme and not as a cata lys t for p r o t e o l y s i s . -55-BIBLIOGRAPHY 1. Berman, N. 1918. The inf luence of carbohydrates on the nitrogen metabolism of b a c t e r i a . J . Bact. 3* 389-2. Castaneda-Agullo, M. 1956. Studies on the biosynthesis of external proteases by b a c t e r i a . I . Senatia marsescens. Synthesis any ge la t in media. J . Gen. P h y s i o l . 39, 369. 3. Castaneda-Agullo, M. 1958. Studies on the biosynthesis of e x t r a c e l l u l a r proteases by bac te f ia . I I I . Ef fec t s of several ava i lab le energy compounds. Revista Latinoamericana de .Microb io log ia , V o l . 1, Num.3, p. 177. 4. Campbell, L . L . 1957 • P u r i f i c a t i o n and propert ies of a pro teo ly t i c enzyme from Baci l lusSlearothermophi lus . Arch . Bioch. Biophys. 70, 432. 5. C l a r k , C.W and M e r r i l l , A . T . (1928). Some condit ions a f f ec t ing the production of gelat inase by proteus b a c t e r i a . J . Bact. 15, p. 267. 6. Damodaran, 1955- The p r o t e o l y t i c system of B a c i l l u s lidnenformis Biochem. Biophys,, Acta , 17, 99-7. Fermi, C. (1890). Die le im und f i b r i n losenden und die d iastat i schen fermente der microorganismen. Arch, fur Hyg. und B a c t . , 10, p. 1. 8. F i s h e r , E . (i960). Some propert ies of a proteinase obtained from P. aeruginosa. Bact . Proc. i960, p. 60. 9. G o r i n i , L . (1950). Le ro le du calcium dans l ' a c t i v i t e et l a s t a b i l i t e de quelques proteinases bacteriennes. Biochim. Biophys. Acta , 6_, p. 237. 10. Guntelberg, A . V . A . (1954). A method for the production of the plakalbumin - forming proteinase from B a c i l l u s  s u b t i l i s . Compt. rend. t rav . l a b . Carlsberg ser . ch im. , 29, 27-35. 11. Hagihara, B. (1954). C r y s t a l l i n e b a c t e r i a l amylase and proteinase . Ann. Rep. S c i . Works, Facul ty of Science, Osaka U n i v . , 2, p. 36. -56-12. Haines, R .B . (1931)- Formation of B a c t e r i a l Proteases. Biochem. Journ. 25, 1849-13. Haines, R .B . (1933)' Further studies on the effect of the medium on the production of b a c t e r i a l ge lat inase . Biochem. J o u r n . , 27, 466. 14. McDonald, I . J . (196l). Proteinase production i n r e l a t i o n to growth of a Micrococcus species . Can. J . M i c r o b i o l . , 7, p. 111. 15. McDonald, I . J . (1963). P r o t e o l y t i c a c t i v i t y of some c o l d -to lerant bacter ia from a r c t i c sediments. Can. J . M i c r o b i o l . , 9, 303. 16. Morihara, K. (1956). Studies on the protease of Pseudo-monas . Part I . The production of the enzyme under various c u l t u r a l condi t ions . B u l l . Agr. Chem. Soc. Japan, 20, Suppl. p. 24-3. 17. Morihara, K. (1956 A ) . Studies on the protease of Pseudo-monas . Part I I . C r y s t a l l i z a t i o n of the protease and i t s physico-chemical and general proper t i e s . B u l l . Agr. Chem. Soc. Japan, 21, No. 1, p. 11. 18. Morihara, K. (1959)- Studies on the protease of Pseudo-monas . Part V. On the role of calcium i n the enzyme production. B u l l . Agr . Soc. Chem. Japan, 23_, No. 1, p. 60. 19. Morihara, K. (1962). Studies on the protease of Pseudo-monas. Part V I I I . Proteinase production of various Pseudomonas species , e spec ia l ly P. aeruginosa. Agr. B i o l . Chem., 26, No.12, p. 842. 20. Morihara, K. (1963). Pseudomonas aeruginosa prote inase . I . P u r i f i c a t i o n and general p r o p r t i e s . Biochim. Biophys. A c t a , 73» P- 113. 21. N o r r i s , F . C . and Campbell, J . J . R . (1949). The intermediate metabolism of Pseudomonas aeruginosa. I . The a p p l i c a t i o n of paper chromatography to the i d e n t i f i -cat ion of gluconic and 2-ketogluconic ac ids , i n t e r -mediates i n glucose ox idat ion . Can. Journ. of Research, 27_, p. 253. 22. Po l lock , M.R. (1956). The cel l -bound p e n i c i l l i n a s e of B a c i l l u s cereus. J . Gen. M i c r o b i o l . , 14, p. 90. 23. Po l lock , M.R. (1961). In The Bac ter ia , V o l . 4, Chap. 12. Exo-Enzymes. ( I . C . Gunsalus & R.Y. Stanier , E d s . ) . New York : Academic Press . -57-2k. Schoenheimer, R. (1946). The Dynamic State of Body- Const i tuents . Harvard Univers i ty Monographs i n Medicine and Publ ic Health No. 3» Cambridge, Massachussetts, Harvard Univers i ty Press , 2nd e d i t i o n . Wilson, E . (1930) Studies i n b a c t e r i a l proteases. I . The / r e l a t i o n of protease production to the cul ture medium, J . Bact, 20, p. kl. Zimmerman, L . N . et a l (1957)' Proteinase biosynthesis by Streptococcus l iquefac iens . I I . Purine , pyrimidine and vitamin requirements. Can. J . M i c r o b i o l . 3, P- 553« 

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