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Hydrologic properties and water balance of the forest floor of a Canadian west coast watershed 1972

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THE CINNAMIC ACID PATHWAY AND HISPIDIN BIOSYNTHESIS IN CULTURES OF POLYPORUS HISPIDUS FRIES by PETER WILLIAM PERRIN B.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In presenting t h i s thesis in 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 shall make it f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying'of this thesis for s c h olarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or p u b l i c a t i o n of this thesis for f i n a n c i a l gain shall not be allowed without my written permission. Department of The University of B r i t i s h Columbia Vancouver 8, Canada Date i ABSTRACT The biosynthesis of h i s p i d i n , 6-(3,4-dihydroxystyryl)- 4-hydroxy-2-pyrone, was examined i n cultures of Polyporus hispidus Fr. C u l t u r a l studies were undertaken to determine the most sui t a b l e medium for in v e s t i g a t i n g the biosynthesis of t h i s pigment. These studies showed that l i g h t was nec- essary for h i s p i d i n formation and that the development of basidiocarps with viable spores could be achieved on agar media. On the l i q u i d medium employed for biochemical studies, the maximum rate of h i s p i d i n production was observed to lag the maximum rate of growth by about f i v e days. Trimethylhispidin, 4-methylhispidin and yangonin were synthesized for comparative purposes and for d i l u t i o n i n tracer experiments. These and numerous other phenolic and aromatic compounds were employed as references i n examinations of nonradioactive culture extracts. P-coumaric, c a f f e i c , £- hydroxybenzoic, protocatechuic and o- and p_-hydroxyphenyl- a c e t i c acids were detected i n extracts of the culture medium. Bis-noryangonin (6-(4-hydroxystyryl)-4-hydroxy-2-pyrone) and other possible styrylpyrones were detected i n extracts of the mycelium. Tracer experiments established that phenylalanine was metabolized to cinnamic, benzoic, p_-hydroxybenzoic and protocatechuic acids. The incorporation of rad i o a c t i v i t ; ' 1 1 i n t o p h e n y l l a c t i c , p h e n y l p y r u v i c , p h e n y l a c e t i c and rj-hydroxy- p h e n y l a c e t i c a c i d s a l s o was observed. Furthermore, p_-coum- a r i c a c i d , c a f f e i c a c i d and h i s p i d i n i n c o r p o r a t e d r a d i o a c t i v - i t y from p h e n y l a l a n i n e . H i s p i d i n a l s o was shown t o i n c o r - p o r a t e r a d i o a c t i v i t y from t y r o s i n e , cinnamic a c i d , p_-coum- a r i c a c i d , c a f f e i c a c i d , malonic a c i d and sodium a c e t a t e . D e g r a d a t i o n o f the l a b e l l e d h i s p i d i n o b t a i n e d from these p r e - c u r s o r s confirms the h y p o t h e s i s t h a t t h i s molecule i s b i o - s y n t h e s i z e d from a phe n y l p r o p a n o i d moiety w i t h the a d d i t i o n o f two e q u i v a l e n t s o f a c e t a t e . Crude and p a r t i a l l y p u r i f i e d p r e p a r a t i o n s o f s e v e r a l enzymes r e l a t e d t o aromatic metabolism were o b t a i n e d . Phen- y l a l a n i n e and t y r o s i n e ammonia-lyase a c t i v i t y were demonstrat- ed i n c e l l - f r e e p r e p a r a t i o n s . Maximum p h e n y l a l a n i n e ammonia- l y a s e a c t i v i t y was o b t a i n e d from c u l t u r e s d u r i n g the l o g a r - i t h m i c phase o f growth. Enzymes capable o f h y d r o x y l a t i n g cinnamic a c i d , b e n z o i c a c i d and b i s - n o r y a n g o n i n a l s o were o b t a i n e d i n v i t r o . i i i TABLE OF CONTENTS PAGE ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES , v i LIST OF FIGURES v i i ACKNOWLEDGMENT i x INTRODUCTION 1 LITERATURE REVIEW 4 I. The acetate-polymalonate pathway i n fungi 4 I I . The shikimic acid pathway i n fungi............ 6 I I I . Aromatic amino ac i d metabolism i n Basidio- mycetes 11 IV. Pigment production i n fungi 16 V. Sporophore formation i n Basidiomycetes......... 17 CHAPTER ONE. CULTURAL STUDIES OF POLYPORUS HISPIDUS... 21 Introduction 21 Materials and Methods 22 I. Sources of cultures 22 I I . Sources of medium constituents 22 I I I . Media employed 23 IV. Culturing techniques 24 V. Measurements of h i s p i d i n production 24 VI. Dry weight determinations 25 Results and Discussion.. 26 I. Comparison of P_. hispidus and P. schwein- i t z i i 26 I I . Growth and pigment production i n P. h i s - pidus 30 I I I . Sporophore formation i n agar cultures of P. hispidus . 45 i v TABLE OF CONTENTS (cont'd) PAGE CHAPTER TWO. CHEMICAL STUDIES OF PHENOLIC ACIDS, STYRYLPYRONES AND RELATED COMPOUNDS 49 Introduction 49 Materials and Methods 49 I. Chemicals 49 I I . - Chromatography. 50 I I I . Spectroscopy 51 IV. Melting points 51 V. Chemical preparations 51 T r i a c e t i c lactone. • 51 6-methyl-4-methoxy-2-pyrone. 52 Trimethylhispidin 53 4-methylhispidin 53 Al k a l i n e hydrolysis of t r i m e t h y l h i s p i d i n . . . 54 3,4-dimethoxycinnamic a c i d . . . . . . . . . . . . . . . . . 55 3,4-bis-(methoxymethoxy)benzaldehyde 55 • Vera t r i e a c i d from t r i m e t h y l h i s p i d i n 56 VI. Radioautography 57 Results and Discussion 58 CHAPTER THREE. RADIOACTIVE FEEDING EXPERIMENTS WITH CULTURES OF POLYPORUS HISPIDUS 70 Introduction 70 Materials and Methods 70 I. Analysis of phenolic acids 70 I I . Preparation and administration of radioac- t i v e compounds 70 I I I . Detection of r a d i o a c t i v i t y 71 IV. Recovery of free amino acids 72 14 V. Methylation of C-l a b e l l e d h i s p i d i n 72 Results and Discussion 73 V TABLE OF CONTENTS (cont'd) PAGE CHAPTER FOUR. PRELIMINARY STUDIES OF ENZYMES ASSO- CIATED WITH AROMATIC METABOLISM 92 I n t r o d u c t i o n 92 M a t e r i a l s and Methods 93 I. P h e n y l a l a n i n e ammonia-lyase........... 93 I I . T y r o s i n e ammonia-lyase.... 94 I I I . Benzoic and cinnamic a c i d - 4 - h y d r o x y l a s e . . . 94 IV. Bis-noryangonin-3-hydroxylase 95 R e s u l t s and D i s c u s s i o n 96 GENERAL SUMMARY AND CONCLUSIONS 100 BIBLIOGRAPHY 104 APPENDICES 113 A. C h a r a c t e r i s t i c o f Wrattan f i l t e r s 113 B. Spray Reagents 114 v i LIST OF TABLES TABLE PAGE I. M y c e l i a l dry weight and pH of medium of cultures of P. hispidus a f t e r sixteen days when grown on DM#1 at various i n i t i a l pH* s 34 I I . Colours of phenolic acids i n long wave u l t r a - v i o l e t l i g h t and i n v i s i b l e l i g h t a f t e r spray- ing with diazotized-p_-nitroaniline reagent „ 60 I I I . C h a r a c t e r i s t i c s of some styrylpyrones and r e l a - ted phenolic compounds chromatographed on c e l l u - lose TLC plates i n solvent system C and sprayed with various reagents (Appendix B) 66 IV. Metabolic products from various aromatic com- pounds administered to P. hispidus 82 V. Incorporation of various precursors i n t o h i s p i d - i n by 17-day-old cultures of P. hispidus 87 v i i LIST OF FIGURES FIGURE PAGE 1* Naturally-occurring styrylpyrones 2 2. Acetate-polymalonate-derived fungal products.... 5 3. Complex polyketides from Basidiomycetes 7 4* Shikimic a c i d pathway to aromatic amino acids... 8 5. Shikimic-acid-derived metabolites of Basidio- mycetes 10 6. Fungal metabolites of mixed biogenesis 12 7. Transformations of cinnamyl compounds i n L. lep- ideus o 14 8. Comparison of the growth of P. hispidus and P. s c h w e i n i t z i i on l i q u i d MYP 27 9. O p t i c a l density measurements of ether extracts of the mycelium of P. s c h w e i n i t z i i 28 10. pH change during growth of P_. hispidus on DM#1.. 32 11. V a r i a t i o n i n colony diameter with temperature of P. hispidus cultures on MYP agar 35 12. E f f e c t of wavelength of l i g h t source on growth and pigment development i n agar cultures 37 13. E f f e c t of malt extract and Soytone concentration on colony diameter a f t e r f i f t e e n days.. 40 14. Growth and h i s p i d i n production of P_. hispidus on GYSS 44 15. Pigment development i n the mycelium of P. h i s - pidus a f t e r ten and f i f t e e n days incubation on GYSS 46 16. Sporocarp of P. hispidus ten days a f t e r i n i t i a - t i o n on MYP agar 46 17. Diagrammatic representation of two-dimensional chromatogram of authentic samples of phenolic and cinnamic a c i d derivatives 59 v i i i LIST OF FIGURES (cont'd) FIGURE PAGE 18. Standard absorbance curve of p_-coumaric a c i d . . . . . 61 19. Standard absorbance curve of c a f f e i c acid 62 20. Standard absorbance curve of t r i m e t h y l h i s p i d i n . . . 63 21. Standard absorbance curve of v e r a t r i c a c i d 64 22. U l t r a v i o l e t spectra of yellow, fluorescent bands from chromatograms of P_. hispidus extracts 68 23. Diagrammatic representation of the compounds de- tected i n chromatographed extracts of the medium using 48 hr induction by replacement medium 75 24. Probable pathways of L-phenylalanine degradation i n P. hispidus 76 25 Probable relationships of radioactive cinnamic ac i d derivatives detected i n cultures of P_. h i s - pidus 78 26. Aromatic amino ac i d metabolism v i a the cinnamate pathway i n P. hispidus 85 27. Biosynthesis and degradation of radioactive h i s - p i d i n . 91 28. Phenylalanine ammonia-lyase a c t i v i t y i n cultures of P. hispidus 97 29. Alternate routes proposed f o r the biosynthesis of h i s p i d i n i n P. hispidus 102 i x ACKNOWLEDGMENT I wish to express my sincere gratitude to Dr. R.J. Bandoni and Dr. G.H.N. Towers with whose f a c i l i t i e s and under whose guidance t h i s work was c a r r i e d out. Their en- couragement, advice and c r i t i c i s m of t h i s manuscript are g r a t e f u l l y acknowledged. The assistance and advice of Dr. C.K. Wat and the f a c i l i t i e s provided by Dr. T. Money were gre a t l y appreciated. To my collegues, for h e l p f u l discus- sions, and to the members of my committee, f o r t h e i r com- ments on t h i s manuscript, I give my thanks. The f i n a n c i a l support of the National Research Council of Canada and of the H.R. MacMillan family i s acknowledged. Also, I would l i k e to thank my wife, Janne, f o r her encouragement throughout t h i s work. I N T R O D U C T I O N 1 Naturally occurring sporophores of Polyporus hispidus Fr., P_. s c h w e i n i t z i i Fr. and several species of Gymnopilus contain the styrylpyrone pigment h i s p i d i n (Fig. l.)(Bu'Lock and Smith 1961, Edwards et a l 1961, Ueno et a l 1964, Hat- f i e l d and Brady 1971). Gymnopilus also contains the s t y r y l - pyrone bis-noryangonin (Fig. 1 . ) ( H a t f i e l d and Brady 1968). Compounds of t h i s type were f i r s t known from higher plants, e s p e c i a l l y Aniba firmula (Nees et Mart.) Mez. and Piper meth- ysticum Forster. From these have been i s o l a t e d 5,6-dehydro- kawain and yangonin (Fig.1.)(Borsche and Gerhardt 1914, Gott- l i e b and Mors 1959). More recently species of A l p i n i a (Kim- ura et a l 1966) and Ranunculus (Shibata et a l 1972) also have been shown to contain these molecules. In higher plants there i s evidence that s t y r y l - pyrones are synthesized v i a the shikimic and cinnamic a c i d pathways with the addition of two acetate units much i n the same manner as the 'A' r i n g of flavonoids i s produced (Wat- k i n et a l 1957, Geissman and Swain 1957, Shibata and Yama- zaki 1957, Birch and Donovan 1953). Also, i n P. s c h w e i n i t z i i i t has been demonstrated that phenylalanine and acetate are incorporated i n t o the h i s p i d i n molecule (Hatfield 1970). Thus there i s preliminary evidence that fungi are capable of elabor- a t i n g upon phenylpropanoid molecules. 2 YANGONIN (Piper, Ranunculus) OH HO- b i s-NORYANGONIN (Gymnopilus) OCH, 5,6-DEHYDROKAWAIN (Aniba, A l p i n i a ) OH HISPIDIN (Polyporus, Gymnopilus) FIG. 1. Naturally-occurring styrylpyrones. 3 This study w i l l present a d d i t i o n a l evidence f o r the biosynthesis of styrylpyrones i n fungi. The intermediary aromatic a c i d metabolism of P. hispidus i s examined with radioactive tracers and the incorporation of s p e c i f i c a l l y l a b e l l e d precursors i n t o the h i s p i d i n molecule i s shown. Also, the presence of c e r t a i n necessary enzymes i s demonstrated i n c e l l - f r e e extracts of the fungus. Bu'Lock (1967) reported that P. s c h w e i n i t z i i produced h i s p i d i n when grown on usual culture media, but that P. hispidus yielded t h i s compound only when grown on wood blocks. In t h i s study, the e f f e c t s of environmental and n u t r i t i o n a l factors on growth and h i s - p i d i n production i n l i q u i d cultures of J?. hispidus i s exam- ined. LITERATURE REVIEW 4 I . The a c e t a t e - p o l y m a l o n a t e pathway i n f u n g i As e a r l y as 1907 ( C o l l i e ) the b i o s y n t h e s i s o f o r s e l - l i n i c a c i d was c o n s i d e r e d t o o c c u r by the c o n d e n s a t i o n o f a c e t a t e u n i t s . In 1953, e x p e r i m e n t a l evidence i n d i c a t e d t h a t C o l l i e ' s t h e o r y was c o r r e c t ( B i r c h and Donovan 1953). Two years l a t e r , evidence was obained f o r the s y n t h e s i s o f 6 - m e t h y l s a l i c y l i c a c i d by P e n i c i l l i u m g r i s e o f u l v u m v i a a p o l y a c e t a t e pathway ( B i r c h e t a l 1955). Evidence f o r the p r o d u c t i o n o f o r s e l l i n i c a c i d and 6 - m e t h y l s a l i c y l i c a c i d by P e n i c i l l i u m u r t i c a e and P. g r i s e o f u l v u m was r e p o r t e d sub- s e q u e n t l y t o i n v o l v e m a l o n i c a c i d as a " b u i l d i n g u n i t " i n the manner o f f a t t y a c i d b i o s y n t h e s i s ( B e n t l e y and K e i l 1961, B i r c h e t a l 1961, Bu*Lock e t a l 1962). More r e c e n t l y , the b i o s y n t h e s i s o f t e t r a c e t i c l a c t o n e and methyl t r i a c e t i c l a c - tone (Fig.2.) by P e n i c i l l i u m s t i p i t a t u m have been shown t o o c c u r by the a c e t a t e - p o l y m a l o n a t e pathway (Bentl e y and Zwitkowits 1967, Tanenbaum e t a l 1969). C e l l - f r e e enzyme p r e p a r a t i o n s o f P e n i c i l l i u m patulum now have been o b t a i n e d which w i l l condense a c e t y l and m a lonyl Coenzyme A i n t o 6- m e t h y l s a l i c y l i c a c i d and t r i a c e t i c a c i d l a c t o n e (Dimroth e t a l 1970). However, t h e r e a r e no r e p o r t s o f c e l l - f r e e p r e - p a r a t i o n s which w i l l extend an a r o m a t i c a c i d p r e c u r s o r as i n f l a v o n o i d o r s t y r y l p y r o n e b i o s y n t h e s i s . 5 O H H 3 C ^ 0 ^ 0 METHYL TRIACETIC LACTONE O H TETRACETIC LACTONE FIG. 2. Acetate-polymalonate-derived fungal products, 6 In addition to simple polyketides, many complex polyketides are produced as secondary metabolites of fungi, although these compounds are r e l a t i v e l y rare i n Basidiomy- cetes (Turner 1971). Cortinarius sanguineus has yi e l d e d the r e l a t e d anthraquinones, emodin, dermoglaucin and dermocybin (Fig. 3.)(Kogl and Postowsky 1925, S t e g l i c h and Austel 1966). These octaketides are among the most complex polyketides known from Basidiomycetes. Several penta- and lower molecular weight ketides also have been i s o l a t e d from Basidiomycetes. Some examples of these are 6-methyl-l,4- naphthoquinone from Marasmius graminum (Fig. 3.)(Bendz 1951) and 8-hydroxy-3-methylisocoumarin from Marasmius ram- e a l i s (Fig. 3.)(Bendz 1959). I I . The shikimic a c i d pathway i n fungi Evidence indicates that i n fungi the shikimic a c i d pathway i s used much less commonly than the acetate-polymal- onate or polyketide pathway f o r the biosynthesis of aromatic secondary metabolites (Bentley 1962). Nevertheless, the shikimic a c i d pathway (Fig. 4.) i s believed to be operative i n aromatic amino a c i d synthesis i n most fungi (Burnett 1968, Turner 1971). Although much of the research i n e l u c i d a t i n g the shikimic a c i d pathway has been c a r r i e d out u t i l i z i n g bac- t e r i a l mutants (Davis 1955, Gibson and Gibson 1964, Sprin- son I960), mutants of Neurospora crassa have been u t i l i z e d 7 R 4 Q R 5 DERMOGLAUCIN OH OH OCH3 H H DERMOCYBIN OH OH OCH3 OH H (Cortinarius sanguinius) O 6-METHYL—l,4-NAPHTHOQUINONE (Marasmius graminum) 0 H 0 8-HYDROXY-3-METHYLISOCOUMARIN (Marasmius ramealis) FIG. 3. Complex Polyketides from Basidiomycetes CHO I HCOH I HCOH CH 2OP0 3H 2 Erythrose 4- phosphate 8 COOH I COPO-H-| 3 2 CH„ COOH I c=o I CH-I 2 HOCH I HCOH I HCOH Phosphoenolpyruvic a c i d CH 2OP0 3H 2 3-deoxy-2-keto-D- arabino-heptulosonic a c i d 7-phosphate COOH COOH HO Shikimic a c i d COOH OH 5-dehydroshikimic a c i d COOH OH H 20 3P 5-phosphoshikimic a c i d NH2 CH2-CH-COOH 3-enolpyruvylshikimic ac i d 5-phosphate O CH2_C-COOH ' r HO COOH 5-dehydroquinic a c i d COOH OH Chorismic a c i d 1 HOOC ' CH2-C-COOH Phenylalanine Phenylpyruvic a c i d Prephenic acid FIG. 5. Shikimic a c i d pathway to aromatic amino acids. 9 extensively i n studies of t h i s pathway i n fungi. A mutant of Neurospora has been shown to possess the enzyme dehydro- shikimic dehydrase catalyzing the conversion of 5-dehydro- shikimic a c i d to protocatechuic a c i d (Gross 1958). Another mutant has been shown to accumulate prephenic ac i d (Metzen- berg and M i t c h e l l 1956). The phosphate of shikimic a c i d has been detected i n cultures of the Basidiomycete Lentinus lepideus (Eberhardt 1956). The production of secondary metabolites from the shik- imic a c i d pathway ei t h e r d i r e c t l y , or by way of aromatic amino acids or cinnamic ac i d derivatives i s well-known i n the Basidiomycetes. Several diphenylbenzoquinones are known from t h i s source. One example i s leucomelone (Fig. 5.) from Polyporus leucomelas (Akagi 1942). X y l e r y t h r i n (Fig..5.) from Peniophora sanguinea (Gripenberg 1965) obviously r e s u l t s from condensation of polyporic a c i d with p-hydroxyphenyl- a c e t i c a c i d . The only known fungal flavone, c h l o r f l a v o n i n (Fig. 6.) which has been i s o l a t e d from cultures of A s p e r g i l - lus candidus (Richards et a l 1969) probably has i t s o r i g i n i n the shikimic a c i d pathway. However, l a t e r studies have been unable to v e r i f y production of t h i s compound i n c u l - tures of the fungus (Towers, personal communication). Many metabolites of higher plants such as the flavon- oids are derived through the shikimic a c i d pathway with 10 LEUCOMELONE (Polyporus leucomelas) XYLERYTHRIN (Peniophora sanguinea) FIG. 5. S h i k i m i c a c i d - d e r i v e d M e t a b o l i t e s o f Basidiomycetes 11 subsequent elaboration by condensation with an acetate-derived chain. In Basidiomycetes, acetate chains more t y p i c a l l y form condensation products with t r i c a r b o x y l i c a c i d cycle i n t e r - mediates or other acetate-derived molecules. Such compounds as a g a r i c i c a c i d (Fig. 6.) from Polyporus o f f i c i n a l i s (Thorns and Vogelsang 1907) are derived by condensation of the car- bonyl group of oxaloacetate with the a-methylene group of a f a t t y a c i d . C o r t i s a l i n (Fig. 6.), which was i s o l a t e d from C y t i d i a s a l i c i n a (Corticium salicinum)(Gripenberg 1952, Mar- s h a l l and Whiting 1957), and c h l o r f l a v o n i n represent the only fungal products reported, i n addition to styrylpyrones, which appear to be formed by polyketide extension of a shikimic acid-derived phenylpropanoid moiety. I I I . Aromatic amino ac i d metabolism i n Basidiomycetes Many fungi have been shown to produce aromatic amino acids v i a the shikimic a c i d pathway, and the conversion of these aromatic compounds to other aromatic compounds i s of considerable i n t e r e s t . Many Basidiomycetes possess phenyl- alanine ammonia-lyase (PAL) a c t i v i t y , e f f e c t i n g the conversion of phenylalanine to cinnamic a c i d (Power et a l 1965, Bandoni et a l 1968). In some of these organisms tyrosine ammonia- lyase (TAL) i s also present, y i e l d i n g p_-coumaric acid from tyrosine. Detailed studies on the metabolism of phenylala- nine and tyrosine have been reported and these have been re- viewed by Towers (1969). CHLORFLAVONIN (Aspergillus candidus) HO~Y 7— ( C H = C H ) 7 — COOH CORTISALIN (Cytidia s a l i c i n a ) HOOC CH_, \ / 3 CH—CH CH 3(CH 2) 1 ( )CH 2 CCOH AGARICIC ACID (Polyporus o f f i c i n a l i s ) FIG. 6 . Fungal metabolites of mixed biogenesis. 13 C u l t u r a l studies with Lentinus lepideus employing radioactive tracers showed that carbon from D-glucose and L-phenylalanine was incorporated i n t o i s o f e r u l i c acid, while acetate was a poor precursor (Power et a l 1 9 6 5 ) . The data fo r L-tyrosine are questionable i n l i g h t of the detected TAL a c t i v i t y and unknown s p e c i f i c a c t i v i t y of the tyrosine fed. E a r l i e r work by Shimazono (1959) with L. lepideus showed that carboxyl-labelled methyl ja-coumarate was converted to methyl p_-methoxycinnamate without scrambling of the l a b e l . The known transformations of cinnamyl compounds i n t h i s organism are summarized i n F i g . 7.(Towers 1 9 6 9 ) . 14 In L. lepideus, tyrosine- C i s converted to p_-hydroxy- phenylacetic a c i d (Power et a l 1965) while i n Sporobolomy- ces roseus £-coumaric a c i d i s formed from L-tyrosine and m-coumaric a c i d i s derived from m-hydroxyphenylalanine feed- ings (Moore et a l 1 9 6 8 ) . An i n v e s t i g a t i o n of L-phenylalanine metabolism by Schizophyllum commune has revealed PAL a c t i v i t y (Moore and Towers 1 9 6 7 ) . In addition, phenylpyruvic a c i d and L ( - ) - 0 - p h e n y l l a c t i c a c i d became r a d i o a c t i v e l y l a b e l l e d when Dlr- 14 phenylalanine- C was administered to cultures of the fun- gus. However, the absence of other l a b e l l e d phenylpropan- o i d molecules i n the fungus suggests that, as i n many 14 C O O H I H « N C H z I C H o (I) C O O H I C H II C H O H (III) O C H . (V) FIG. 7 . Transformations of Cinnamyl Compounds i n L. lepideus (I)phenylalanine; (Il)cinnamic acid; (Ill)rj-coumaric acid; (IV)phloretic acid; (V)rj-methoxycinnamic acid; (VI) i s o f e r u l i c acid; ( V I I ) c a f f e i c a c i d . 15 Basidiomycetes, the enzymes PAL and TAL function p r i m a r i l y i n providing a degradative route to carbon dioxide. Benzofurans have been i d e n t i f i e d as metabolites of phenylalanine and tyrosine i n Stereum subpileatum (Bu'Lock et a l 1971). In t h i s organism, the phenylpropanoid moiety appears to undergo 8-cleavage before a c e t y l a t i o n and incor- poration i n t o the benzofuran molecules. In Polyporus tum- ulosus, the p r i n c i p a l radioactive phenolic compounds iden- 14 t i f i e d a f t e r long-term feeding of glucose-U- C, shikimic 14 14 acid-U- C and acetate-1- C indicated that tyrosine was degraded v i a p_-hydroxyphenylpyruvic a c i d to carbon dioxide (Crowden 1967). No other phenolic phenylpropanoid molecules were observed. Recently, i n our labor a t o r i e s , a survey was made of flavonoid and phenolic a c i d production i n thirty-one d i f - ferent species of Basidiomycetes (Saleh,Bandoni and Towers, personal communication). These organisms were grown i n Roux bot t l e s on a l i q u i d medium containing glucose as the major carbon source and were examined a f t e r two weeks incubation at 23°C. Flavonoid compounds were not detected i n any of the cultures examined, while o-hydroxyphenylacetic a c i d was produced by Fomes subroseus and Poria w e i r i i . In addition/ cultures of Polyporus f i b r i l l o s u s and Poria xantha produced c a f f e i c a c i d . 16 IV. Pigment production i n fungi Great variations i n the e f f e c t s of pH, temperature, C/N r a t i o , nutrients and l i g h t on pigment production are found among d i f f e r e n t fungi. These v a r i a t i o n s w i l l depend on the nature of the pigment and on the organism i t s e l f . C a r l i l e (1965) has reviewed the photobiology of fungi, and i t i s apparent that the q u a l i t a t i v e e f f e c t of l i g h t on the production of many d i f f e r e n t pigments may be of considerable importance. Light stimulation of carotenoid synthesis has been reported for Pyronema confluens ( C a r l i l e and Friend 1956), Phycomyces blakesleeanus (Garton et a l 1951), Neurospora crassa (Zalokar 19 54) and Dacryopinax spathularia ( V a i l and L i l l y 1968). I t i s also observed i n other Dacrymycetaceous fungi. However, l i g h t appears to i n h i b i t the synthesis of carotenoids i n B l a s t o c l a d i e l l a emersonii (Cantino and Horen- s t e i n 1956). S i m i l a r l y , l i g h t has been shown to stimulate melanin production i n Aureobasidium pullulans (Lingappa et a l 1963) and i n h i b i t the production of melanin i n a mutant of Neurospora crassa (Schaeffer 1953). The mutant of Neuro- spora provided an example i n which l i g h t stimulated caro- tenoid synthesis but i n h i b i t e d melanin production. The major e f f e c t s of l i g h t may be a t t r i b u t a b l e to photooxidation. The photooxidation of one pigment to another 1 7 has been r e p o r t e d f o r P e n i c i l l i u m h e r q u e i (Reidhart and Por- t e r 1958). The e x t i n c t i o n o f p h o t o i n d u c t i o n o f c a r o t e n o i d s y n t h e s i s by r e d u c i n g substances and the s u b s t i t u t i o n o f hydrogen p e r o x i d e f o r l i g h t has been demonstrated i n Fusarium aqueductium (Thiemer and Rau 1970). In C e r c o s p o r i n a k i k u c h i i i t has been demonstrated t h a t l i g h t i s n e c e s s a r y f o r both pigment and p r e c u r s o r f o r m a t i o n , and i t has been suggested t h a t l i g h t a f f e c t s the e n t i r e m e t a b o l i c pathway r a t h e r than e f f e c t i n g c o n v e r s i o n a t the l e v e l o f the m e t a b o l i t e (Mat- sueda 1969). In some cases i t has been demonstrated t h a t c e r t a i n wavelengths o f l i g h t a r e more e f f e c t i v e than o t h e r s w i t h r e - sp e c t t o pigment p r o d u c t i o n . The e f f e c t i v e n e s s o f l i g h t near 360 - 380 nm i n s t i m u l a t i n g c a r o t e n o i d s y n t h e s i s i n Phyco- myces (Delbruck and S h r o p s h i r e 1960) and P i l o b o l u s k l e i n i i (Page 1965) i n d i c a t e s t h a t a f l a v i n o r f l a v o p r o t e i n may be the p h o t o s e n s i t i v e compound i n these f u n g i . V. Sporophore fo r m a t i o n i n Basidiomycetes B a s i d i o c a r p p r o d u c t i o n i n c u l t u r e s o f w o o d - r o t t i n g Basidiomycetes has been i n v e s t i g a t e d many times (notably, Badcock 1943, Lohwag 1952, Nobles 1948, Tamblyn and Dacosta 1958). In a d d i t i o n , more d e t a i l e d s t u d i e s o f morphogenetic f a c t o r s a f f e c t i n g sporophore f o r m a t i o n have been c a r r i e d out w i t h some Basidiomycetes. A review by Taber (1966) 18 outlines the studies determining the roles of l i g h t , temper- ature, pH, humidity, gases and various substrate c o n s t i t - uents. I t has been suggested that reproduction i s i n i t - i a t ed by factors that check the growth of an established mycelium without d r a s t i c a l l y poisoning i t s metabolism (Coch- rane 1958). In general, reproduction occurs over a narrow- er range of environmental conditions than does growth, but few other g e n e r a l i t i e s can be made concerning sporocarp production i n Basidiomycetes. Cochrane (1958) describes the three p r i c i p a l respon- ces to l i g h t as inductive, i n h i b i t o r y and t r o p i c . The re- quirement of l i g h t f or the induction of basidiocarps i n many Basidiomycetes i s well established (Taber 1966), but the amount of l i g h t , e f f e c t i v e wavelengths and time of stim- ulus required are v a r i a b l e . While Polyporus v e r s i c o l o r requires 1000 lux per day for sporocarp production to occur (Koch 1958), Poria ambigua w i l l r e t a i n a l i g h t stimulus i n i t s mycelium and form basidiocarps i n subsequent darkness (Robbins and Hervey 1960). In some fungi, darkness i s requis- i t e f or sporulation to occur ( L i l l y and Barnett 1951). Research indicates that the e f f e c t i v e wavelengths f o r bas- idiocarp induction are i n the blue region of the v i s i b l e spectrum (Cochrane 1958). While short wavelength u l t r a v i - o l e t l i g h t shows i n h i b i t o r y or l e t h a l e f f e c t s , near u l t r a - 19 v i o l e t i s often stimulatory to reproduction (Leach 1962). Posi t i v e phototropism i s also important i n Basidiomycete reproduction. Plunkett (1958)/ working with Polyporus brum- a J J L s , found that under normal conditions, response to l i g h t brings the s t i p e t i p in t o the a i r . The s t i p e i s both pos- i t i v e l y phototropic and negatively geotropic, and when both s t i m u l i are present, the phototropic stimulus takes preced- ence . In general, the temperature range permitting repro- duction i s narrower than that for vegetative growth. While exploring the r e l a t i o n s h i p of temperature to sporulation i n non-light s e n s i t i v e fungi i s r e l a t i v e l y straighforward, defining the r e l a t i o n s h i p of l i g h t to sporulation and the i n t e r a c t i o n of l i g h t and temperature i s considerably more complex (Leach 1967). In some cases higher temperatures w i l l p a r t i a l l y replace a l i g h t requirement fo basidiocarp formation ( C a r l i l e 1965). I t i s commonly observed that basidiocarp formation i s reduced i n closed containers (Badcock 1943). Although t h i s may, i n part, be caused by oxygen deficiency, the ac- cumulation of carbon dioxide and other v o l a t i l e waste prod- ucts i s also involved. Levels of v o l a t i l e s t o l e rated or required varies considerably and l i t t l e quantitative work has been done. Although the pH range f o r basidiocarp f o r - 20 mation may be narrower than f o r growth, few g e n e r a l i z a t i o n s can be made. There i s evidence, however, t h a t pH i s impor- t a n t t o s p o r u l a t i o n i n some f u n g i . E a r l y workers r e p o r t e d t h a t r e p r o d u c t i o n i n some f u n g i i s most l i k e l y t o o c c u r when a v i g o r o u s l y growing my- c e l i u m exhausts i t s n u t r i e n t s , o r i s t r a n s f e r r e d t o a medium low i n n u t r i e n t s (Klebs 1900). In a d d i t i o n , the c o n c e n t r a - t i o n o f the carbon source i s more d e c i s i v e than t h a t o f o t h e r n u t r i e n t s (Cochrane 1958). In l a r g e Basidiomycetes, owing t o the g r e a t e r mass o f the sporocarp, h i g h e r n u t r i e n t l e v e l s must be ma i n t a i n e d f o r s p o r u l a t i o n t o o c c u r (Plun- k e t t 1953). The s p e c i f i c i t y o f d i f f e r e n t n u t r i e n t r e q u i r e - ments i n s o f a r as d i f f e r e n t s p e c i e s a re concerned, i s o b v i o u s . In g e n e r a l , h i g h n i t r o g e n l e v e l s tend t o suppress s p o r u l a - t i o n . The optimum n i t r o g e n source and i t s c o n c e n t r a t i o n may v a r y w i t h d i f f e r e n t c o n d i t i o n s , and i n p a r t i c u l a r , the carbon source and i t s c o n c e n t r a t i o n w i l l i n f l u e n c e t h i s op- timum. Although d i f f e r e n t Basidiomycetes undoubtedly have d i f - f e r e n t v i t a m i n and microelement requirements, t h e r e i s l i t - t l e e vidence t h a t q u a l i t a t i v e d i f f e r e n c e s e x i s t i n these r e - quirements f o r v e g e t a t i v e growth and r e p r o d u c t i o n . Evidence a l s o i n d i c a t e s t h a t c e r t a i n substances i n l i m i t i n g o r exces- s i v e q u a n t i t i e s w i l l e x e r t much g r e a t e r e f f e c t s on r e p r o - d u c t i o n than on v e g e t a t i v e growth (Hawker 1966). CHAPTER ONE CULTURAL STUDIES OF POLYPORUS HISPIDUS 21 Introduction At the commencement of t h i s study, h i s p i d i n had been i s o l a t e d only from Polyporus hispidus and P. s c h w e i n i t z i i and Bu'Lock (1967) had reported that while P. s c h w e i n i t z i i would produce h i s p i d i n on usual culture media, i t was neces- sary to grow P. hispidus on wood blocks i n order to achieve h i s p i d i n production. Therefore, before h i s p i d i n biosynthesis could be investigated i t was necessary to e s t a b l i s h c u l t u r i n g conditions i n which j?. hispidus would produce r e a d i l y i s o l - able quantities of h i s p i d i n on a l i q u i d medium. I n i t i a l l y , comparisons of growth and pigment formation also were made with an i s o l a t e of P. s c h w e i n i t z i i . The growth of the two species (dry weight) was compared on sev- e r a l d i f f e r e n t media and t h e i r pigment production was com- pared on a n u t r i e n t - r i c h undefined medium by monitoring the production of mycelial constituents with absorbance at 370 nm. A f t e r evaluating the comparative study, i t was decided that only P_. hispidus would be examined further i n c u l t u r a l i n v e s t i g a t i o n s . In these, the e f f e c t s of various c o n s t i t - uents of the medium and of d i f f e r e n t environmental conditions on growth and pigment production were recorded. In t h i s way, a medium and l i g h t and temperature conditions 22 appropriate to biochemical studies were established. In view of the natural occurrence of h i s p i d i n i n the sporophores of the fungus, the production of sporophores i n culture was examined a l s o . Materials and Methods I. Sources of Cultures? The culture of Polyporus hispidus was obtained from Dr. M.K. Nobles of the Mycology Section, Central Experiment- a l Farm, Ottawa. This culture, F2037, was given the UBC Culture C o l l e c t i o n number UBC 513. The culture of P. schwein' i t z i i (UBC 686) was i s o l a t e d from a sporophore c o l l e c t e d on the U n i v e r s i t y of B r i t i s h Columbia Endowment Lands. I I . Sources of Medium Constituents: A l l chemicals employed i n the media were obtained from Fisher S c i e n t i f i c Company or J . T. Baker Chemical Com- pany and were of C. P. grade or were designated as meeting ACS or USP standards. Vitamin- and s a l t - f r e e casein hydrol- ysate was obtained from N u t r i t i o n a l Biochemical Corporation, Cleveland, Ohio and agar was from K & S Laboratories, Van- couver, B.C. Malt extract, yeast extract, and Soytone (an enzymatic hydrolysate of soybean meal) were obtained from Difco Laboratories, Detroit, Michigan. A l l water used i n the media was g l a s s - d i s t i l l e d . 23 I I I . Media Employed: A. Malt-Yeast-Soytone (MYP): malt extract 7g, yeast extract 0.75g, Soytone l.Og per l i t r e of water. S o l i d medium was prepared by adding 1.5% agar. B. Modifications of Czapek's Medium: KF^PO^ l g , MgS0 4 . 7H20 0.5g, KC1 0.5g, FeS0 4 . 7H20 O.Olg plus 10 g of glucose, sucrose, maltose or xylose and one of NaNO^ 2.0g, NH 4N0 3 0.94g, (NH 4) 2S0 4 1.55g or casein hydrolysate 2.0g per l i t r e of water C. Defined Medium #1 (DM#1): KH 2P0 4 l.Og, MgS0 4 . 7H 20 0.25g, NaCl 0.05g, C a C l 2 . 2H 20 0.2g, glucose 10g, casein hydrolysate 2.0g and water 1 l i t r e . D. Four Vitamin Stock Solution (VSS): b i o t i n 0.5 mg, thiamine 10 mg, pyridoxine 10 mg, i n o s i t o l 0.5g per 100 ml of 20% (vol.) aqueous ethanol. The stock s o l u t i o n was employed at a concentration of 1 ml per l i t r e of medium. E. Glucose-Yeast-Soytone-Salts (GYSS): glucose 15g, yeast extract 0.5g, soytone l.Og, s a l t stock s o l - ution 10 ml and water 1 l i t r e . F. S a l t Stock Solution: KH 2P0 4 5g, MgSC>4 „ 7H 20 2.5g, NaCl 0.5g, CaCl . 2H O 0.2g and 100 ml of water. 24 IV. C u l t u r i n g Techniques: L i q u i d c u l t u r i n g was performed u s i n g Roux b o t t l e s . Each b o t t l e c o n t a i n e d 100 ml o f medium and was i n o c u l a t e d by p i p e t t i n g 10 ml o f c u l t u r e homogenate i n t o the b o t t l e . The homogenate was p r e p a r e d by b l e n d i n g a two-week-old MYP agar c u l t u r e w i t h 250 ml o f s t e r i l e d i s t i l l e d water. The organisms were m a i n t a i n e d on MYP agar, and these c u l t u r e s were p r e p a r e d by p i p e t t i n g 1 ml o f the homogenate i n t o each s t a n d a r d 10 cm P e t r i d i s h c o n t a i n i n g a p p r o x i m a t e l y 25 ml o f agar medium. Agar c u l t u r e s f o r s t u d i e s i n v o l v i n g c o l o n y diameter measurements were i n o c u l a t e d near the c e n t e r . The inoculum was a 2 mm cube c u t from the agar near the hy- p h a l t i p s o f two-week-old c o l o n i e s . The cubes were p l a c e d mycelium-side down on the new agar s u r f a c e . V. Measurements o f H i s p i d i n P r o d u c t i o n : H i s p i d i n , which has a s t r o n g absorbance peak near 370 nm i n a l c o h o l i c s o l u t i o n , was e x t r a c t e d from the mycel- ium. The medium and mycelium were . . a c i d i f i e d w i t h 6 N HC1, the medium- f i l t e r e d o f f , and~the mycelium e x t r a c t e d by g r i n - d i n g i n a mortar w i t h - s i l i c a and d i e t h y l e t h e r . The e t h e r was removed i n vacuo and the r e s i d u e taken up i n 5 ml o f 95% e t h a n o l . A Unicam SP.800 r e c o r d i n g spectrophotometer was employed f o r the o p t i c a l d e n s i t y measurements. An a l t e r n a t i v e procedure was employed f o r the 25 q u a n t i t a t i v e d e t e r m i n a t i o n o f h i s p i d i n p r o d u c t i o n i n P. h i s p i d u s . The medium was removed by f i l t r a t i o n and the my- c e l i u m washed w i t h d i s t i l l e d water and f r o z e n i n an acetone- dr y i c e b a t h . The f r o z e n mycelium was then l y o p h i l i z e d and the d r y weight o f t i s s u e determined b e f o r e e x t r a c t i o n . The d r i e d mycelium was then e x h a u s t i v e l y e x t r a c t e d w i t h methanol by g r i n d i n g w i t h A l 2 ° 3 ^ n a m o r t a r . The e x t r a c t was reduced i n volume and banded on 500 micron A v i c e l c e l l u l o s e t h i n l a y e r p l a t e s . The p l a t e s were developed i n a s o l v e n t system con- s i s t i n g o f t o l u e n e : e t h y l f o r m a t e : f o r m i c a c i d (5/4/1 by v o l . ) and the band c o r r e s p o n d i n g t o h i s p i d i n was e l u t e d . E l u t i o n was performed by powdering the r e c o v e r e d band and p l a c i n g the powder i n a 1 cm column which was e l u t e d w i t h methanol. The u l t r a v i o l e t spectrum o f the r e c o v e r e d m a t e r i a l was observed t o c o r r e s p o n d c l o s e l y t o t h a t o f h i s p i d i n , and the Molar ex- t i n c t i o n c o e f f i c i e n t a t 366 nm was used t o c a l c u l a t e the y i e l d o f h i s p i d i n . VI. Dry Weight D e t e r m i n a t i o n s : The medium was removed from the mycelium by f i l t e r i n g through c h e e s e c l o t h and washing w i t h 100 ml o f d i s t i l l e d water. I f the mycelium were t o be examined f o r p h e n o l i c compounds a f t e r d r y weight measurement the m a t e r i a l was l y o p h i l i z e d as d e s c r i b e d above, otherwise d r y i n g was performed i n an oven a t o 90 C. A l l weighings were performed a t room temperature and h u m i d i t y . 26 R e s u l t s and D i s c u s s i o n I. Comparison o f Polyporus h i s p i d u s and P. s c h w e i n i t z i i The growth o f P. h i s p i d u s and P. s c h w e i n i t z i i on MYP a t 22-24°C i n dark cupboards were compared ( F i g . 8.). Meas- urements o f the d r y weight p e r Roux b o t t l e ( t r i p l i c a t e ) i n d i - c a t e d t h a t w h i l e P. s c h w e i n i t z i i matured more r a p i d l y , a g r e a t e r t o t a l d r y weight o f mycelium was produced by P. h i s - pidus . I t was observed a l s o t h a t the c u l t u r e s o f P. schwein- i t z i i showed y e l l o w p i g m e n t a t i o n and subsequent browning e a r - l i e r and more s t r o n g l y than c u l t u r e s o f P. h i s p i d u s . I t was suspected t h a t the y e l l o w c o l o u r a t i o n was asso- c i a t e d w i t h the development o f h i s p i d i n , and as a crude a s s e s s - ment o f t h i s , o p t i c a l d e n s i t y measurements a t 370 nm o f e t h e r e x t r a c t s o f the mycelium were r e c o r d e d . These measurements f o r P. s c h w e i n i t z i i a re p r e s e n t e d ( F i g . 9.). The v a l u e s o b t a i n e d f o r P. h i s p i d u s were more than f i f t y times s m a l l e r than those o b t a i n e d f o r P. s c h w e i n i t z i i , a l t h o u g h a maximum v a l u e o f 0.6 absorbance u n i t s p e r gram o f d r y mycelium was re c o r d e d f o r P. h i s p i d u s a f t e r 21 days. The maximum absor- bance a t 370 nm f o r c u l t u r e s o f P. s c h w e i n i t z i i was o b t a i n e d a f t e r t e n days o f growth. The o p t i c a l d e n s i t y measurements r e p o r t e d can be ex- pe c t e d o n l y t o g i v e an i n d i c a t i o n o f s t y r y l p y r o n e s y n t h e s i s s i n c e o t h e r a c i d i c , e t h e r s o l u b l e compounds may absorb a t t h i s 2 7  wavelength. However, the great difference i n o p t i c a l den- s i t y readings between P_. s c h w e i n i t z i i and P. hispidus prob- ably confirms the evidence (Bu 1Lock 1967) that P. schwein- i t z i i i s capable of much better h i s p i d i n production i n c u l - ture than i s P. hispidus. Also, under these c u l t u r a l con- d i t i o n s , maximum h i s p i d i n production might be expected ear- l i e r i n P. s c h w e i n i t z i i (10-11 days) than i n P. hispidus (ca. 21 days). The observed decrease i n o p t i c a l density measurements and concommitant browning of the medium and mycelium i n cultures of P. s c h w e i n i t z i i a f t e r ten days might be the r e s u l t of increased oxidative a c t i v i t y . This speculation i s further substantiated by the decrease i n dry weight observed a f t e r 18 days (Fig. 8.). Attempts were made to compare the growth of the two organisms on simple modifications of Czapek"s medium employ- ing d i f f e r e n t carbon and nitrogen sources described i n Mat- e r i a l s and Methods. While P. hispidus grew well on a l l def- ined media examined, the dry weights per Roux b o t t l e f o r P. s c h w e i n i t z i i were less than 0.05 g a f t e r two weeks. I t was hoped that the comparative experiments would help i n e s t a b l i s h i n g c u l t u r i n g conditions for P. hispidus that would r e s u l t i n s u i t a b l e h i s p i d i n production. However, i t soon became apparent that the two organisms were very 30 d i f f e r e n t i n t h e i r growth. Although both fungi produce h i s p i d i n , many p h y s i o l o g i c a l differences are well-known. P. hispidus i s a white-rot fungus, one which c h a r a c t e r i s t i c - a l l y shows strong l i g n i n decomposition when growing on wood. P_. s c h w e i n i t z i i , on the other hand, i s a t y p i c a l brown-rot fungus, deriving i t s energy l a r g e l y through c e l l u l o l y t i c enzymes. I t also has been suggested that h i s p i d i n biosyn- thesis i n P. hispidus may proceed r e a d i l y only i f phenyl- propanoid moieties from l i g n i n decomposition are present (Bu'Lock 1967). Thus, conditions most favourable for p i g - ment formation i n one organism are u n l i k e l y to be e f f e c t i v e i n the other. I I . Growth and pigment production i n Polyporus hispidus I n i t i a l l y , a simple, defined l i q u i d culture medium was sought for the growth of P_. hispidus. The comparative studies with P_. s c h w e i n i t z i i had already shown that P_. h i s - pidus would grow well on such a medium. A defined medium would f a c i l i t a t e the study of h i s p i d i n biosynthesis by en- suring that, large l y , only known kinds and quantities of phenolic compounds or possible h i s p i d i n precursors were be- ing introduced into the culture medium. The four d i f f e r e n t carbon sources ( f i l t e r - s t e r i l i z e d ) and four d i f f e r e n t nitrogen sources that were employed i n the modifications of Czapek's medium were supplemented 31 w i t h s a l t s t o c k s o l u t i o n (10 ml/1) and were examined i n a l l combinations a t a f i x e d c o n c e n t r a t i o n which mai n t a i n e d a f a i r - l y c o n s t a n t C/N r a t i o . Each sugar and each n i t r o g e n source were r a t e d on the b a s i s o f v i s u a l p i g m e n t a t i o n and d e n s i t y o f the m y c e l i a l mat i n t r i p l i c a t e c u l t u r e s a f t e r two weeks i n c u b a t i o n a t 23°C i n nominal darkness (dark cupboards opened d a i l y f o r i n s p e c t i o n o f the c u l t u r e s ) . While g l u c o s e and maltose were both good carbon sources f o r growth, g l u c o s e gave b e t t e r pigment p r o d u c t i o n . Of the n i t r o g e n s o u r c e s , c a - s e i n h y d r o l y s a t e s t i m u l a t e d both growth and p i g m e n t a t i o n b e s t . The medium which c o n t a i n e d g l u c o s e , c a s e i n h y d r o l y - s a t e and s a l t s t o c k s o l u t i o n was d e s i g n a t e d as D e f i n e d Medium #1 (DM#1). S i n c e i t seemed p o s s i b l e t h a t the enhanced growth and p i g m e n t a t i o n w i t h c a s e i n h y d r o l y s a t e might be a t t r i b u t a b l e t o b u f f e r i n g a c t i o n , the change i n pH o f the c u l t u r e medium (DM#1) was examined i n t r i p l i c a t e c u l t u r e s over a f i v e week p e r i o d o f growth o f P_. h i s p i d u s . The c u l t u r e s were grown a t 23°C i n darkened cupboards. The pH was observed t o f a l l r a p - i d l y from about pH 7 t o pH 5 d u r i n g the l o g a r i t h m i c phase o f growth o f the fungus ( F i g . 10.). D u r i n g the d e c e l e r a t i o n stage, however, the pH i n c r e a s e d r a p i d l y t o about pH 6, where i t remained f o r the d u r a t i o n o f the experiment. Buf- f e r i n g a c t i o n was not apparent, and the data were s i m i l a r 0 5 10 15 20 25 30 AGE OF CULTURE (Days) to those of Crewther and Lennox (1953) for Aspergillus or- yzae. In t h e i r experiment, the pH declined with reduction of the t o t a l nitrogen, phosphate and carbon i n the medium, but began to increase again as the amount of protein n i t r o - gen i n the medium increased. In my pH-growth study of P. hispidus (Fig. 10.), strong and rapid browning of the med- ium and mycelium began i n the cultures a f t e r f i f t e e n days. At the same time, the dry weight of the mycelium showed a s i g - n i f i c a n t decrease i n growth rate. The observations of co- incident decrease i n dry weight, increase i n pH and onset of browning are consistent with the observation of Crewther and Lennox (1953) that the amount of p r o t e i n nitrogen i n the medium i s increasing, e s p e c i a l l y i f much of t h i s p r o t e i n i s represented by oxidative enzymes. I t seems that strong oxidative a c t i v i t y i n the cultures should be avoided i n or- der to achieve higher y i e l d s of h i s p i d i n from the fungus. In a second experiment employing DM#1, quadruplicate cultures were adjusted p r i o r to autoclaving to approximately pH 4, 5, 6, 7 and 8 with 1% ^2S°4 o r 1 % KOH* A f t e r t h e c u l _ tures had grown for sixteen days at 23°C i n darkened cup- boards the mycelial dry weights and pH of the medium were measured (Table I . ) . In every instance the pH a f t e r s i x - teen days was lower than the i n i t i a l pH. Although only small differences i n mycelial dry weights were observed i n 34 cultures i n i t i a t e d at pH 4, 5, 6, and 7, the dry weight of mycelium was more than 20% greater than these, i n cultures i n i t i a t e d at pH 8. Unfortunately, v i s i b l e pigmentation was not enhanced i n cultures i n i t i a t e d at pH 8 and browning was more noticeable. TABLE I. Myce l i a l dry weight and pH of medium of cultures of P. hispidus a f t e r sixteen days when grown on DM#1 at various i n i t i a l pH's. I n i t i a l M y c e l i a l Dry Wt. pH of Medium pH a f t e r 16 days (g) a f t e r 16 days 3~9 0.28 375 4.9 0.30 4.3 6.0 0.29 4.9 7.0 0.31 5.5 8.3 0.38 5.8 Higher dry weights of mycelium (Fig.8.) and more i n - tense v i s i b l e pigmentation were observed i n cultures employ- ing MYP than were recorded for growth on DM#1 (Fig. 10.) . Further c u l t u r i n g studies to determine the optimum tempera- ture, e f f e c t i v e i l l u m i n a t i o n and the e f f e c t of o x i d i z i n g and reducing substances i n the medium were performed employ- ing MYP. Subsequent modification of t h i s medium permitted the formulation of the medium for biochemical studies. P. hispidus was grown on MYP agar and the colony diameters of four r e p l i c a t e s were measured a f t e r two weeks at f i v e d i f f e r e n t temperatures (Fig. 1 1 . ) . While the o p t i - mum temperature for increase i n colony diameter was near 35 FIG. 11. V a r i a t i o n i n c o l o n y diameter w i t h temperature o f P_. h i s p i d u s c u l t u r e s on MYP agar. 36 26°C, the browning o f the c u l t u r e s , which was p r o b a b l y ox- i d a t i v e , was g r e a t l y enhanced a t temperatures above 23°C and, t h e r e f o r e , 23°C was chosen f o r f u r t h e r s t u d i e s . The e f f e c t o f the wavelength o f the l i g h t source on growth and pigment development was i n v e s t i g a t e d i n MYP agar c u l - t u r e s o f P. h i s p i d u s . In t h i s study, Wrattan f i l t e r s w i t h known s p e c t r a l c h a r a c t e r i s t i c s (Appendix A) were f i t t e d t o the tops o f P e t r i d i s h e s . A l l but f i l t e r e d l i g h t was excluded from c u l t u r e s i n c u b a t e d a t 23°C w i t h 24 h i l l u m i n a t i o n p e r day. Growth was as s e s s e d by measurement o f the c o l o n y d i a - meter a f t e r t e n days, and m y c e l i a l p i g m e n t a t i o n was e v a l u - a t e d e m p i r i c a l l y on a f i v e - p o i n t s c a l e ( F i g . 12.). From t h i s study, i t i s apparent t h a t l i g h t i s important i n both growth and pigment development. While b l u e l i g h t o r f u l l l i g h t s t i m u l a t e pigment development, growth i s slower under these c o n d i t i o n s . I t seemed p o s s i b l e t h a t p h o t o - o x i d a t i o n was r e s p o n s i b l e f o r both observed e f f e c t s . I t was a l s o noted t h a t c u l t u r e s grown f o r t e n days i n r e d l i g h t o r darkness, dev- e l o p e d p i g m e n t a t i o n w i t h i n 24 h when exposed t o f u l l l i g h t . R e t u r n i n g c u l t u r e s so exposed t o darkness d i d not immediate- l y a r r e s t f u r t h e r c o l o u r development i n the mycelium. I t i s p r o b a b l e t h a t photoinduced enzymes remain a c t i v e i n the mycelium f o r s e v e r a l days. As a r e s u l t o f t h i s study the f l u o r e s c e n t i l l u m i n a t i o n i n the i n c u b a t o r s was changed H o o i — i 1 1 1 r g FULL BLUE GREEN RED RED DARKNESS J LIGHT & y YELLOW COLOUR OF LIGHT RECEIVED BY CULTURES 38 from 40 watt Cool White lamps to 40 watt Daylight lamps to provide approximately twice as much i l l u m i n a t i o n i n the blue region of the v i s i b l e spectrum. Also, i l l u m i n a t i o n was provided for two hours per day for a l l future studies. In an attempt to ascertain i f the role of l i g h t was oxidative, small quantities of hydrogen peroxide or hydrox- ylamine hydrochloride were added to MYP agar cult u r e s . Both substances were added a s e p t i c a l l y a f t e r the medium had been autoclaved. Hydrogen peroxide caused s l i g h t oxidative browning i n the dark when employed at a concentration of -2 -2 10 M/l. At 3*10 M/l no growth of the fungus occurred, _ 3 while at a concentration of 10 M/l no oxidative e f f e c t was noticed. No yellow pigmentation was observed i n cultures containing hydrogen peroxide when they remained i n darkness. The e f f e c t of hydroxylamine hydrochloride at a con- _2 centration of 10 M/l was almost n e g l i g i b l e , and the higher concentrations employed produced unusual or l e t h a l e f f e c t s , and d i d not appreciably a r r e s t pigment development i n the l i g h t . These r e s u l t s indicate that hydrogen peroxide i s un- able to replace l i g h t i n pigment production i n t h i s fungus, although oxidative e f f e c t s were observed. S i m i l a r l y , hydrox- ylamine hydrochloride i s unable to bring about the extinc- t i o n of photoinduction as i t has been reported to do i n 39 Fusarium (Thiemer and Rau 1970). Although photoinduction of pigment development i s apparent i n P. hispidus, i t i s s t i l l uncertain that t h i s i s an oxidative process. Having established suitable i l l u m i n a t i o n conditions and temperature for growth and pigment production on MYP, various modifications of t h i s medium were evaluated i n order to obtain, i f possible, a better medium for studying h i s - p i d i n biosynthesis. Variations i n the concentration of malt extract, yeast extract and soytone were examined. Also, the e f f e c t on colony diameter and pigmentation was noted when various combinations of defined nutrients were s u b s t i t - uted for these three undefined medium constituents. T r i p l i c a t e agar cultures of P_. hispidus were incu- bated for f i f t e e n days on a medium containing yeast extract 0.5 g/1, Soytone 1.0 g/1 and variable amounts of malt extract. Increases i n malt extract concentration beyond 7 g/1 effected much smaller r e l a t i v e increases i n colony diameter than did the same increases i n malt extract below 7 g/1 (Fig. 13). However, concentrations of malt extract of 14 - 15 g/1 resulted i n much stronger yellow pigmentation of the mycelium than did lower concentrations. In a s i m i l a r experiment, the concentrations of yeast extract, malt extract and Soytone were varied, and i n some cultures, defined nutrients were substituted for one or more of these ingredients. Exclusion of yeast extract had l i t t l e 40 4 0 - CONCENTRATION OF MALT EXTRACT (g/l) FIG. 13. E f f e c t o f malt e x t r a c t and Soytone c o n c e n t r a t i o n on c o l o n y diameter a f t e r f i f t e e n days. 41 apparent e f f e c t on c o l o n y diameter so l o n g as e i t h e r malt e x t r a c t o r Soytone was p r e s e n t i n the medium. However, when pure sugars and v i t a m i n - f r e e amino a c i d s were used t o r e p l a c e the malt e x t r a c t and Soytone, the e x c l u s i o n o f y e a s t e x t r a c t r e s u l t e d i n a f u r t h e r 25% r e d u c t i o n i n c o l o n y diameter. M y c e l i a l p i g m e n t a t i o n was l e s s i n t e n s e i n the absence o f y e a s t e x t r a c t . Colony diameters o f c u l t u r e s c o n t a i n i n g e i t h e r 0.5 o r 1.0 g/1 o f Soytone a t v a r i o u s c o n c e n t r a t i o n s o f malt e x t r a c t were r e c o r d e d a f t e r 15 days ( F i g . 13.). Colony diameters a t 0.5 g/1 Soytone were 1 - 5 mm s m a l l e r than those a t 1.0 g/1 Soytone a t a l l c o n c e n t r a t i o n s o f malt e x t r a c t . In one experiment, the e f f e c t on c o l o n y diameter and pi g m e n t a t i o n a f t e r f i f t e e n days was observed when malt e x t r a c t was r e p l a c e d w i t h o t h e r n u t r i e n t s . The agar medium c o n t a i n e d y e a s t e x t r a c t 0.5 g/1, Soytone 1.0 g/1 and e i t h e r malt ex- t r a c t 14 g/1 o r maltose 10 g/1, g l u c o s e 4 g/1 and s a l t s t o c k s o l u t i o n 10 ml/1 o r g l u c o s e 14 g/1 and s a l t s t o c k s o l u t i o n 10 ml/1 o r g l u c o s e 14 g/1. Of these combinations, the r e p l a - cement o f malt e x t r a c t w i t h g l u c o s e p l u s s a l t s was s e l e c t e d as the most d e s i r e a b l e . Although the c o l o n y diameter was s m a l l e r than w i t h malt e x t r a c t alone, the p i g m e n t a t i o n was more i n t e n s e without showing browning and the d e f i n e d carbon source was c o n s i d e r e d b e n e f i c i a l f o r b i o c h e m i c a l s t u d i e s . 42 In MYP, malt extract i s c l e a r l y the major source of n u t r i t i o n a l carfcxm, and when present i n greater than 7 - 1 0 g/1, other components of the medium become more l i m i t i n g than the carbon supply for growth as expressed by the colony diameter. Components of malt extract which enhance pigment- ation of the mycelium continue to do so beyond concentrations of 10 g/1 of malt extract. Yeast extract i s r i c h i n amino acids and low molecu- l a r weight pep tidies and also supplies a number of s o l - uble B vitamins. Although P. hispidus grows w e l l on DM#1 i n the absence of yeast extract, t h i s complex vitamin source undoubtedly enhances growth and pigment development. Soy- tone supplies carbohydrates i n addition to vitamins, proteins and amino acids. Because the replacement of Soytone with casein hydrolysate i n MYP r e s u l t s i n a 50% loss i n colony diameter, i t seems probable that Soytone i s supplying impor- tant vitamins or cofactors to the medium. Soytone concen- t r a t i o n affected pigment production v a r i o u s l y with d i f f e r e n t l e v e l s of malt extract and d i f f e r e n t carbon sources, aug- menting pigmentation i n one instance, and diminishing i t i n another. These e f f e c t s did not appear to co r r e l a t e with C/N r a t i o s and an explanation was not found. The use of glucose plus s a l t s for replacement of malt extract or as employed i n DM#1 had the desireable e f f e c t of 43 augmenting the y e l l o w c o l o r a t i o n - o f the mycelium without enhancing the browning a c t i v i t y . With r e s p e c t t o t h i s e v a l - u a t i o n , the medium, GYSS, was developed f o r b i o c h e m i c a l s t u d - i e s . In o r d e r to s u b s t a n t i a t e the v i s u a l evidence o f p i g - ment p r o d u c t i o n and t o e v a l u a t e the growth o f the fungus on the newly-developed GYSS, c u l t u r e s were examined over a f o r t y day i n c u b a t i o n p e r i o d ( F i g . 14.). The d r y weights were averaged f o r f i v e d i f f e r e n t s e t s o f c u l t u r e s and the h i s p i d i n assay was performed on o n l y two o f t h e s e . Dry weight measurements were made a f t e r o v e n-drying o r l y o p h i l i z a t i o n . H i s p i d i n p r o d u c t i o n was assayed by the chromatographic-spec- t r o p h o t o m e t r y technique d e s c r i b e d . The data show t h a t the maximum r a t e of h i s p i d i n p r o - d u c t i o n lagged the maximum r a t e o f growth by a p p r o x i m a t e l y f i v e days. The maximum d r y weight a t t a i n e d p e r Roux b o t t l e was between 0.5 and 0.6 g a f t e r 21 days i n c u b a t i o n . The maximum h i s p i d i n p r o d u c t i o n r e c o r d e d was a p p r o x i m a t e l y 5 mg per gram o f dry mycelium o r 0.5% a t 24 days. The data f o r h i s p i d i n p r o d u c t i o n c o r r e l a t e w e l l w i t h v i s u a l o b s e r - v a t i o n s o f the mycelium. The mycelium remained white d u r i n g the f i r s t t e n days o f growth. Y e l l o w c o l o r a t i o n developed r a p - i d l y a f t e r t h i s ( F i g . 15.) w i t h browning a p p e a r i n g s l o w l y 'SSAO uo snpTdsxu; "a jo uox^onpoad uTpxdsTu; put? q ^ o a o 'pj 'OIJ DRY WEIGHT PER BOTTLE ( g ) co A cji J HISPIDIN PRODUCTION (mg/g dry wt) 45 a f t e r two weeks. Af t e r three weeks of growth the cultures began to darken more r a p i d l y but by four weeks l i t t l e further change was noted. I t i s probable that the strong catechol oxidase a c t i v i t y which resulted i n darkening of the f r u i t - body and polymerization and c e l l - w a l l - b i n d i n g of the phen- ols (Bu 1Lock and Smith 1961) i s also present i n the cultures of the fungus. This would account f o r the sharp decrease i n extractable h i s p i d i n and the strong browning observed i n cultures of the fungus a f t e r 24 days. I I I . Sporophore Formation i n Agar Cultures of P. hispidus Sporophore i n i t i a t i o n had been observed i n 3 - 4 week-old agar cultures grown for previous experiments. By employing GYSS and MYP with various i n o c u l a t i o n techniques and incubation conditions, sporophore formation was a- chieved. Inoculation was made with 2mm agar cubes transferred from 2-week-old MYP agar cultures or with one ml of the homo- genate prepared by blending a two-week-old MYP agar culture with 100 ml of s t e r i l e d i s t i l l e d water. In one experiment, 1, 2, 3, 5 or 10 agar cubes were evenly spaced mycelium-side down on the agar surface of GYSS and MYP. In one set, these agar cubes were taken from the region of hyphal t i p s of the inoculum culture and i n another they were taken from about 1 cm away from the center of the cult u r e . In the second FIG. 16. Sporocarp o f P. h i s p i d u s ten days a f t e r i n i t i a t i o n on MYP agar. 4 7 experiment 1 ml of homogenate was evenly spread on the sur- face of MYP and GYSS agar. Cultures i n both experiments were incubated under each of three d i f f e r e n t conditions: 20°C with 8 hr i l l u m i n a t i o n per day from Cool White f l u o r - o o escent lamps; 23 C with the same i l l u m i n a t i o n and 22 - 24 C with fluorescent room l i g h t i n g (ambient). Basidiocarp i n i t i a t i o n with formation of basidia and v i a b l e basidiospores was observed three times i n these cultur e s . This occurred on both media under ambient con- d i t i o n s , where the inoculum was 1 or 2 agar cubes, and the culture age at the onset of sporulation was 3 - 4 weeks. The appearance of a ten-day-old sporocarp which developed on MYP agar i s shown i n F i g . 16. Spores from th i s b a s i d i o - carp germinated without treatment a f t e r s i x months on MYP agar i n sealed P e t r i dishes. However, within three days of germ tube i n i t i a t i o n the agar on which the spores germinated showed signs of r a p i d l y drying and i t was suspected that slow desiccation together with a c r i t i c a l water tension of the substrate were important i n spore germination. When o l d mycelium or homogenate was — employed as i n - oculum f o r the cultures, oxidative browning was observed much e a r l i e r than i n cultures inoculated with mycelial t i p s . Sporocarp formation took place only i n cultures which re- mained light-coloured u n t i l almost three weeks incubation. 48 The u l t r a v i o l e t absorbance spectrum o f an e t h a n o l i c s o l u t i o n o f a d i e t h y l e t h e r e x t r a c t o f a six-week-old b a s i d i o c a r p c l e a r l y showed the presence o f h i s p i d i n , p r o b a b l y i n d i c a t i n g f a i r l y low c a t e c h o l oxidase a c t i v i t y . I t appears t h a t c u l - t u r a l c o n d i t i o n s t h a t are f a v o u r a b l e t o sporophore f o r m a t i o n are a l s o f a v o u r a b l e to h i s p i d i n b i o s y n t h e s i s and r e t a r d the fo r m a t i o n o f o x i d a t i v e enzymes. CHAPTER TWO CHEMICAL STUDIES OF PHENOLIC ACIDS, STYRYL- PYRONES AND RELATED COMPOUNDS 49 Introduction The synthesis of h i s p i d i n and re l a t e d styrylpyrones has been performed many times by d i f f e r e n t workers. Borsche and h i s coworkers (1914 - 1933) prepared yangonin and sever- a l other styrylpyrones i n t h e i r studies on the components of the roots of Piper methysticum, however, they assigned a gamma-pyrone structure to the molecule. In a study of the chemistry of styrylpyrones Macierewicz (1939, 1950) e l u c i - dated the correct a-pyrone structure for yangonin, 5,6-de- hydrokawain and a number of other re l a t e d pyrones. More recently, employing unequivocal synthetic procedures, yang- onin (Bu'Lock and Smith 1960), h i s p i d i n (Edwards and Wilson 1961) and twenty-three analogues of h i s p i d i n (Edwards and Mir 1967) have been synthesized. To f a c i l i t a t e the i n v e s t i g a t i o n of h i s p i d i n biosyn- thesis i n P. hispidus, the chemistry of a number of phen- o l i c compounds has been examined. Also, t r i m e t h y l h i s p i d i n and r e l a t e d molecules were synthesized for comparative pur- poses and for d i l u t i o n i n degradative studies with radio- active compounds. Materials and Methods I. Chemicals: A l l chemicals employed i n synthetic work were of C.P. 50 grade. Phenolic compounds and c r i t i c a l reagents were recrys- t a l l i z e d before use. Ethanol and methanol were dried, where necessary, by r e f l u x i n g with magnesium and subsequently d i s - t i l l i n g o f f the dried solvent. Acetone was dried by passing through a 2 X 20 cm column of a c t i v i t y grade I Woelm Alumina. Protocatechualdehyde was r e c r y s t a l l i z e d from toluene u n t i l white c r y s t a l s were obtained. Chloromethyl-methyl ether was prepared by saturating a mixture of 210 ml of 36% aqueous formaldehyde and 100 ml of methanol with HCl gas at 10°C. The chloromethyl-methyl ether was dried over anhydrous CaCl^ and the d i s t i l l a t e b o i l i n g between 55 - 62°C was employed (Marvel and Porter 1929). In the absense of a cyli n d e r of HCl gas, the gas was generated by dropping concentrated ^SO^ into concentrated aqueous HCl. C a f f e i c a c i d was re- c r y s t a l l i z e d from water f o r preparation of the standard absorbance curve. Veratrie a c i d was p u r i f i e d by vacuum sublimation and r e c r y s t a l l i z a t i o n from water for preparation of the standard absorbance curve. I I . Chromatography Paper chromatography employing solvent system A: benzene/acetic a c i d / water (10:7:3 by. v o l . (upper phase)) i n the f i r s t dimension and solvent system B: 2% aqueous f o r - mic acid (by vol.) i n the second dimension was used for the separation of phenolic acids. Thin layer chromatography 51 (TLC) employing 500 micron A v i c e l c e l l u l o s e layers and s o l - vent system C: toluene/ethylformate/formic a c i d (5:4:1 by vol.) was used for the separation of s t y r y l pyrones. Phen- o l i c compounds were detected by spraying with d i a z o t i z e d p_-nitroaniline reagent (Appendix B) or f e r r i c chloride rea- gent (Appendix B). Styrylpyrones were detected with modi- f i e d E h r l i c h ' s reagent (Appendix B) or p_-dimethylaminocin- namaldehyde reagent (Appendix B). Acids were detected with Bromocresol green reagent (Appendix B). I I I . Spectroscopy: U l t r a v i o l e t spectra were obtained using a Unicam model SP.8O0 recording spectrophotometer. Infrared (IR) spectra were taken i n a Unicam SP.200G spectrophotometer employing nujol mulls or KBr d i s c s . Nuclear magnetic res- onance (NMR) spectra were obtained on a Varian 60 mHz model spectrophotometer. IV. Melting Points: Melting points were taken on a hot-stage melting point apparatus or i n a Thomas, Hoover c a p i l l a r y melting point ap- paratus. The values obtained were uncorrected. V. Chemical Preparations: Preparation of t r i a c e t i c lactone (Berson 1952) Th i r t y grams of dehydroacetic a c i d was dissolved i n 50 ml of 90% H_SO. i n a 100 ml round bottom f l a s k . The f l a s k 52 and contents were immersed i n a 150 °C o i l bath while the fl a s k was flushed continuously with a stream of nitrogen. The reaction mixture was raised to and maintained at 130 - 140 °C for f i v e minutes and the r e s u l t i n g deep reddish-brown so l u t i o n was cooled r a p i d l y i n an i c e bath. The sol u t i o n was then added to 200 ml of i c e water. C r y s t a l l i z a t i o n was considered to be complete i n f i v e minutes. The o f f - white p r e c i p i t a t e was c o l l e c t e d i n a Buchner funnel and washed with 100 ml of i c e water. The y i e l d was 15 g of crude t r i a c e t i c lactone which upon r e c r y s t a l l i z a t i o n from e t h y l acetate gave M.P. 191-192.5 °C / A M e 0 H 283 nm, NMR spectrum max S MeOH-d4 2.25 (s, 3H,-CH3), 5.34 (d, IH, H3,.J=2.4 cps), 5.63 (m, IH, H , coupled to H 3 and -CH 3), 5.98 (s, IH, -OH). Preparation of 6-methyl-4-methoxy-2-pyrone (Bu 1Lock and Smith 1960) Twenty grams of dimethyl sulphate was added to a 500 ml round bottom flask containing 70 g of anhydrous potassium carbonate and 20 g of t r i a c e t i c lactone i n 300 ml of dry acet- one. The mixture was refluxed i n a heating mantle with magnetic s t i r r i n g f o r s i x hours while maintaining anhydrous conditions. The reaction mixture was then allowed to cool, f i l t e r e d on a Buchner funnel and evaporated under reduced pressure. R e c r y s t a l l i z a t i o n from petroleum ether (60 - 110 °C) gave a t o t a l y i e l d of 17.3 grams of white c r y s t a l s M.P. 89 - 90 °C, X M e 0 H 280 nm, NMR spectrum &CCl. 2.20 (s, 3H, max 4 -CH 3) / 3.81 (s, 3H, -OCH3) , 5.29 (d, 1H, H. 3 , J=2.4 cps),'.5.65 (m, 1H, H , . , coupled to H 3 and -CH 3). Preparation of t r i m e t h y l h i s p i d i n (Edwards et a l 1961) 4g of magnesium turnings were dissolved i n 100 ml of dry methanol with magnetic s t i r r i n g at room temperature under anhydrous conditions. Ten grams of 6-methyl-4-meth- oxy-2-pyrone and 10 g of veratraldehyde were introduced, and the reaction mixture was s t i r r e d at room temperature f o r sixteen hours. 2 - 3 ml of a c e t i c a c i d were then added to dissolve the magnesium hydroxide present, and the p r e c i p i - tated product was c o l l e c t e d i n a Buchner funnel, washed with a few ml of methanol and dried. On r e c r y s t a l l i z a t i o n from methanol 6.5 grams of lemon yellow c r y s t a l s were ob- o Me OH tained, M.P. 164 - 166 C, A 362 nm, IR spectrum 1704 ' ' max * (VC=0), 1555 and 1643 cm~ 1(Vpyrone C=C), NMR spectrum SCDC1 3 3.82 (s, 3H, pyrone-OCH 3), 3.95 (d, 6H, aromatic-0CH 3), 5.46 (d, IH, H 3, J=2.4 cps), 5.91 (d, IH, Hg, J=2.4 cps), 6.44 (d, IH, H Q / J=16 cps), 6.73 - 7.23 (m, 3H, aromatic), o 7.45 (d, IH, H ?, J=16cps). Preparation of 4-methylhispidin (Benton and D i l l o n 1942) 0.5 g of t r i m e t h y l h i s p i d i n was dissolved i n 50 ml of methylene chloride i n a round bottom f l a s k . Flask and con- tents were then cooled i n a bath of acetone and dry i c e a f t e r which 0.3 ml of boron tribromide was pipetted i n t o the 54 reaction vessel. The reaction mixture was then allowed to warm to room temperature with occasional a g i t a t i o n by hand. A f t e r standing at room temperature overnight, 50 ml of water was added to the reaction f l a s k . The mixture was s t i r r e d thoroughly and allowed to stand an a d d i t i o n a l h a l f hour. The mixture was then f i l t e r e d on a Buchner funnel and the greenish-yellow p r e c i p i t a t e c o l l e c t e d . A f t e r r e c r y s t a l l i - zation from 6 0 % ethanol, 0.22 g was c o l l e c t e d with melting o MeOH point 252 - 255 C as fine pale yellow c r y s t a l s , ^ m a x 375 nm, IR spectrum 1682 cm" 1C=C). 25 nm bathochromic s h i f t i n u l t r a v i o l e t absorption on addition of sodium borate, no s h i f t observed on addition of sodium acetate. A l k a l i n e hydrolysis of t r i m e t h y l h i s p i d i n (Bu*Lock et a l 1962) F i f t y mg of t r i m e t h y l h i s p i d i n was dissolved i n 12 ml of 10% NaOH and the s o l u t i o n was refluxed on an o i l bath for 1 hour. At the end of t h i s time the reaction mixture was cooled i n an ice bath and the mixture f i l t e r e d i n a Buchner funnel. The f i l t r a t e was then a c i d i f i e d by f i r s t adding i c e , then concentrated HCl. Upon a c i d i f i c a t i o n a white p r e c i p i t a t e formed which was c o l l e c t e d . The f i l t r a t e was further extracted with ethyl acetate, the solvent removed and the residue combined with the p r e c i p i t a t e . R e c r y s t a l l - i z a t i o n from benzene yielded approximately 6 mg of 3,4-di- methoxy cinnamic acid as white c r y s t a l s , M.P. 181 - 183°C. Preparation of 3,4-dimethoxycinnamic ac i d 0.5 g of f e r u l i c acid, 3.5 g of anhydrous potassium carbonate and 1 ml of dimethyl sulphate were placed i n 15 ml of anhydrous acetone and refluxed with s t i r r i n g under anhydrous conditions i n a 50 ml round bottom f l a s k . The reac- t i o n mixture was allowed to cool, f i l t e r e d and evaporated under reduced pressure. An orange o i l was obtained to which was added 2 ml of IN NaOH. This mixture was allowed to stand for 16 hr and an ad d i t i o n a l 0.2 g of NaOH was added to bring the pH to n e u t r a l i t y . R e a c i d i f i c a t i o n with 6 drops of 4N HC1 caused intense p r e c i p i t a t i o n . This p r e c i p i t a t e was c o l - l ected (M.P. 70 - 71 °C) and the f i l t r a t e washed with 3 X 10 ml of d i e t h y l ether. The ether extract was dried over anhydrous sodium sulphate and taken to dryness under reduced pressure. This was combined with the p r e c i p i t a t e to give 0. 45 g of o f f - white c r y s t a l s . These c r y s t a l s were then heated under r e f l u x i n a sol u t i o n of 50% aqueous NaOH for 2 hr. The sol u t i o n was allowed to cool and a c i d i f i e d with concentrated HC1 r e s u l t i n g i n a white p r e c i p i t a t e which was col l e c t e d , washed with water and dried . This material was r e c r y s t a l l i z e d from water to aff o r d white c r y s t a l s M.P. 181 - 183 °C, >y M e 0 H 293, 319; y i e l d 0.25 g. max 3 Preparation of 3,4-bis-(methoxymethoxy)-benzaldehyde (Edwards and Wilson 1961) 56 To protocatechualdehyde (20 g) i n s t i r r e d b o i l i n g t o l u e n e (250 ml), sodium ethoxide (from 10 g sodium and 120 ml e t h a n o l ) was added d u r i n g t h i r t y , minutes, the a l c o h o l meanwhile b e i n g removed i n a stream o f n i t r o g e n . The r e - maining a l c o h o l was d i s t i l l e d o f f and the r e s i d u e was c o o l e d t o 0°C and t r e a t e d w i t h chloromethyl-methyl e t h e r (27 ml) d u r i n g t h i r t y minutes, s t i r r e d a t room temperature f o r 4 h r and shaken w i t h 40 ml o f 2N sodium h y d r o x i d e . The t o l u e n e l a y e r was separated, washed w i t h water, d r i e d over anhydrous sodium s u l p h a t e and evaporated. The r e s i d u e brown o i l was d i s t i l l e d a t 2 mm and the f r a c t i o n b o i l i n g up to 152°C was c o l l e c t e d . The p r o d u c t was d i s s o l v e d i n e t h a n o l and water was added u n t i l an o i l began t o s e p a r a t e . The mixture was then c o o l e d to -20°C whereupon white c r y s t a l s were o b t a i n e d , M.P. 58 - 60°C; y i e l d 6.4 g. O x i d a t i o n o f t r i m e t h y l h i s p i d i n t o v e r a t r i c a c i d Two mg o f t r i m e t h y l h i s p i d i n were d i s s o l v e d i n 5 ml o f dr y acetone. To the acetone s o l u t i o n was added 50 mg o f potassium permanganate and the mixture was allowed to r e a c t a t room temperature f o r two h r w i t h o c c a s i o n a l a g i t a t i o n by hand. At the end o f t h i s time the acetone was removed under reduced p r e s s u r e and 1.0 ml o f IN HCl and 1.0 ml o f t e c h n i c a l grade sodium b i s u l p h i t e (10% aqueous) were added t o the r e a c t i o n f l a s k and a g i t a t e d t o o b t a i n a c o l o u r l e s s s o l u t i o n . This solution was extracted with 3 X 2 ml of d i e t h y l ether and the ether solu t i o n transferred to the sub- limation vessel where i t was taken to dryness with gentle warming and sublimed at 0.3 mm of mercury at 110°C. the recovery of v e r a t r i c a c i d was calculated from the standard absorbance curve to be approximately 40%. VI. Radioautography Two-dimensional chromatograms were placed i n l i g h t - t i g h t X-ray f i l m exposure holders with a 14 X 19 inch sheet of Kodak Blue Brand Medical X-ray f i l m (Estar Base) f o r three to four weeks. At the end of the exposure period the films were removed and developed under a red s a f e l i g h t . 58 Results and Discussion In order to become f a m i l i a r with phenolic and c i n - namic acid derivatives l i k e l y to be encountered, a compos- i t e two-dimensional paper chromatogram of authentic compounds was prepared (Fig. 17.). This was developed i n solvent systems A and B and the spots were v i s u a l i z e d under long wave u l t r a v i o l e t l i g h t , by spraying with diazotized p_-nitro- a n i l i n e reagent or by radioautography. The colours observed for the phenolic spots are recorded i n Table I I . Radio- 14 active C-labelled samples were employed f o r those compounds not r e a d i l y detected with spray reagents. I t was observed that less t a i l i n g of spots i n the f i r s t dimension was en- countered when the chromatogram was developed opposite to the machine d i r e c t i o n i n manufacture of the paper,, To f a c i l i t a t e the determination of s p e c i f i c a c t i v i t i e s 14 14 of samples of p_-coumaric acid-2- C and c a f f e i c acid-2- C which were to be employed i n feeding experiments, standard absorbance curves for these two acids were prepared. The absorbance of p_-coumaric a c i d was determined at 310 nm (Fig. 18.) and the absorbance of c a f f e i c a c i d was determined at 324 nm (Fig. 19.). Both acids were dissolved i n methanol for preparation of the curves. i t was necessary also to prepare standard absorbance curves for t r i m e t h y l h i s p i d i n and v e r a t r i c a c i d to be used i n the determination of s p e c i f i c m FIG. 17. Diagrammatic representation of two-dimensional chromatogram of authentic samples of phenolic and cinn- amic a c i d d e r i v a t i v e s . I m-hydroxymandelic acid, II p_-hydroxymandelic acid, III p_-hydroxybenzyl alcohol, IV p h e n y l l a c t i c acid, V p_-hydrox- yphenylacetic acid, VI o-hydroxyphenylacetic acid, VII p_-hydroxybenzaldehyde, VIII c i s p_-coumaric acid, IX c i s c a f f e i c acid, X p_-hyroxybenzoic acid, XI protocatechuic acid, XII trans p_-coumaric acid, XIII trans c a f f e i c acid, XIV phenylacetic acid, XV c i s cinnamic acid, XVI benzoic acid, XVII trans cinnamic a c i d . 60 TABLE I I . C o l o u r s o f p h e n o l i c a c i d s i n l o n g wave u l t r a v i - o l e t l i g h t and i n v i s i b l e l i g h t a f t e r s p r a y i n g w i t h d i a z o t i z e d p _ - n i t r o a n i l i n e reagent. Compound C o l o u r i n l o n g wave UV C o l o u r a f t e r S p r a y i n g m-hydroxymandelic a c i d r e d p_-hydroxymandelic " p i n k rj-hydroxybenzyl a l c o h o l p i n k p_-hydroxyphenylacetic a c i d p u r p l e o - h y d r o x y p h e n y l a c e t i c " wine-red p_-hydr oxybenza ldehyde brown p_-coumaric a c i d ( c i s and t r a n s ) b l u e f l u o r e s c e n c e a f t e r NH^OH vapours b l u e c a f f e i c a c i d ( c i s and t r a n s ) b l u e f l u o r e s c e n c e green (fades t o brown) p r o t o c a t e c h u i c a c i d green ( " ) p_-hydroxybenzoic a c i d p i n k a c t i v i t i e s o f these compounds o b t a i n e d from the m e t h y l a t i o n and d e g r a d a t i o n o f h i s p i d i n i s o l a t e d from c u l t u r e s o f the fungus t h a t had been f e d r a d i o a c t i v e p r e c u r s o r s . The absor- bance o f t r i m e t h y l h i s p i d i n was determined a t 365 nm ( F i g . 20.) and t h a t o f v e r a t r i c a c i d a t 253 nm ( F i g . 21.). These d e t e r m i n a t i o n s a l s o were made i n methanol. The c h e m i c a l s y n t h e s i s o f t r i m e t h y l h i s p i d i n was a- c h i e v e d employing a s t a n d a r d method (Edwards e t a l 1961) and the pro d u c t was c r i t i c a l l y c h a r a c t e r i z e d because o f i t s importance f o r d i l u t i o n i n t r a c e r s t u d i e s . The p h y s i c a l •pfoe oxaeumoo-cT 3 0 e/ano eoueqaosqp paepue^s * 8 T 9IJ T9 •p-uoe oTa jgeo 3 0 sAano aoueqaosqe pjtepuet+s * 6 T 'Did Z9 • U T P T C T S T U _ T A U _ H \ 9 U I T J I ^ J O 5AJ.no a o u e q j o s q n p a e p u e ^ s '02 'Did Z9 •p-cop D T J ^ P I S A 3 0 a A a n o aoueqaosqe paepue^s "IZ 'Did fr9 65 c o n s t a n t s determined and s p e c t r a l data o b t a i n e d conformed c l o s e l y t o those r e p o r t e d (Edwards and M i r 1967, B a r t l e e t a l 1967). Yangonin a l s o was prepared from a n i s a l d e h y d e and 6-methyl-4-methoxy-2-pyrone by the same procedure f o r use as a r e f e r e n c e compound. The m e l t i n g p o i n t (155 - 157 ° C ) , u l t r a v i o l e t absorbance spectrum ) \ M e O H 358 nm and IR ' max spectrum 1725 (vC=0), 1258 (VC-O) c o n f i r m the i d e n t i t y o f the p r o d u c t (Bu*Lock and Smith 1960, Edwards and M i r 1967). A sample o f a u t h e n t i c b i s - n o r y a n g o n i n f o r r e f e r e n c e pur- poses was o b t a i n e d through the c o u r t e s y o f Dr. G.M. Hat- f i e l d . The f o u r r e f e r e n c e s t y r y l p y r o n e s and a sample o f h i s p i d i n o b t a i n e d from the mycelium o f P_. h i s p i d u s were compared c h r o m a t o g r a p h i c a l l y i n s o l v e n t system C on c e l - l u l o s e TLC p l a t e s . C a f f e i c a c i d , p_-coumaric a c i d and v e r a t r i c a c i d were a l s o examined i n t h i s chromatographic system. The Rf v a l u e s o f these compounds and t h e i r c o l o u r r e a c t i o n s w i t h the spray reagents (Appendix B) were r e c o r d e d (Table I I I . ) . V e r a t r i c a c i d was d e t e c t e d w i t h bromocresol green reagent as a y e l l o w spot on a b l u e background. The Rf o f t h i s compound i n s o l v e n t system C was 0.82. Although the c o l o u r r e a c t i o n s w i t h spray reagents (Table I I I . ) and the Rf v a l u e o f 0.19 f o r the methanol-ex- t r a c t a b l e , u l t r a v i o l e t f l u o r e s c e n t (yellow-green) component 66 TABLE I I I . C h a r a c t e r i s t i c s of some styrylpyrones and related phenolic compounds chromatographed on c e l l u l o s e TLC plates i n solvent system C and sprayed with various reagents (Appendix B). Compound Rf X 100 Reagent 1 Reagent 3 Reagent 4 tr i m e t h y l h i s p i d i n 97 yellow yel-gr yel-gr 4-methylhispidin 44 orange yel-gr green h i s p i d i n 19 pink-brown blue-gr gr-blue bis-noryangonin 44 wine red blue-gr gr-blue yangonin 100 pale brown l t - b l u e pale gr p_-coumaric a c i d 60 blue purp-blue bleached c a f f e i c a c i d 36 green l t - b l u e bleached of P. hispidus provided preliminary evidence that t h i s sub- stance was h i s p i d i n , a d d i t i o n a l characterization was obtained. H i s p i d i n was i s o l a t e d according to the procedure given i n Part V., Materials and Methods, Chapter one. This substance MeOH gave UV spectrum 366, 251, 221 and IR spectrum 1661 N C=0), 1669, 1564 (V pyrone C=C), 1608 and 1530 cm"1 (̂  aromatic C=C). A f t e r methylation of the pigment with dimethyl sulphate (Edwards et a l 1961), the product was found to correspond to the authentic sample upon chromatography and examination of the UV spectrum. A f t e r r e c r y s t a l l i z i n g h i s p i d i n three times from ethanol-water, M.P. 254 - 257°C was obtained ( L i t . M.P. 259°C). Chromatographic examination of methanol extracts of the fungus revealed f i v e yellow-coloured components i n 67 addition to h i s p i d i n . Under long wave UV l i g h t these sub- stances fluoresced shades of yellow, yellow-green and blue- green. The three most prominent of these bands obtained i n solvent system C at Rf 0.62 (Band I ) , 0.37 (Band II) and Rf 0.00 (Band IV) were removed from the plates and saved f o r future examination. Their u l t r a v i o l e t spectra (Fig. 22.) suggest that they are also styrylpyrones. Another of these components which was present only i n trace amounts also was examined. This band fluoresced blue- green i n long wave UV l i g h t and moved at Rf 0.44 i n solvent system C. I t was scraped from the TLC plates and eluted from the powdered c e l l u l o s e with methanol i n a 1 cm column. This substance cochromatographed with bis-noryangonin i n solvent system C on both c e l l u l o s e and s i l i c a g e l G TLC p l a t e s . I t gave i d e n t i c a l colour reactions to bis-noryang- onin with spray reagents 1,3 and 4 (Table III.) and was considered to be the same substance. The presence of b i s - noryangonin i n P. hispidus i s very important as a possible precursor i n h i s p i d i n biosynthesis. Very i n t e r e s t i n g i s the a d d i t i o n a l fact that bis-noryangonin could not be de- tected i n Polyporus s c h w e i n i t z i i (Hatfield 1970). This could suggest that an alternate pathway or more than one pathway i s operative i n P. hispidus. The p o s s i b i l i t y that Band I or another of the yellow fluorescent components observed 68 FIG. 22. U l t r a v i o l e t spectra of yellow, fluorescent bands from chromatograms of P. hispidus extracts. 69 i s 6-styryl-4-hydroxy-2-pyrone p r o v i d e s f u r t h e r c r e d i b i l i t y t o t h i s s u g g e s t i o n . CHAPTER THREE RADIOACTIVE FEEDING EXPERIMENTS WITH CULTURES OF POLYPORUS HISPIDUS 70 Introduction Preliminary examinations of P_. hispidus cultures grown on GYSS established that numerous phenolic acids were present i n the medium a f t e r two weeks incubation. Phenylalanine-3- 14 . . . C was then fed to these cultures under various conditions to e s t a b l i s h the best procedure for e l u c i d a t i n g the metabol- 14 ism of t h i s and other aromatic compounds. A v a r i e t y of re- l a b e l l e d compounds was fed to the fungus and the metabolic products examined with radioautography and s c i n t i l l a t i o n techniques. Materials and Methods I. Analysis of phenolic acids: The medium was removed with f i l t r a t i o n , a c i d i f i e d to pH 2 with 2N HCl and extracted three times i n a separatory funnel with a t o t a l of 200 ml of d i e t h y l ether. The ether extract was analysed by paper chromatography employing au- thentic samples as references(Fig. 17.). I I . Preparation and administration of radioactive compounds: 14 14 Phenylalanine-3- C, phenylalanine-2- C, 3,4-dihydroxy- 14 14 phenylalanine-2- C, phenylacetic acid-2- C, sodium acetate 14 14 -2- C and malonic acid-2- C were obtained from New England Nu- 14 . 14 cl e a r Corporation. Cinnamic acid-2- C and tyrosine-3- C were obtained from International Chemical and Nuclear Corporation and 14 benzoic a c i d - r i n g . C (U) was obtained from the Radiochemical 71 14 Center, Amersham, England. P-coumaric acid-2- C and c a f f e i c 14 . . acid-2- C were prepared by condensation of the appropriate 14 benzaldehyde with malonic acid-2- C i n pyridine with a trace of p i p e r i d i n e (Austin and Meyers 1965). Usually, 2 ̂ uCi of the radioactive compound was administered on the appropriate day, d i r e c t l y into the culture medium of each Roux b o t t l e . The compound was dispersed by b r i e f a g i t a t i o n and the c u l - tures incubated with the radioisotopes f o r 24 hours. I l l , Detection of r a d i o a c t i v i t y : Sprayed chromatograms were used for radioautography. Unsprayed duplicate chromatograms were examined quantitat- i v e l y by cutt i n g t'ae phenolic a c i d spots from the chromato- grams and counting them d i r e c t l y by s c i n t i l l a t i o n employ- ing a Nuclear-Chicago 720 series or Unilux II l i q u i d s c i n - t i l l a t i o n spectrometer. Dual channel counting permitted c a l c u l a t i o n of e f f i c i e n c y from a quench curve prepared for each instrument employing a series of v a r i a b l y quenched samples. The s c i n t i l l a t i o n f l u i d employed for most samples consisted of 4 g 2,5-diphenyloxazole (PPO) and 30 mg p_- bis 2-(5-phenyloxazolyl)-benzene (POPOP) i n one l i t r e of toluene. Aquasol (New England Nuclear Corporation) was used for aqueous samples and methanolic samples of trimethylhis- p i d i n and v e r a t r i c a c i d . 72 IV. Recovery of free amino acids: Free amino acids i n the culture medium were recovered by removing the mycelium with f i l t r a t i o n and passing the medium onto a column of Dowex 50W - X8 strongly c a t i o n i c exchange r e s i n . The amino acids were eluted with 5% aqueous NH^OH and the ammonia was removed by evaporation i n vacuo. Af t e r reducing i t s volume by h a l f , the amino ac i d s o l u t i o n was frozen and l y o p h i l i z e d . The residue was taken up i n 70% ethanol and chromatographed one-dimensionally on What- man No. 1 paper i n the upper phase of n-butanol/acetic a c i d / water (4:1:5 by volume). The tyrosine band was eluted and rechromatographed on 500 micron A v i c e l (cellulose) t h i n layer plates employing the same solvent system. The tyro- sine band recovered from TLC was d i s t i n c t and the radio- a c t i v i t y was determined by s c i n t i l l a t i o n . For t h i s purpose, the c e l l u l o s e scraped from the plates was suspended i n the s c i n t i l l a t i o n f l u i d with Cabosil (Cabot Corp.). 14 V. Methylation of C-labelled h i s p i d i n : H i s p i d i n obtained a f t e r chromatography of fungal ex- tracts was dried under vacuum. The t o t a l amount recovered from each radioactive feeding was dissoved i n ten ml dry acetone and to the solu t i o n was added 200 mg anhydrous pot- assium carbonate and 0.1 ml of dimethyl sulphate. The s o l u t i o n was refluxed for 24 hr over a steam bath employing a condenser f i t t e d with drying tube. The reaction mixture was subsequent- l y reduced i n volume i n vacuo and banded d i r e c t l y onto four 500 micron A v i c e l TLC plates (20 X 20 cm). The plates were dev- eloped i n solvent system C and the t r i m e t h y l h i s p i d i n band (Rf 0.97) was recovered from the plates i n the same manner as the h i s p i d i n band. The recovered t r i m e t h y l h i s p i d i n showed ident- c a l chromatographic behaviour and gave the same colour reaction to spray reagents (Table III.) as the authentic material. Results and Discussion Cultures of P_. hispidus were examined a f t e r 14 and 21 days incubation. These cultures were fed phenylalanine- 14 . 3- C and were examined a f t e r 6, 12 and 24 hr incubation with the radioactive compound. In one set of cultures, the medium was replaced with 0.5% phenylalanine s o l u t i o n 48 hr p r i o r to the feeding of 2.5 ;uCi (0.21/wM) of phenylalanine- 14 3- C per 100 ml of culture medium. In the second set of c u l - tures, the same amount of radioactive phenylalanine was added to the medium without replacement. Two-week-old cultures which were incubated on phenyl- alanine s o l u t i o n for 48 hr p r i o r to feeding showed incor- poration of r a d i o a c t i v i t y i n t o phenylpyruvic, p h e n y l l a c t i c , phenylacetic, cinnamic, benzoic and p_-hydroxybenzoic acids. 74 Two other u n i d e n t i f i e d compounds were also radioactive. In addition, p_-hydroxyphenylacetic, p_-coumaric, c a f f e i c and protocatechuic acids and one other u n i d e n t i f i e d compound were present on the chromatograms without l a b e l . Figure 23. shows a diagrammatic representation of the detected compounds on these chromatograms. Spot number 1. which gave a v i o l e t colour reaction with spray reagent 1. (p_-NA) and spot number 2. (brownish with p_-NA) could not be deter- mined from a v a i l a b l e reference compounds. Figure 24. shows the probable i n t e r r e l a t i o n s h i p s of the determined radioactive compounds. Examination of three-week-old cultures which also were incubated with nonradioactive phenylalanine p r i o r to feeding showed less consistent r e s u l t s , although the pat- tern of compounds was s i m i l a r to that observed for two- week-old cultures. P-hydroxyphenylacetic acid and proto- catechuic a c i d showed incorporation of l a b e l a f t e r 6, 12 and 24 hours incubation with radioactive phenylalanine. Also, l a b e l i n phenylacetic a c i d was not detected and radio- a c t i v i t y i n two spots which chromatographed very close to c i s and trans c a f f e i c acids was detected a f t e r 6 and 12 hour feeding periods. No spots i n addition to those ob- served i n two-week-old cultures were detected. The suspected r e l a t i o n s h i p of protocatechuic a c i d i n the degradation of 75 Solvent Front (system A) FIG. 23. Diagrammatic representation of the compounds detected i n chromatographed extracts of the medium using 48 hr induction by replacement medium. « • - detected by radioautography .JtfL - were radioactive with three-week-old cultures only 7 6 FIG. 24. Probable pathways of L-phenylalanine degradation i n P.hispidus. I L-phenylalanine, II cinnamic acid, III Benzoic acid, IV p_-hydroxybenzoic acid,V p h e n y l l a c t i c acid, VI phenyl- pyruvic acid, VII phenylacetic acid, VIII p_-hydroxyphen- y l a c e t i c acid, IX protocatechuic a c i d . phenylalanine i s included i n Figure 24. In cultures which were fed radioactive phenylalanine without p r i o r incubation on replacement medium, a new lab- e l l i n g pattern emerged. Although the recoverable quantities of phenolic compounds from three-week old cultures was con- siderably smaller than those from two-week-old cultures, r a d i o a c t i v e l y - l a b e l l e d p_-coumaric and c a f f e i c acids were present i n both. Extracts of two-week-old cultures con- tained radioactive c a f f e i c and protocatechuic acids only a f t e r 24 hr incubation with the radioactive phenylalanine. While no radioactive p_-hydroxyphenylacetic acid was detected i n extracts of two-week-old cultures, i t was detected a f t e r 24 hr feeding of three-week-old cultures. In other respects, the compounds and l a b e l l i n g detected i n these cultures was s i m i l a r to those which were grown on replacement medium. The probable relationships of the newly-detected radioactive compounds are shown (Fig. 25.). The r e s u l t s of t h i s experiment indicate that degrad- ative pathways to carbon dioxide as well as the cinnamate pathway are operative i n phenylalanine metabolism i n P_. h i s - pidus cultures. The degradative products are very s i m i l a r to those found by Moore and Towers (1967) i n Schizophyllum commune. No hydroxylated cinnamic acid derivatives were found i n that fungus, however, i n Lentinus lepideus (Power et a l 1965) s i m i l a r hydroxylations have been shown to occur 78 L- PHENYLALANINE COOH CINNAMIC ACID FIG. 25. Probable r e l a t i o n s h i p of radioactive cinnamic a c i d derivatives detected i n cultures of P_. hispidus. 79 (Fig. 7.). When cultures were incubated on phenylalanine medium p r i o r to radioactive feeding, the hydroxylated c i n - namic ac i d derivatives, although e a s i l y detected on the chromatograms, were non-radioactive. However, when the re- placement medium was not used, these compounds were radio- a c t i v e l y l a b e l l e d . This has been interpreted as i n d i c a t i n g that when replacement medium i s used the cinnamate pathway becomes saturated and the phenolic products act to repress the pathway, probably at cinnamic acid-4-hydroxylase. These same products may also derepress the degradative pathways to carbon dioxide. A n o t h e r m o r e u n l i k e l y . p o s - s i b i l i t y i s that the hydroxylated cinnamic acids are a r i s i n g from phenylalanine through tyrosine and that phenylalanine- 4-hydroxylase or tyrosine ammonia-lyase are being repressed. In any case, i t appeared that i n order to follow the c i n - namate pathway and examine h i s p i d i n biosynthesis, cultures should not be incubated on replacement medium p r i o r to r a - dioactive feeding and concentrations of l a b e l l e d compounds fed to the cultures should be kept as small as possible to avoid repressive e f f e c t s . Two-week-old cultures were selected as the most suitable for feeding radioactive compounds, since larger quantities of phenolic compounds were recover- able from extracts of the medium. The lower recovery a f t e r three weeks i s l i k e l y the r e s u l t of oxidative enzyme a c t i v i t y . 80 Because radioactive dihydroxy-compounds normally were not recovered from the medium except a f t e r 24 hr feedings, t h i s length of time was chosen for future feeding studies. Cultures of P. hispidus grown under the conditions established i n Chapter One , were fed 5^Ci (0.42 ̂ M)of DL- 14 phenylalanine-3- C i n addition to 1 mg non-labelled phenyl- alanine per Roux bottle.Teh-day-old cultures were examined a f t e r 18 hours to determine i f r a d i o a c t i v i t y had become incorpor- ated into the free tyrosine pool i n the medium. The free amino acids were recovered and the tyrosine p u r i f i e d by the method described . S c i n t i l l a t i o n counting of the recovered tyrosine detected 3,800 disin t e g r a t i o n s per minute (dpm) i n the t o t a l tyrosine obtained from one Roux b o t t l e . This value was considered s i g n i f i c a n t l y high to conclude that enzymes produced by the fungus were capable of hydroxylating phenylalanine to tyrosine and that hydroxylated cinnamic ac i d derivatives could a r i s e from phenylalanine through tyrosine. To obtain more quantitative and more extensive e v i - dence of aromatic metabolism i n P. hispidus, several radioactive compounds were fed to cultures at various stages of growth and the incorporation of r a d i o a c t i v i t y i n t o phen- o l i c acids and t h e i r precursors was examined with s c i n t i l - 14 14 l a t i o n techniques. DL-phenylalanine-3- C, DL-tyrosine-3- C, 81 14 14 cinnamic acid-2- C, p_-coumaric acid-2- C, 3,4-dihydroxy- 14 14 phenylalanme-2- C and benzoic acid-ring C (U) were i n - cubated with the cultures for 24 hr. Two microcuries of each compound was administered to each Roux bo t t l e except 14 for p_-coumaric acid-2- C, of which 0.5 yuCi was administered. The r a d i o a c t i v i t y detected i n metabolic products was recorded (Table IV.). In t h i s experiment, as i n most tracer experiments which y i e l d quantitative data, great caution must be exer- cised i n in t e r p r e t i n g the r e s u l t s . Many factors are unknown and d i f f i c u l t to determine. The most s i g n i f i c a n t of these are, perhaps, turnover rate, pool s i z e and permeability of the fungus to the compound i n question. Turnover rate of any compound w i l l depend p a r t i a l l y on the pool si z e as well as on the number of pathways leading to and from t h i s compound and the a c t i v i t y of the enzymes regulating these pathways. The permeability of the hyphae may vary with the age of the organism, the concentration of the com- pound being considered, the concentration of another compound or for many other reasons. In an examination such as t h i s , both the i n t e r n a l and external pools w i l l a f f e c t the data obtained. The pool size may also vary with the age of the organ- ism. However, s c i n t i l l a t i o n counting has proved to be a more se n s i t i v e technique than radioautography for detection of 82 w P p o CJ « W S3 o -H CO N O O N c •H ^ W J •H > TJ o -H TS CO o 4-> •H CN cy •H rH EH W o <3 u O •H ft I o m >1 ro • >H 0) < o rH ft 0 •H CD u TSL o £ A A / ^ \ f NP O N N O O N CO CN o • \0 O N N? cn •H o VO ^-^ '—^ ^— •— — • m-H o o o o I H U o o o o o • * ro < o o cn N* CJ CN NO 53 rH rH CN H rH H -—V •—v ^—. ^-^ NP Q N N O O N £ CJ ON NP ON N O ON CN N O O N ft -H CO cn cn CO CO i n -cs SH rH —' ro TJ ' S -H O o o o o 3 o O o o o o o EH o <c O CM N0 LO f> o H o N* > i , rH CN CN N t i n N H CN N* N t EH CJ Ti —^ s—v , , , ^ 0s- N O ON. N O O N N O O N N O O N o O N? t> o o H o < CN ID cn rH CO CN Q -H e o « fO -H o O o o O O fl o O O o o o o a N O cn i n LO ro o •H CJ ». u cu cn N * o cn cn CO ffl rH rH tr; EH W cn S O >, H N* O o CO o <d rH rH CN rH rH H OS EH U CJ rH rH 1 1 cn CN 1 1 W a p 55 H H u 53 < o Q < w u H o u >; w g H cu u vp ON CO O O cn 0"N o co o o 0 0 CO N? CJ o -H •p ro ̂ "Q. co X cn O 6N o o rH U H m I W 55 H o dN CN o O N* I CN I < o p CJ •H o N C CU o i l o o CO CN O O in co •NT H u s H £ I P H CJ < CJ H o 53 W TABLE IV. M e t a b o l i c products from v a r i o u s aromatic compounds a d m i n i s t e r e d t o P_. h i s p i d u s . Percentages i n b r a c k e t s i n d i c a t e the f r a c t i o n o f the t o t a l r a d i o a c t i v i t y r e c o v e r e d on the chromatogram r e p r e s e n t e d by the p r e c e d i n g f i g u r e . 83 radioactive substances on chromatograms and i t i s for t h i s reason that i t i s employed here i n a q u a l i t a t i v e manner. The data obtained from feeding phenylalanine were very s i m i l a r to those from the experiment just described. P_- hydroxybenzoic a c i d was radioactive on chromatograms of a l l three growth stages examined, representing 22% of the re- covered r a d i o a c t i v i t y a f t e r 11 days, 7% a f t e r 14 days and 5% a f t e r 20 days. Protocatechuic a c i d showed no r a d i o a c t i v i t y a f t e r 11 days and represented less than 1% of the recovered r a d i o a c t i v i t y a f t e r 14 and 20 days. Cinnamic ac i d showed the anticipated conversion to p_-coumaric ac i d and c a f f e i c acid, i n d i c a t i n g that cinnamic acid-4-hydroxylase and p-coumaric acid-3-hydroxylase are op- erative i n the fungus. A much higher recovery of c a f f e i c a c i d was observed i n cultures which were fed a f t e r 18 days growth compared to the recovery from younger cultur e s . Sim- i l a r l y , p_-coumaric ac i d fed to two-week-old cultures was metabolized to c a f f e i c a c i d . The conversion of tyrosine to p_-coumaric a c i d i n d i - cates that tyrosine ammonia-lyase a c t i v i t y i s present i n two-week-old cultures. In addition, the s i g n i f i c a n t con- version of tyrosine to p_-hydroxyphenylacetic a c i d suggests that a degradative pathway s i m i l a r to that reported f o r Polyporus tumulosus (Crowden 1967) might also be operative 84 i n t h i s organism. While radioactive p_-hydroxyphenylacetic ac i d was also previously detected i n three-week-old cultures fed radioactive phenylalanine a f t e r 48 hr on replacement medium, i t might have been formed v i a tyrosine since the conversion of phenylalanine to tyrosine also has been ob- served. The detected conversion of 3,4-dihydroxyphenylala- nine (DOPA) to c a f f e i c a c i d requires a more thorough exam- in a t i o n . The background r a d i o a c t i v i t y detected on the chrom- atograms of the medium extracts was s i g n i f i c a n t l y higher than that detected i n the c a f f e i c a c i d spots, thus lowering the accuracy of the method. In addition, DOPA ammonia-lyase a c t i v i t y i s not known i n fungi. Nevertheless, there could be some b i o l o g i c a l importance f o r t h i s a c t i v i t y i n a para- s i t e of higher plants, as a number of higher plants are known to produce DOPA as an intermediate i n the formation of DOPA-melanin. When benzoic acid was fed to two-week-old cultures of the fungus, the only i d e n t i f i a b l e product was p_-hydroxy- benzoic a c i d . However, the absense of radioactive proto- catechuic a c i d on the chromatograms does not indicate that ring cleavage does not occur. Later studies showed that large quantities of radioactive carbon dioxide were released when benzoic a c i d was fed to the organism (Nambudiri, personal 85 COOH H2NCH H2NCH COOH I CH II CH OH COOH COOH OH COOH FIG. 26. Aromatic amino acid metabolism v i a the cinnamate pathway i n P_. hispidus. Dashed l i n e s refer to interconversions for which no d i r e c t evidence has been obtained. -^jr has been shown but requires a d d i t i o n a l examination (see t e x t ) . 86 communication). The d e t a i l s of aromatic amino aci d metabolism elucidated by these studies are shown i n Figure 26. A " g r i d - l i k e " net- work of interconversions of these aromatic compounds i s postulated; ultimately leading to the formation of carbon dioxide. In the next feeding experiment the conversion of many of the same compounds and others to h i s p i d i n was exa- mined. The numbers (1 - 5) i n figure 26. r e f e r to some of the enzymes examined i n P. hispidus. These w i l l be discussed i n Chapter Four. The rate of h i s p i d i n production i s highest i n 14- to 24-day-old cultures (Fig. 14). Therefore, 17-day-old cultures were selected for examination of incorporation of various precursors into the h i s p i d i n molecule. Two micro- 14 curies of each of DL-phenylalanine-2- C, DL-phenylalanine- 14 14 . . . 14 . 3- C, DL-tyrosine-3- C, cinnamic acid-2- C, malonic acid- 14 14 . . . 14 2- C and sodium acetate-2- C, 1 JACI of c a f f e i c acid-2- C 14 and 0.5 ̂ C i of p_-coumaric acid-2- C were fed to each of eight cultures. A f t e r 24 hr the h i s p i d i n was recovered as described i n Chapter One, the trimethyl ether was prepared and i t s s p e c i f i c a c t i v i t y determined. In turn, the trimethyl- h i s p i d i n from each feeding was degraded to v e r a t r i c a c i d and the s p e c i f i c a c t i v i t y of th i s compound also was deter- mined. In th i s way i t was possible to ascertain i f these 87 ft >H H H • > C H E H ^ H O W U H CJ>HCJ H>En <xN C J H r f -H co<> o H EH D H Q CO CJ r^ H — j f n H . i H > E H I U H H -H W E - t g O ft U H CO < « — 5s Q - 9 ON  K W P O CO ft H 2 O H CJ 2 Q < CJ CJ CO 1 O rH 1 o (Ti 1 •H cn cn m • i o o i o rH rH rH 1 X X X 1 1 H o cn • • • H H cn o O O o o O O o VO m cn o CN m CN cn cn l l 1 o o o o rH rH rH rH X X X X 00 rH rH • • • • rH rH VO m o • • • • CN CO o <N CJ o CN CO • • • m CN 1 • 1 1 T i n m 1 l I O o o H rH rH X X X 1 1 o VO 1 1 • • • CN cn VO O o o o o rH o O CN cn rH O I I CJ cn I cj VO rH I o o o o rH rH H rH X X X X o CO 00 i n • • • • 00 i n H CO vo rH rH o • • • • o o CN 1 <tf u rH CJ rH rH rH CN rH 1 1 1 I | rH CN rH CN cn CN 1 1 1 | l U | - H CN CD CN CD CD O | +> 1 C rH - H ro TS ro TJ - H • H 1 CJ •H -P • H C cn ro O CJ <D CJ rcj ro I • H rfl O ro rH rH CD O U ro ro ro a • H ro CJ CJ rH H •H e • H P • H >1 >i CO ro 3 CD 3 c C C 0 o m • H o a» CD u G CJ m rH Xi Xi - H i, ro o ra ft ft O ft CJ CO TABLE V. Incorporation of various precursors i n t o h i s p i d i n by 17-day-old cultures of P. hispidus. 88 compounds were l a b e l l i n g the h i s p i d i n , and i f so, whether the phenylpropanoid moiety was being incorporated i n t a c t . The data obtained from these feedings are reported i n Table V. Radioactivity was incorporated into h i s p i d i n from a l l the compounds administered and, i n general, the data substantiated the hypothesis that the phenylpropanoid precursors were incor- porated without scrambling of the l a b e l and that acetate primar- i l y was incorporated into the pyrone portion of the molecule. In view of the s t a t i s t i c a l nature of radioactive decay, the 95% confidence l i m i t s for the data were calculated and these showed that the s p e c i f i c a c t i v i t i e s reported i n Table V. might vary as much as + 7% and that the fractio n s reported i n the l a s t column of Table V. might vary by twice t h i s amount. This var- i a t i o n represents the maximum found i n any sample. No values were obtained for the s p e c i f i c a c t i v i t y of 14 v e r a t r i c a c i d from the phenylalanine-2- C or the malonic a c i d - 14 2- C feedings. These samples were l o s t by overoxidation of the tri m e t h y l h i s p i d i n . A maximum oxidation time of two hours should be s t r i c t l y adhered to with the oxidation conditions employed. Nevertheless, i n view of the data obtained from feedings of other compounds, the l a b e l l i n g pattern i s apparent. D i l u t i o n values for the various precursors must be i n t e r - preted c a r e f u l l y . As explained previously, pool sizes, turnover rates and permeabilities w i l l a f f e c t t h i s data. The d i l u t i o n 89 14 14 values for phenylalanine-2- C and phenylalanine-3- C indicate the v a r i a t i o n that might be expected for the same compound. How- ever, the d i l u t i o n f or cinnamic acid i s s i g n i f i c a n t l y higher than that obtained for p_-coumaric and c a f f e i c acids. Although t h i s might seem u n l i k e l y at f i r s t , i t appears more acceptable i n l i g h t of the previous experiments which suggested that cinna- mic a c i d 4-hydroxylase was a regulatory enzyme and although 14- day-old cultures r e a d i l y converted cinnamic ac i d to p_-coumaric acid (Table IV.) the recovery of hydroxylated cinnamic acids from 18-day-old cultures was appreciably lower. Also, a larger amount of unconverted cinnamic ac i d was recovered from the medium of 18-day-old cultures (Table IV.). In addition, i t has been shown that when cinnamic ac i d i s present i n the culture medium of P_. hispidus at a concentration of 0.1%, no growth i s observed i n the culture s . The data are consistent with a phenylpropanoid-acetate (malonate) biogenesis of h i s p i d i n . Both types of molecules are r e a d i l y incorporated into h i s p i d i n and the r a d i o a c t i v i t y i s l o - cated i n the appropriate portion of the molecule (Fig. 28.). When phenylalanine (I) or tyrosine (II) l a b e l l e d i n the t h i r d carbon (b*) are administered to the fungus, both the trimethy- h i s p i d i n (VII) and the v e r a t r i c acid (VIII) obtained by oxidation show approximately the same s p e c i f i c a c t i v i t y (Table V.). How- ever, when cinnamic ac i d (III), p_-coumaric ac i d (IV), c a f f e i c a c i d (V) or acetate l a b e l l e d i n the second carbon (a*, c*) are 90 administered almost no r a d i o a c t i v i t y i s found i n the v e r a t r i c a c i d while t r i m e t h y l h i s p i d i n shows good incorporation of radio- a c t i v i t y (Table V.). 91 COOH COOH FIG. 27. Biosynthesis and degradation of radioactive h i s p i d i n . CHAPTER FOUR PRELIMINARY STUDIES OF ENZYMES ASSOCIATED WITH AROMATIC METABOLISM Introduction The conversion of phenylalanine to tyrosine and cinnamic and p_-coumaric acids i s an a b i l i t y l a r g e l y r e s t r i c t e d to higher plants and c e r t a i n fungi (Camm and Towers 1969). The non-oxida- t i v e deamination of phenylalanine also has been detected i n Strep- tomyces (Bezanson et a l 1970) and i s implicated i n some ba c t e r i a . Of the fungi, p r i m a r i l y Basidiomycetes have been shown to possess phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) a c t i v i t y (Power et a l 1965, Bandoni et a l 1968). I t i s these en- zymes i n Polyporus hispidus which i n i t i a t e the sequence of enzym- a t i c conversions leading to h i s p i d i n from the aromatic amino acids. Those enzymes which e f f e c t the hydroxylation of the arom- a t i c r i n g are also c r i t i c a l to h i s p i d i n biosynthesis. I t seems l i k e l y that hydroxylation i n P. hispidus may occur both at the l e v e l of cinnamic a c i d and i t s derivatives and at the l e v e l of styrylpyrone, bis-noryangonin and h i s p i d i n . This i s indicated by the presence of trace amounts of bis-noryangonin and strong bis-noryangonin 4-hydroxylase a c t i v i t y i n fungal extracts. No work has been reported on enzymes which are capable of mediating hydroxylation at the l e v e l of the styrylpyrones, although exam- inations of enzymes which accomplish hydroxylation of cinna- mic acids have been reported for higher plants (Hasegawa and Maier 1972, Hahlbrock et a l 1971) and micro-organisms (Nam- b u d i r i 1971, and references c i t e d t h e r e i n ) . The a b i l i t y of many fungi to hydroxylate benzoic a c i d to p_-hydroxybenzoic 93 acid and protocatechuic a c i d i s well-known (Cain et a l 1968, H a l s a l l et a l 1969). This a c t i v i t y t y p i c a l l y leads to r i n g cleavage. C e l l - f r e e preparations of Polyporus hispidus were ex- amined for PAL and TAL a c t i v i t y . PAL a c t i v i t y was i n v e s t i g a - ted i n cultures of d i f f e r e n t ages and these data are reported. In addition, a number of c e l l - f r e e preparations were obtained which effected hydroxylation of benzoic acid, cinnamic acids and the styrylpyrone, bis-noryangonin. Materials and Methods I. Phenylalanine ammonia-lyase Cultures were examined at 2 - 4 day i n t e r v a l s for phen- ylal a n i n e ammonia-lyase a c t i v i t y . The cultures were f i l t e r e d , the mycelium b l o t t e d with paper towels and the extracted by grinding with an equal weight (equal to the wet weight of mycelium) of aluminum oxide, and twice t h i s weight of 0.05 N Tris-HCl buffer, pH 8.6. The s l u r r y was c e n t r i - fuged at 18,00 g for ten minutes i n a r e f r i g e r a t e d c e n t r i - fuge and 5 ml of the supernatent was passed onto a 2 cm X 20 cm column of Sepadex G - 25 (Pharmacia, Uppsala, Swe- den) prepared with the same buffer, for removal of the low molecular weight molecules. Five ml of eluate was c o l l e c t e d and assayed spectrophotometrically at 290 nm a f t e r combining a portion with 1 ml of 2.5 yJA phenylalanine 94 i n buffer solution and s u f f i c i e n t Tris-HCl buffer to make a t o t a l of three ml i n the spectrophotometer cuvette. The incubation i n the cuvette was performed at 30°C, and changes i n o p t i c a l density were recorded at 5 min i n t e r v a l s for 20 min. A l l procedures were performed at 0 - 5°C u n t i l the assay was begun and each assay was performed with at l e a s t two d i f f e r e n t quantities of the enzyme preparation. Protein was not determined, and s p e c i f i c a c t i v i t i e s were reported with respect to dry weight of fungus. I I . Tyrosine ammonia-lyase Three ml of the enzyme preparation employed for the 14-day phenylalanine ammonia-lyase assay was also examined for tyrosine ammonia-lyase a c t i v i t y . To t h i s was added an a d d i t i o n a l 5 ml of the supernatant which'had not been passed through the Sephadex G-25 column. To th i s t o t a l of eight ml 14 was added 1yaCi of tyrosine-3- C plus three drops of 5% 2-mercaptoethanol to prevent oxidation. The mixture was i n - cubated for 14 hr at room temperature, a c i d i f i e d , and extracted with d i e t h y l ether. The ether extract was chromatographed 2-dimensionally on c e l l u l o s e TLC plates i n solvent systems A and B. I I I . Benzoic and cinnamic acid-4-hydroxylase The fungus was harvested and macerated as described above, employing 0.1 M phosphate buffer, pH 7.5. The s l u r - ry was centrifuged at 27,000 g for ten minutes and the sup- 95 ernatant at 40,000 g for a further 10 min. This supernatant was then centrifuged at 105,000 g for 90 min to obtain a microsomal p e l l e t . Both the p e l l e t and the supernatant were examined for cinnamic acid-4-hydroxylase a c t i v i t y . The microsomal p e l l e t was suspended i n two 2 ml a l i - quots of 0.1 M phosphate buffer pH 7.5. To one p e l l e t sus- 14 pension and the supernatant were added cinnamic acid-2- C O.S^uCi, non-labelled cinnamic a c i d 0.15 mg, 2-mercaptoeth- anol 0.24 mg and three ml of the NADPH + H + generating sys- tem. To the second p e l l e t suspension was added benzoic acid - 14 (UL) (RL)- C 2/^.Ci, 2-mercaptoethanol 0.24 mg and three ml + + of the NADPH + H generating system. To provide NADPH + H for the enzymes, a mixture of 1.6 mg NADP+, 1.4 mg glucose- 6-phosphate and 0.4 units glucose-6-phosphate dehydrogenase i n three ml 0.1 M phosphate buffer, pH 7.5, was prepared and incubated at 30°C for ten minutes before addition to the assay mixture. The assay mixture was incubated with a g i t - o ation for 90 min at 30 C. The reaction was terminated by the addition of hydrochloric a c i d . Radioactive p_-coumaric and p_-hydroxybenzoic acids were examined for, employing paper chrom- atography i n solvent systems A and B. IV. Bis-noryangonin-3-hydroxylase Cultures grown for 14 days on GYSS supplemented with cinnamic a c i d (3yxg/ml for 13 days and 0.1% for l a s t 24 hr) 96 were extracted i n a mortar with powdered glass and 0.1 M citrate-phosphate buffer, pH 7.0. The crude extract was cen- t r i f u g e d at 10,000 g for twenty minutes and passed onto a 2 cm X 20 cm column of Sephadex G - 25 prepared i n the same buffer. Twice the volume applied to the column was c o l l e c t e d and used as the crude enzyme preparation. Two ml of crude enzyme, 1.5 ml O^uMoles) ascorbate i n buffer and 0.2 ml 0.1 M c i t r a t e - phosphate buffer, pH 7.0 were mixed i n a t e s t tube and incuba- ted at 30°C for 1 hour. At the end of the reaction time the mix- ture was treated with 0.5 ml 5 N HCl and extracted with d i e t h y l ether (15 ml t o t a l ) . The ether extract was reduced i n volume and banded onto a c e l l u l o s e TLC plate and developed i n solvent system C. A control to which the enzyme was added a f t e r incu- bation was s i m i l a r l y examined. Results and Discussion In the assay for phenylalanine ammonia-lyase, an increase i n absorbance i s recorded at 290 nm as phenylalanine i s con- verted to cinnamic a c i d . The rate of increase i s a meas- ure of the PAL a c t i v i t y . By examining two aliquots of crude enzyme of d i f f e r e n t sizes and employing the difference i n rate with respect to the difference i n a l i q u o t s i z e as the measure of a c t i v i t y , errors are minimized. Three d i f f e r - ent sets of cultures were analyzed for PAL a c t i v i t y and a l l sets showed maximum PAL a c t i v i t y at the same culture age. 97 B T l l O a U3d 1HOI3M AUG ( I M Ajp B / u!iu / 0 6 3 a O v ) A1IAI10V "IVd F I G . 28. P h e n y l a l a n i n e a m m o n i a - l y a s e a c t i v i t y i n c u l t u r e s o f P_. h i s p i d u s . 98 Maximum PAL a c t i v i t y was observed i n 14-day-old cultures (Fig. 29.) which compares favourably with the observed con- version of phenylalanine to cinnamic acid i n vivo (Table IV.). However, P. hispidus d i f f e r s from Rhodotorula glutinus and Sporobolomyces roseus i n the time of maximum PAL a c t i v i t y . While the l a t t e r organisms showed maximum PAL a c t i v i t y as the cultures entered the stationary phase of growth (Ogata et a l 1967, Camm and Towers 1969), maximum PAL a c t i v i t y i n P. hispidus occurs near the end of the logarithmic phase of growth. Radioactive p_-coumaric acid from the tyrosine ammonia- lyase assay mixture was detected by autoradiography. While t h i s confirms that the enzyme i s active i n c e l l - f r e e prep- arations of 14-day-old cultures, no attempt was made to deter- mine i f th i s was the same enzyme which demonstrated PAL a c t i v i t y , or i f two d i f f e r e n t enzymes were responsible for these a c t i v i t i e s as has been shown i n Ustilago (Subba Rao et a l 1967) and i n the higher plant Ipomoea (Minimikawa and U r i t a n i 1965). Both cinnamic acid and benzoic a c i d were hydroxylated i n the para p o s i t i o n by the microsomal p e l l e t preparation. However, no cinnamic acid-4-hydroxylase a c t i v i t y was observed i n the supernatant preparation from the 105,000 g c e n t r i - fugation. While benzoic a c i d was r e a d i l y converted to 99 p_-hydroxy-benzoic acid (12%), l i t t l e more than 1% conversion to p_-coumaric ac i d from cinnamic ac i d was effected. Further studies of cinnamic acid 4-hydroxylase i n crude enzyme prepar- ations from J>. hispidus have f a i l e d to show more than 1 - 2 % conversion i n a 90 minute incubation period (Vance, personal communication). A c r i t i c a l study of the factors which con- t r o l the a c t i v i t y of t h i s enzyme i s required. The bis-noryangonin 4-hydroxylase preparation converted approximately 2yUmoles of bis-noryangonin to h i s p i d i n i n 1 hour. No h i s p i d i n was detected i n the c o n t r o l . This suggests that h i s p i d i n can also a r i s e from cinnamic ac i d through hydroxyla- t i o n of styrylpyrone. The presence of bis-noryangonin i n crude fungal extracts adds further support to t h i s claim. I t has not been determined whether the enzymes responsible for the hydroxylation of these various aromatic compounds are a c t u a l l y d i f f e r e n t . Since many fungi produce hydroxylated derivatives of benzoic ac i d without y i e l d i n g hydroxylated cinnamic acids and s t i l l other fungi w i l l hydroxylate cinnamate, i t seems l i k e l y that d i f f e r e n t enzymes are involved. GENERAL SUMMARY AND CONCLUSIONS 100 General Summary and Conclusions C u l t u r a l conditions amenable to examining the cinnamate pathway and h i s p i d i n biosynthesis i n Polyporus hispidus were established. A medium containing glucose, yeast extract, an enzymatic hydrolysate of soybean meal and several s a l t s and having an i n i t i a l pH of 7 was the most sui t a b l e found for o these studies. An incubation temperature of 23 C was s e l - ected for optimum pigment production. Light was shown to be necessary for pigment formation and t h i s requirement could not be replaced by hydrogen peroxide or hydroxylamine hydrochloride. Blue l i g h t was most e f f e c t i v e i n stimulating pigment development. The maximum rate of h i s p i d i n production under the es- tablished c u l t u r a l conditions was observed to lag the max- imum growth rate by about f i v e days. As the cultures became senescent, the y i e l d of h i s p i d i n was observed to f a l l rapid- l y . I t i s proposed that t h i s decline i s associated with oxidative a c t i v i t y r e f l e c t e d i n browning of the c u l t u r e s . P. hispidus was observed to sporulate i n agar c u l - tures when vigorously-growing hyphal t i p s i n agar blocks were employed as inoculum. Viable basidiospores were ob- tained from the developing sporocarp and these germinated af- ter s i x months. It i s suggested that a c r i t i c a l l y low mois- ture l e v e l i n the substrate triggered the germination. 101 P_-coumaric, c a f f e i c , p_-hydroxyphenylacetic, p_-hydroxy- benzoic and protocatechuic acids were observed to occur n a t u r a l l y i n cultures of the fungus. By feeding r a d i o a c t i v e l y - l a b e l l e d compounds to these cultures, i t was possible to e- lucidate the d e t a i l s of the cinnamate pathway proposed i n Figure 26. The degradation of phenylalanine was also pro- posed from the evidence obtained (Fig. 2 4 . ) . In other feeding experiments the incorporation of various radioactive molecules i n t o h i s p i d i n was examined. These r e s u l t s provide good evidence for the hypothesis that fungal styrylpyrones are biosynthesized through the conden- sation of two units of a c e t i c a c i d with cinnamic acids (possibly as esters of Coenzyme A). In addition, bis-nor- yangonin was detected i n cultures of the fungus. The presence of t h i s compound^ together with obtaining a crude enzyme preparation capable of hydroxylating i t to h i s - p i d i n , suggests that more than one pathway to h i s p i d i n i s operative i n Polyporus hispidus (Fig. 30.). Other crude enzyme preparations have been obtained from cultures of P. hispidus. Although no attempts were made to p u r i f y and characterize the enzymes, demonstrating t h e i r presence provides the best evidence for a biochemical con- version. Both PAL and TAL a c t i v i t y were detected i n fungal extracts with maximum PAL a c t i v i t y recorded during the COOH 102 COOH COOH o II C-SCoA HO T V I I I I OH O il J-SCoA V H V I I I OH I I I OH OH OH OH HO. 'OH OH F I G . 29. Alternate routes proposed for the biosynthesis of h i s p i d i n i n Polyporus hispidus. I cinnamic acid, I I p_-coumaric acid, I I I c a f f e i c acid, I V cinnamoylCoenzyme A, V p_-coumaroylCoenzyme A, V I caf- feoylCoenzyme A, V I I s t y r y l pyrone, V I I I bis-noryangonin, DC h i s p i d i n . 103 logarithmic phase of growth. Enzymes capable of hydroxy- l a t i n g cinnamic acid, benzoic a c i d and bis-noryangonin were present i n c e l l - f r e e preparations. Also, l a t e r e v i - dence has shown that a crude enzyme preparation from th i s fungus w i l l hydroxylate p_-coumaric a c i d to c a f f e i c a c i d (Nam- b u d i r i , pers. comm.). Although the maximum y i e l d of h i s p i d i n from cultures of P. hispidus was only 10% of that from cultures of P. sc h w e i n i t z i i (Hatfield 1970), j?. hispidus has proved to be an e x c i t i n g organism for biochemical study. Cultures of t h i s fungus perform numerous enzymatic conversions of aromatic compounds reported from only a few other Basidiomycetes, and i t has been shown that active extracts of several of these enzymes can be obtained r e a d i l y . The implications of obtain- ing sporophores i n culture are important with respect to studying the biochemistry of thi s fungus. An examination of single-spore i s o l a t e s could provide important d e t a i l s concerning genetic control i n aromatic amino acid metabolism and h i s p i d i n biosynthesis. F i n a l l y , a number of aromatic compounds detected i n cultures of P. hispidus remain to be i d e n t i f i e d . Some of these are poss i b l y styrylpyrones, i n t e r - mediates i n h i s p i d i n biosynthesis, or derivatives of h i s p i d i n and i t s precursors. BIBLIOGRAPHY 104 BIBLIOGRAPHY Akagi, M. 1942. Leucomelone from Polyporus leucomelas. J . Pharm. Soc. Japan, 62:129. Austin, D.J. and M.B. Meyers. 1965. 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Ch a r a c t e r i s t i c s of Wrattan f i l t e r s employed i n experiments determining the ef f e c t s of the wavelength of the l i g h t source on growth and pigment development. Minimum 10% T F i l t e r No. Colour of l i g h t passed Bandpass (nm) H-45 Blue 430 - 540 B-58 Green 500 - 620 G-15 Red & Yellow 520 - 700+ A-25 Red 590 - 700+ 114 APPENDIX B. Spray reagents employed i n the detection of various compounds separated by paper and thin - l a y e r chromatographic techniques 1. Diazotized p_-nitroaniline reagent (Ibrahim and Towers 1960) : 5 ml of 0.3% p_-nitroaniline, 1 ml of 5% sodium n i t - r i t e and 15 ml of 20% sodium acetate were combined, i n that order, just p r i o r to use. A f t e r spraying with t h i s s o l u t i o n the chromatogram was allowed to dry b r i e f l y and then oversprayed with a 5% aqueous so l u t i o n of NaOH. Phenolic compounds appear as variously coloured spots. 2. F e r r i c chloride reagent: 2% f e r r i c chloride i n 95% ethanol; detects ortho- dihydroxy phenolic compounds as greenish or brownish spots. 3. Modified E h r l i c h 1 s reagent: 2% £-dimethylaminobenzaldehyde i n ethanol-HCl (3:1) detects styrylpyrones as shades of blue, green or yellow. 4. p_-dimethylaminocinnamaldehyde reagent: 0.5% p_-dimethylaminocinnamaldehyde i n 0.5N HC1 detects styrylpyrones v a r i o u s l y as purples, greens or b l u i s h greens. 5. Bromocresol green reagent: 0.04% i n 96% ethanol with the addition of 0.1 N NaOH u n t i l the soluti o n just turns blue. Detects a c i d i c compounds as yellow spots on a blue background. A l l traces of a c i d i c solvents must be removed from chromatograms before spraying.

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