Chapter 34
Oxidative Enzymes from the Lignin-Degrading Fungus Pleurotus sajor-caju Robert Bourbonnais and Michael G. Paice Pulp and Paper Research Institute of Canada, 570 St. John's Boulevard, Pointe Claire, Quebec H9R 3J9, Canada
Two extracellular oxidase enzymes proposed to play a role in lignin depolymerisation, laccase (polyphenol oxidase) and veratryl alcohol oxidase (VAO), were isolated from ligninolytic cultures of Pleurotus sajor-caju. The enzymes were produced in agitated, mycological broth cultures and were isolated after 12 days from supernatants by precipitation and chromatography. Two purified VAO enzymes had very similar physical and biochemical properties. They oxidised a variety of aromatic primary alcohols to aldehydes with reduction of oxygen to hydrogen peroxide. Sequential treatment of the laccase substrate ABTS with laccase and then VAO and veratryl alcohol producedfirstappearance and then disappearance of characteristic colors. A reduction-oxidation cycle is proposed for the two enzymes in depolymerisation of phenolic substructures of lignin. W h i t e - r o t f u n g i p l a y a key role i n biodégradation o f w o o d y m a t e r i a l s . T h e y are often the i n i t i a l colonizers, a n d are p r o b a b l y the o n l y m i c r o o r g a n i s m s t h a t extensively degrade l i g n i n (1). T h e m e c h a n i s m o f l i g n i n c a t a b o l i s m b y Phanerochaete chrysosporium has been s t u d i e d intensively, a n d a p i c ture is n o w emerging o f the e n z y m o l o g y i n v o l v e d . L i g n i n peroxidase, first recognized i n 1983 (2,3), abstracts a n electron f r o m l i g n i n s u b s t r u c t u r e s a n d subsequent free-radical t r a n s f o r m a t i o n s result i n Co>C/?, /?-ether a n d a r o m a t i c r i n g cleavage (4,5). T h e reactions w h i c h are dependent o n h y drogen peroxide c a n consume oxygen b y a d d i t i o n o f m o l e c u l a r oxygen t o r a d i c a l intermediates (6). M a n g a n e s e peroxidase (7,8) is also p r o d u c e d b y P. chrysosporium a n d abstracts electrons f r o m l i g n i n s u b s t r u c t u r e s w i t h lower redox p o t e n t i a l s such as phenolic u n i t s . T h e e n z y m e d i r e c t l y o x i dised Μ η II t o Μ η I I I as t h e i n i t i a l step i n l i g n i n a t t a c k . O t h e r enzymes 0097-6156/89/0399-0472$06.00/0 © 1989 American Chemical Society
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i m p l i c a t e d i n l i g n i n c a t a b o l i s m b y P. chrysosporium are glucose oxidase (9,10) a n d g l y o x a l oxidase (11) for h y d r o g e n peroxide p r o d u c t i o n , a n d c e l lobiose quinone oxidoreductase (12) for q u i n o n e r e d u c t i o n . Since l i g n i n s c a n be b o t h d e p o l y m e r i z e d a n d p o l y m e r i z e d b y l i g n i n peroxidase (2,13), other enzymes are p r o b a b l y r e q u i r e d for c a t a b o l i s m in vivo. T h e question arises as t o whether other w h i t e - r o t f u n g i e m p l o y the same c a t a b o l i c p a t h w a y s as P. chrysosporium. Also, lignin degradation by P. chrysosporium occurs o n l y d u r i n g secondary m e t a b o l i s m , a n d e m p l o y s v e r a t r y l a l c o h o l , w h i c h is synthesized de novo b y the fungus, as a m e d i a t o r (14) or enzyme i n d u c e r (15). A r e these c o n d i t i o n s required b y other f u n g i ? It n o w appears t h a t l i g n i n peroxidase is p r o d u c e d b y Coriolus versicolor (16) a n d perhaps b y Pleurotus ostreatus (17). However these two w h i t e rot f u n g i produce laccase isoenzymes w h i c h , like manganese peroxidase, c a n a b s t r a c t electrons f r o m p h e n o l i c s u b s t r u c t u r e s of l i g n i n . T h u s there appears to be no obvious requirement for manganese peroxidase i n these f u n g i . I n a d d i t i o n , i t appears t h a t P. ostreatus c a n degrade l i g n i n effectively d u r i n g p r i m a r y m e t a b o l i s m , i.e. i n a h i g h n i t r o g e n m e d i u m (18), a l t h o u g h t h i s has been d i s p u t e d (19). Pleurotus sajor-caju differs f r o m P. ostreatus i n not r e q u i r i n g c o l d shock for f r u c t i f i c a t i o n (20). P. sajor-caju selectively degrades l i g n i n i n w h e a t - s t r a w under c e r t a i n c o n d i t i o n s (21,22), b u t t h i s s e l e c t i v i t y is lost w h e n f r u i t i n g bodies f o r m (23). C h l o r o l i g n i n c a n be m e t a b o l i s e d b y P. sajor-caju (24). W a l d n e r et al. (17) observed o x i d a t i o n o f v e r a t r y l a l c o h o l to v e r a t r a l d e h y d e b y P. ostreatus. W e have recently r e p o r t e d the discovery of a v e r a t r y l a l c o h o l oxidase f r o m P. sajor-caju (25). W e now propose a role for the e n z y m e , i n c o m b i n a t i o n w i t h laccase, i n the d e p o l y m e r i z a t i o n o f p h e n o l i c moieties o f l i g n i n . Experimental Enzyme Production and Isolation. T h e production and isolation of veratryl a l c o h o l oxidase ( V A O ) was described earlier (25). Laccase p r o d u c e d f r o m the same 12-day c u l t u r e (8 litres) was isolated f r o m the s u p e r n a t a n t b y p r e c i p i t a t i o n at 0 ° C w i t h a m m o n i u m sulfate ( 8 0 % s a t u r a t i o n ) . T h e p r e c i p i t a t e was suspended i n 0.05 M N a acetate buffer, p H 5.0 a n d d i a l y s e d overnight against 4 litres of buffer. T h e soluble m a t e r i a l was c o n c e n t r a t e d b y u l t r a filtration ( A m i c o n P M 1 0 ) to a b o u t 60 m L a n d a p p l i e d to a D E A E - B i o - g e l A c o l u m n (2.5 c m χ 35 c m ) . T h e c o l u m n was washed w i t h 20 m L o f the same buffer, t h e n e l u t e d w i t h a linear g r a d i e n t f r o m 0 to 0.6 M N a C l ( t o t a l v o l u m e 550 m L ) . F r a c t i o n s were m o n i t o r e d for V A O a n d laccase a c t i v i t y as described below. Enzyme Assays. Laccase a c t i v i t y was d e t e r m i n e d b y o x i d a t i o n o f 2,2azinobis-(3-ethylbenzthiazoline-6-sulfonate) ( A B T S ) . T h e reaction of suit a b l y d i l u t e d e n z y m e was d e t e r m i n e d at 420 n m i n the presence of 0.03% A B T S a n d 100 m M s o d i u m acetate buffer, p H 5.0. T h e e x t i n c t i o n coeffi cient o f A B T S is ε ο = 3.6 χ 1 0 M " c m ' (26). V A O a c t i v i t y was measured w i t h a m i x t u r e of 1 m M v e r a t r y l a l c o h o l , 250 m M s o d i u m t a r t r a t e buffer, p H 5.0 a n d e n z y m e . O x i d a t i o n to v e r a 4 2
4
1
1
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t r a l d e h y d e was d e t e r m i n e d at 310 n m (£310 = 9300 M " c m ) . L i g n i n peroxidase was measured b y v e r a t r y l a l c o h o l o x i d a t i o n at p H 3.0 i n the presence of 0.4 m M H 2 O 2 (2). A l l e n z y m e u n i t s are / / m o l e of p r o d u c t formed/min. T h e c o m b i n e d effect of laccase a n d V A O o n A B T S was d e t e r m i n e d as follows. T h e reagent (0.03%) was oxidised w i t h p u r i f i e d laccase f r o m P. sajor-caju (0.004 U / m L ) . A f t e r the appearance of the green ( A B T S ) colour, V A O (0.07 U / m L ) a n d v e r a t r y l a l c o h o l (1 m M ) was added a n d the decrease i n colour was observed v i s u a l l y . O x y g e n u p t a k e d u r i n g substrate o x i d a t i o n was measured w i t h a C l a r k oxygen electrode ( R a n k B r o t h e r s , C a m b r i d g e , U . K . ) at r o o m t e m p e r a t u r e w i t h 1 m M substrate i n 0.25 m M s o d i u m t a r t r a t e buffer, p H 3.0 (3 m L ) . R a t e s are expressed relative to v e r a t r y l a l c o h o l o x i d a t i o n . 1
- 1
+ e
Results and Discussion Enzyme Production. P. sajor-caju, when g r o w n i n a n u t r i e n t - r i c h m e d i u m under aerated, a g i t a t e d c o n d i t i o n s , was able to m i n e r a l i z e p a r t i a l l y CD H P l i g n i n , as s h o w n i n F i g u r e 1 (25). L i g n i n peroxidase a c t i v i t y , (i.e., peroxide-dependent o x i d a t i o n of ver a t r y l a l c o h o l at p H 3) was not detected over the 30 days tested, w h i l e laccase appeared at day 7. C u l t u r e m e d i u m f r o m d a y 7 onwards c o u l d also oxidize v e r a t r y l a l c o h o l to aldehyde w i t h c o n c o m i t a n t conversion o f oxygen to hydrogen peroxide. T h i s a c t i v i t y , w h i c h was o p t i m a l at p H 5.0, was n a m e d v e r a t r y l a l c o h o l oxidase ( V A O ) . T h e e x t r a c e l l u l a r o x i d a t i v e e n z y m e a c t i v i t i e s (laccase a n d v e r a t r y l a l c o h o l oxidase) c o u l d be separated b y ion-exchange c h r o m a t o g r a p h y ( F i g u r e 2). F u r t h e r c h r o m a t o g r a p h y o f the coincident laccase a n d v e r a t r y l a l c o h o l oxidase (peak 2), as described elsewhere (25) resulted i n the s e p a r a t i o n of two v e r a t r y l a l c o h o l oxidases f r o m the laccase. 1 4
Enzyme Properties. T h e two isolated v e r a t r y l a l c o h o l oxidases h a d very s i m i l a r properties ( T a b l e I). T h e difference i n isoelectric p o i n t s m i g h t be accounted for b y aspartate content; a l l other a m i n o a c i d contents except g l y c i n e were the same w i t h i n e x p e r i m e n t a l error (5%). T h e specific a c t i v i ties ( v e r a t r y l a l c o h o l as substrate) were significantly different, b u t b o t h e n zymes contained a flavin prosthetic group (25) a n d converted one molecule of oxygen to one molecule of hydrogen peroxide d u r i n g a l c o h o l o x i d a t i o n . T a b l e I. C o m p a r i s o n of V A O I a n d II ( D a t a s u m m a r i z e d f r o m ref. 25)
M o l e c u l a r weight Isoelectric p o i n t Glycosylated Asp/mole Gly/mole Specific a c t i v i t y , I U / m g
VAO I
V A O II
71,000 3.8 yes 83 77 35
71,000 4.0 yes 92 66 31
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0
5
10
15
20
25
475
30
Time, days F i g u r e 1. T i m e course of of l i g n i n o l y t i c a c t i v i t y (conversion of r i n g - l a b e l l e d C - D H P to C C > 2 ) , b i o m a s s c o n c e n t r a t i o n , V A O a c t i v i t y a n d laccase act i v i t y d u r i n g g r o w t h o f Pleurotus sajor-caju i n agitated mycological broth c u l t u r e s . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 25, © 1988, B i o c h e m i c a l Society). 1 4
14
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Fraction no. F i g u r e 2. E l u t i o n profile of P . sajor-caju s u p e r n a t a n t f r o m D E A E - B i o - g e l c o l u m n , s h o w i n g resolution of laccase a n d V A O peaks of a c t i v i t y .
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p-Methoxybenzyl alcohol was oxidised fastest of the substrates tested. Some relative rates of oxidation, measured as oxygen consumption, are shown in Figures 3 and 4. Methoxy substituted benzyl alcohols showed wide variations in their relative rates of oxidation (Figure 3) which are not easily explained by electron availability. Para-hydroxyl substitution severely inhibited oxidation (Figure 4), even in α, β unsubstituted aryl al cohols. Lignin model dimers, both phenolic and non-phenolic, and kraft lignin were not oxidised to any detectable extent. The enzyme properties reported above are similar to those of an aro matic alcohol oxidase from Polystictus versicolor (27). However, the latter enzyme had a different substrate specificity and the cultures did not pro duce laccase. Possible Role in Lignin Biodégradation. Oxidised products of veratryl alcohol have been proposed to play a key role in lignin peroxidase-mediated oxidation of lignin, either as one-electron carriers (14) or through further reaction which generates reactive oxygen species (28). Veratryl alcohol oxidases do not directly oxidize lignin, but they have a broad substrate range for monomeric aromatic alcohols and could therefore be involved in the final cat abolie steps following depolymerization. It seems unlikely however, that the resulting aromatic aldehydes would per se oxidize lignin itself. A possible reductive role for veratryl alcohol oxidase is proposed in Figure 5. Laccases from C. versicolor cam produce both polymerization and depolymerization of lignin (29). In phenolic lignin model dimers, laccase can perform the same electron abstraction and subsequent bond cleavage as found for lignin peroxidase (30). The phenolic radical is however likely to polymerize unless the quinoid-type intermediates can be removed, for example by reduction back to the phenol. Veratryl alcohol oxidase, in
CH OH 2
200 CH2OH
CH OH 2
100 CH OH 2
CH OH
CH OH
50
20
2
CH OH 2
2
CH OH 2
OCH3
Figure 3. Relative oxidation rates of various methoxyl-substituted benzyl alcohols, measured by oxygen consumption/min. (veratryl alcohol = 100). No oxidation of the lower four alcohols was detected.
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PLANT C E L L W A L L
POLYMERS
Figure 4. Relative oxidation rates of various aromatic alcohols. Para-hydroxyl substitution eliminates substrate oxidation.
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the presence of veratryl alcohol or other cosubstrate, could provide this reducing power, as has been proposed previously for glucose oxidase (31) and cellobiose quinone oxidoreductase (12). In support of this hypothesis, we find that veratryl alcohol oxidase in the presence of veratryl alcohol will decolorize the oxidised products produced by laccase from ABTS. A cknowledgment s We thank F. Lafortune for excellent technical contribution. This work was supported in part with financial assistance from the National Research Council of Canada under Contribution number 949-6-0011. Literature Cited 1. Kirk, T . K.; Cowling, Ε. B. The Chemistry of Solid Wood; Amer. Chem. Soc.: Washington, D.C., 1984, p. 455-87. 2. Tien, M.; Kirk, T . K. Science 1983, 221, 661-63. 3. Glenn, J . ; Morgan, Μ. Α.; Mayfield, M . B.; Kuwahara, M.; Gold, M . H. Biochem. Biophys. Res. Commun. 1983, 114, 1077-83. 4. Schoemaker, H. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J . M . FEBS Lett. 1985, 183, 7-12. 5. Kersten, P. J.; Tien, M.; Kalyanaraman, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 2609-12. 6. Miki, K.; Renganathan, V.; Gold, M . H. Biochemistry 1986, 26, 479096. 7. Kuwahara, M.; Glenn, J . K.; Morgan, Μ. Α.; Gold, M . H. FEBS Lett. 1984, 169, 247-50. 8. Paszczynski, Α.; Huynh, V. B.; Crawford, R. Arch. Biochem. Biophys. 1986, 244, 750-65. 9. Ramasamy, K.; Kelley, R. L.; Reddy, C. A. Biochem. Biophys. Res. Commun. 1985, 131, 436-41. 10. Eriksson, Κ. E.; Pettersson, B.; Vole, J.; Musilek, V. Appl. Microbiol. Biotechnol. 1986, 23, 257-62. 11. Kersten, P. J.; Kirk, T . K. J. Bacteriol. 1987, 169, 2195-2201. 12. Westermark, U.; Eriksson, Κ. E. Acta Chem. Scand. 1987, B29, 41924. 13. Haemmerli, S. D.; Leisola, M . S. Α.; Fiechter, A. FEMS Microbiol. Lett. 1986, 35, 33-36. 14. Harvey, P. J.; Schoemaker, Η. E.; Palmer, J . M . FEBS Lett. 1986, 195, 242-46. 15. Faison, B. D.; Kirk, T . K.; Farrell, R. L. Appl. Environ. Microbiol. 1986, 52, 251-54. 16. Dodson, P. J.; Evans, C. S.; Harvey, P. J.; Palmer, J . M . FEMS Mi crobiol. Lett. 1987, 42, 17-22. 17. Waldner, R.; Leisola, M.; Fiechter, A. Proceeding Biotechnology in Pulp and Paper Industry; 3rd Intl. Conf.; Stockholm 1986, 50-153. 18. Leatham, G . F.; Kirk, T . K. FEMS Microbiol. Lett. 1982, 16, 65-67. 19. Commanday, F.; Macy, J . M . Arch. Microbiol. 1985, 142, 61-65. 20. Mueller, J. C.; Gawley, J . R. Mushroom Newsletter Tropics 1983, 4, 3-12.
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21. Mueller, J. C.; Troesch, W. Appl. Microbiol. Biotechnol. 1986, 24, 18085. 22. Zadrazil, F. Eur. J. Appl. Microbiol. 1975, 1, 327-335. 23. Tsang, L. J.; Reid, I. D.; Coxworth, E . C. Appl. Environ. Microbiol. 1987, 53, 1304-06. 24. Bourbonnais, R.; Paice, M . G . J. Wood Chem. Tech. 1987, 7, 51-64. 25. Bourbonnais, R.; Paice, M . G . Biochem. J. 1988, 255, 445-50. 26. Wolfenden, B. S.; Willson, R. L. J. Chem. Soc. Perkin Trans. II 1982, 805-12. 27. Farmer, V.; Henderson, Μ. Ε. K.; Russell, J . D. Biochem. J. 1960, 257-62. 28. Haemmerli, S. D.; Schoemaker, H. E.; Schmidt, H. W. H.; Leisola, M . S. A. FEBS Lett. 1987, 220, 279-87. 29. Morohoshi, N.; Nakamura, M.; Katayama, Y.; Haraguchi, T . Pulping and Wood Chemistry Conf. Proceedings; Paris, 1987, 305-15. 30. Kawai, S.; Umezawa, T.; Shimada, A. M . ; Higuchi, T.; Koide, K.; Nishida, T.; Morohoshi, N.; Haraguchi, T . Mokuzai Gakkaishi 1987, 33, 792-97. 31. Green, T . R. Nature 1977, 268, 78-80. RECEIVED March 17, 1989
