Aromatic Ring Cleavage by Lignin Peroxidase - ACS Symposium

Chapter 36

Aromatic Ring Cleavage by Lignin Peroxidase Toshiaki Umezawa and Takayoshi Higuchi

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 19, 2016 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch036

Research Section of Lignin Chemistry, Wood Research Institute, Kyoto University, Uji, Kyoto 611, Japan

Aromatic ring cleavages by white-rot fungi and by the enzyme lignin peroxidase are reviewed. Intact cells of white-rot fungi cleave aromatic rings of lignin sub structure model compounds as well as polymeric lignin. Lignin peroxidase of Phanerochaete chrysosponum cat alyzes the ring cleavage of β-0-4 lignin substructure model compounds and synthetic lignin (DHP). A mech anism for the ring cleavage by the enzyme is described. Lignin biodégradation has been s t u d i e d by two c o m p l e m e n t a r y approaches: (i) d e g r a d a t i o n o f p o l y m e r i c lignins (such as the d e h y d r o g e n a t i o n p o l y m e r of coniferyl a l c o h o l ( D H P ) , m i l l e d w o o d l i g n i n , or w o o d per se) (1); a n d (ii) d e g r a d a t i o n o f l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s (2,3). I n the early 1980's, analysis o f l i g n i n isolated f r o m w o o d decayed b y w h i t e - r o t b a s i d iomycetes h a d p r o v i d e d a general o u t l i n e o f l i g n i n biodégradation. F o r e x a m p l e , cleavage o f side chains a n d a r o m a t i c rings o c c u r r e d d u r i n g degrad a t i o n o f p o l y m e r i c l i g n i n b y the f u n g i (1). L i g n i n is a c o m p l e x a n d heterogeneous p o l y m e r consisting o f p h e n y l propane u n i t s connected v i a m a n y C - C a n d C - O - C linkages. T h u s , the e l u c i d a t i o n o f specific reactions i n v o l v e d i n l i g n i n biodégradation has been p e r f o r m e d m a i n l y w i t h l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s as s u b s t r a t e for f u n g a l d e g r a d a t i o n . B y the early 1980's, s u b s t r u c t u r e m o d e l studies identified m a n y o f the degradative reactions suggested f r o m p o l y m e r i c l i g n i n biodégradation such as C a - C / ? cleavage o f p r o p y l s i d e - c h a i n a n d cleavage of β-0-4 bonds (2,3). O n the other h a n d , a r o m a t i c r i n g cleavage p r o d u c t s o f l i g n i n s u b s t r u c ture models b y w h i t e - r o t basidiomycetes were n o t identified u n t i l 1985, a l t h o u g h earlier studies o f the p o l y m e r i c l i g n i n d e g r a d a t i o n suggested t h e i n v o l v e m e n t o f r i n g cleavage reactions (1). W e identified for t h e first t i m e a r o m a t i c r i n g cleavage p r o d u c t s o f a β-0-4 l i g n i n s u b s t r u c t u r e m o d e l d i m e r

0097-6156/89/0399-0503$06.00/0 © 1989 American Chemical Society

Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

PLANT C E L L W A L L

504

POLYMERS


p r o d u c e d b y i n t a c t cells of the w h i t e - r o t b a s i d i o m y c e t e , Phanerochaete chrysosporium (4,5). Subsequently, we showed t h a t the a r o m a t i c r i n g cleav age of the d i m e r was c a t a l y z e d by a n e x t r a c e l l u l a r e n z y m e of the fungus, l i g n i n peroxidase (ligninase) (6-8), a n d proposed a m e c h a n i s m o f the r i n g cleavage b y the e n z y m e (9). T h e purpose of the present p a p e r is to describe the a r o m a t i c r i n g cleav age o f l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y w h i t e - r o t basidiomycetes a n d b y l i g n i n peroxidase of P. chrysosponum. T h e a r o m a t i c r i n g cleavage of s y n t h e t i c l i g n i n ( D H P ) b y the e n z y m e w i l l also be d e s c r i b e d . A r o m a t i c R i n g Cleavage iomycetes

b y Intact

Cells of W h i t e - R o t

Basid

E a r l i e r studies of fungus-degraded l i g n i n i s o l a t e d f r o m decayed w o o d s u g gested cleavage of a r o m a t i c rings of l i g n i n (1,10-15). However, the p r o d ucts o f a r o m a t i c r i n g cleavage of l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y w h i t e - r o t basidiomycetes were not identified u n t i l 1985 (4), w h i l e p r o d ucts of s i d e - c h a i n cleavage of l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y the f u n g i were identified earlier (2,3). D u r i n g t h i s s t u d y , we i d e n t i f i e d for the first t i m e a p r o d u c t o f a r o m a t i c r i n g cleavage o f β-0-4 l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d 1 b y P. chrysosponum, n a m e l y the ^ , γ - c y c l i c c a r b o n a t e of a r y l g l y c e r o l 8 (4) ( F i g . 1). Subsequently, several esters o f a r y l g l y c e r o l were identified as p r o d u c t s of a r o m a t i c r i n g cleavage of β-0-4 l i g n i n s u b s t r u c t u r e m o d e l d i m e r s 2 a n d 3 b y the fungus: a , / ? - c y c l i c c a r b o n a t e of a r y l g l y c e r o l 9, 7 - f o r m a t e of a r y l g l y c e r o l 11, a n d m e t h y l oxalate of a r y l g l y c e r o l 10 ( F i g . 1) (5). C - t r a c e r experiments w i t h l,3-dihydroxy-l-(4-ethoxy-3-methoxyphenyl)-2-[U-ring- C](2-methoxyphenoxy)propane and l,3-dihydroxy-l-(4ethoxy-3-methoxyphenol)-2-[U-ring- C](2,6-dimethoxyphenoxy)propane as substrates confirmed t h a t the p r o d u c t s were r i n g cleavage p r o d u c t s . 1 3

13

13

F o r m a t i o n of these a r o m a t i c r i n g cleavage p r o d u c t s was not l i m i t e d to P. chrysosporium, b u t c o u l d be m e d i a t e d b y other w h i t e - r o t b a s i d iomycetes, Coriolus versicolor (16) a n d Coriolus hirsutus (17). I n the d e g r a d a t i o n of 1, 2 a n d 4 b y C. versicolor, 8, 9, a n d 11 were i d e n t i fied as r i n g cleavage p r o d u c t s . A s for C. hirsutus, 8 a n d 9 were identified as r i n g cleavage p r o d u c t s f r o m d e g r a d a t i o n of 1 ( F i g . 1). A r o m a t i c R i n g C l e a v a g e o f /?-0-4 L i g n i n D i m e r s by L i g n i n Peroxidase

Substructure

Model

L e i s o l a et al. r e p o r t e d the a r o m a t i c r i n g cleavage of a m o n o m e r i c a r o m a t i c c o m p o u n d , v e r a t r y l a l c o h o l , b y l i g n i n peroxidase of P. chrysosporium (18), a l t h o u g h they s t a t e d t h a t the p r o d u c t s were o n l y t e n t a t i v e l y i d e n t i f i e d . W e showed, o n the basis of firm i d e n t i f i c a t i o n of the p r o d u c t s w i t h s y n t h e t i c a u t h e n t i c samples, t h a t l i g n i n peroxidase i s o l a t e d f r o m the fungus b y a m o d i f i c a t i o n of the m e t h o d of T i e n a n d K i r k (19), c a t a l y z e d the a r o m a t i c r i n g cleavage of β-0-4 l i g n i n s u b s t r u c t u r e m o d e l d i m e r s (6-8). A r o m a t i c r i n g cleavage p r o d u c t s formed i n the i n t a c t c u l t u r e of the fungus ( F i g . 1)



36.

UMEZAWA & HIGUCHI

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Aromatic Ring Cleavage by Peroxidase

505

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F i g u r e 1. β-0-4 l i g n i n s u b s t r u c t u r e m o d e l d i m e r s 1-4 a n d t h e i r d e g r a d a t i o n b y w h i t e - r o t f u n g i , Phanerochaete chrysosporium, Coriolus versicolor, a n d Coriolus hirsutus. T h e ether b o n d between t h e C/? a n d t h e B - a r o m a t i c nucleus is referred t o as "β-0-4 b o n d " i n l i g n i n c h e m i s t r y .


506

PLANT CELL WALL POLYMERS

were also p r o d u c e d b y t h e e n z y m e . A r o m a t i c r i n g cleavage p r o d u c t s w i t h the e n z y m e were c y c l i c carbonates o f a r y l g l y c e r o l s 8 a n d 9 , f o r m a t e o f a r y l g l y c e r o l 11, m e t h y l oxalates o f arylglycerols 10 a n d 1 0 - E t , a n d a novel p r o d u c t , muconate o f a r y l g l y c e r o l 1 2 - E t ( F i g . 2 ) . A l l t h e p r o d u c t s were confirmed t o b e a r o m a t i c r i n g cleavage p r o d u c t s b y u s i n g r i n g - C labeled substrates. Subsequently, M i k i et al. r e p o r t e d f o r m a t i o n o f a r o m a t i c r i n g cleavage p r o d u c t s i n v o l v i n g cyclic carbonates s i m i l a r t o 8 a n d 9 i n t h e d e g r a d a t i o n o f a β-0-4 l i g n i n m o d e l c o m p o u n d b y t h e e n z y m e (20). T h e muconate o f a r y l g l y c e r o l 1 2 - E t retains a l l s i x c a r b o n a t o m s o f the B - r i n g o f the substrate 1 - E t . Hence, t h i s p r o d u c t seemed t o be a p p r o p r i a t e t o e x a m i n e m e c h a n i s m s for t h e r i n g cleavage. B a s e d o n t h e results of tracer e x p e r i m e n t s , we proposed a r i n g cleavage m e c h a n i s m for t h e e n z y m e (7,9), as now described. D e g r a d a t i o n o f l , 3 - d i h y d r o x y l - l - ( 4 - e t h o x y 3 - m e t h o x y p h e n y l ) - 2 - ( 2 - [ O C H ] m e t h o x y p h e n o x y ) propane 1 - D b y t h e e n z y m e showed t h a t t h e m e t h y l group o f m e t h y l ester o f oxalate 1 0 - D w a s d e r i v e d f r o m the m e t h o x y l group o f the B - r i n g o f the s u b s t r a t e 1 - D ( F i g . 3) (7). T h i s result i n d i c a t e d t h a t d e m e t h y l a t i o n (or d e m e t h o x y l a t i o n ) w h i c h was p r e v i o u s l y p o s t u l a t e d for the r i n g cleavage b y t h e fungus (13) is n o t a prerequisite for the a r o m a t i c r i n g cleavage b y the enzyme, a n d t h a t the r i n g cleavage b y t h e enzyme is completely different f r o m the r i n g cleavage c a t a l y z e d b y c o n v e n t i o n a l dioxygenases (21). β-0-4 l i g n i n m o d e l c o m p o u n d s 2, 2 - M e , 2 - E t a n d 1 - E t were degraded under H 0 or 0 ( F i g . 4 ) , a n d G C - M S analysis o f the products showed t h a t one o f t h e c a r b o n y l oxygen a t o m s o f muconate 1 2 - E t a n d oxalates 10, 1 0 - M e a n d 1 0 - E t was d e r i v e d f r o m H 2 O a n d t h e other f r o m O 2 . A s for cyclic carbonates 8, 8 - M e a n d 9 a n d formates 11 a n d 1 1 - M e , the c a r b o n y l oxygen a t o m s were d e r i v e d f r o m H 2 O ( F i g . 4 ) . B a s e d o n these results, we proposed a m e c h a n i s m for a r o m a t i c r i n g cleavage by the enzyme w h i c h involves single electron o x i d a t i o n of the B - r i n g t o the corresponding c a t i o n r a d i c a l , followed b y n u c l e o p h i l i c a t t a c k o f H 2 O a n d r a d i c a l c o u p l i n g w i t h O2 (or a r a d i c a l species d e r i v e d f r o m 0 ( F i g . 5) (9).


1 3

2

3

2

1 8

1

8

2

2

S i m i l a r m e c h a n i s m s were also proposed for the r i n g cleavage o f v e r a t r y l a l c o h o l (22,23) a n d a β-0-4 l i g n i n s u b s t r u c t u r e m o d e l (24) b y the e n z y m e . A s described i n the previous section, the f o r m a t i o n o f the r i n g cleavage p r o d u c t s 8, 9 a n d 11 f r o m β-0-4 l i g n i n s u b s t r u c t u r e models were also m e d i a t e d b y C. versicolor (16) a n d C. hirsutus (17). It is most l i k e l y t h a t the a r o m a t i c r i n g cleavage b y b o t h f u n g i is m e d i a t e d b y a n e n z y m e s i m i l a r to l i g n i n peroxidase o f P. chrysosporium. Recently, i t w a s s h o w n t h a t C. versicolor produces a n enzyme s i m i l a r t o P. chrysosporium l i g n i n peroxidase (25), a l t h o u g h concrete evidence t h a t the enzyme catalyzes the r i n g cleavage has n o t been s h o w n . A r o m a t i c R i n g Cleavage o f a (/?-0-4)-(/?-0-4) L i g n i n S u b s t r u c t u r e M o d e l T r i m e r b y L i g n i n Peroxidase M e c h a n i s m s w h i c h involve t h e c a t i o n r a d i c a l i n t e r m e d i a t e were also p r o posed for t h e cleavage o f C a - C / ? a n d β-0-4 b o n d s o f β-0-4 l i g n i n s u b s t r u c ture models b y t h e enzyme (26,27). T h u s , m e c h a n i s m s for most o f t h e



E

t

o

ï

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OCHj

F i g u r e 2. β-0-i l i g n i n s u b s t r u c t u r e m o d e l d i m e r s 1, 2, 1 - E t a n d 2 - E t , a n d their d e g r a d a t i o n b y l i g n i n peroxidase of Phanerochaete chrysosporium.

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PLANT

CELL

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POLYMERS

F i g u r e 3. M e t h y l g r o u p of m e t h y l oxalate was derived f r o m m e t h o x y l g r o u p of the B - r i n g of β-0-4 l i g n i n m o d e l d i m e r 1-D.


UMEZAWA & HIGUCHI



36.


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F i g u r e 5. P r o p o s e d mechanisms for a r o m a t i c r i n g cleavage o f β-0-4 l i g n i n s u b s t r u c t u r e m o d e l dimers b y l i g n i n peroxidases.

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UMEZAWA & HIGUCHI


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d e g r a d a t i v e reactions of β-0-4 l i g n i n s u b s t r u c t u r e models b y the e n z y m e can be e x p l a i n e d o n the basis o f single electron o x i d a t i o n of a r o m a t i c r i n g s . O n the other h a n d , a r o m a t i c r i n g cleavage o f p o l y m e r i c l i g n i n by the e n z y m e has not been r e p o r t e d . T h e f o r m a t i o n o f B - r i n g cleavage p r o d u c t s of β-0-4 l i g n i n s u b s t r u c t u r e models b y the e n z y m e ( F i g . 2) was e x p e c t e d t o be a n i n d i c a t o r of a r o m a t i c r i n g cleavage i n p o l y m e r i c l i g n i n b y the e n z y m e . However, the β-0-4 l i g n i n m o d e l c o m p o u n d s used i n the m o d e l studies have no p r o p y l s i d e - c h a i n o n the B - r i n g as s h o w n i n F i g u r e s 1 a n d 2, w h i l e the B - r i n g s present i n p o l y m e r i c l i g n i n have p r o p y l s i d e - c h a i n s . F u r t h e r m o r e , p r e v i o u s i n v e s t i g a t i o n s showed t h a t the a r o m a t i c s u b s t i t u e n t s s u c h as f o r m y l or m e t h o x y l groups influenced d r a s t i c a l l y the d e g r a d a b i l i t y of the β-0-4 l i g n i n s u b s t r u c t u r e models b y the e n z y m e (6,26,27). H e n c e , we e x a m i n e d the effect of the p r o p y l s i d e - c h a i n of the B - r i n g o n the B - r i n g cleavage b y the e n z y m e u s i n g a l i g n i n s u b s t r u c t u r e m o d e l t r i m e r c o m p o s e d of two β-0-4 s u b s t r u c t u r e s 5 ( F i g . 6) (28). Identified d e g r a d a t i o n p r o d u c t s b y the e n z y m e were as follows ( F i g . 6): C y c l i c c a r b o n a t e s a n d f o r m a t e of a r y l g l y c e r o l as B - r i n g cleavage p r o d u c t s 8 , 9 a n d 11; a r y l g l y c e r o l 13 a n d i t s α - c a r b o n y l d e r i v a t i v e 14 as cleavage p r o d u c t s of the β-0-4 b o n d between A - a n d B - r i n g s ; 4 - e t h o x y - 3 - m e t h o x y b e n z a l d e h y d e 15 as a C a - C / ? cleavage p r o d u c t between A - a n d B - r i n g s ; a n d l , 3 - d i h y d r o x y - l - ( 4 - e t h o x y 3 - m e t h o x y p h e n y l ) - 2 - ( 4 - f o r m y l - 2 - m e t h o x y p h e n o x y ) p r o p a n e 3 as a p r o d u c t of C a - C / ? cleavage between B - a n d C - r i n g s . T h e results showed t h a t the p r o p y l s i d e - c h a i n o f the B - r i n g of 5 d i d not influence, at least q u a l i t a t i v e l y , the a r o m a t i c r i n g cleavage b y the e n z y m e . T h e results suggested t h a t p o l y m e r i c l i g n i n is degraded t h r o u g h the cleavage r e a c t i o n of a r o m a t i c rings by the e n z y m e . I n fact, a r o m a t i c r i n g cleavage o f D H P ( s y n t h e t i c l i g n i n ) b y the e n z y m e has now been p r o v e n as discussed i n the f o l l o w i n g s e c t i o n . A r o m a t i c R i n g Cleavage of D H P by L i g n i n Peroxidase R e a c t i o n m e c h a n i s m s for d e g r a d a t i o n of β-0-4 l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y l i g n i n peroxidase are b e i n g clarified r a p i d l y , w h i l e the ac t i o n of the e n z y m e o n p o l y m e r i c l i g n i n is not f u l l y e l u c i d a t e d . T i e n a n d K i r k (29) r e p o r t e d d e g r a d a t i o n of m e t h y l a t e d spruce l i g n i n b y the e n z y m e . T h e y showed f o r m a t i o n of low m o l e c u l a r weight d e g r a d a t i o n p r o d u c t s o n the basis of gel f i l t r a t i o n w i t h Sephadex L H - 2 0 , a n d detected a C a - C / ? cleavage p r o d u c t b y a n isotope t r a p p i n g m e t h o d (29). O n the other h a n d , a c c o r d i n g to H a e m m e r l i et α/., w h e n p o l y m e r i c l i g n i n , p h e n o l i c h y d r o x y l groups of w h i c h are not a l k y l a t e d , were t r e a t e d b y the e n z y m e , p o l y m e r i z a t i o n o f the l i g n i n o c c u r r e d o n the basis o f gel filtration a n a l y s i s o f the d e g r a d a t i o n p r o d u c t s (30). T h e y m o n i t o r e d the c h r o m a t o g r a p h i c profile b y a U V - d e t e c t o r . T h e results were confirmed b y O d i e r et ai (31) b y u s i n g C - l a b e l l e d D H P as s u b s t r a t e . T h u s , for the d e g r a d a t i o n of p o l y m e r i c l i g n i n by the e n z y m e , t w o m a j o r questions were left: (i) C a n l i g n i n peroxidase, b y itself, d e p o l y m e r i z e p o l y m e r i c l i g n i n w i t h o u t r e p o l y m e r i z a t i o n or n o t ? (ii) C a n l i g n i n p e r o x idase cleave a r o m a t i c rings a n d β-0-4 b o n d s o f p o l y m e r i c l i g n i n , or n o t ? 1 4


Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989. 15

OCH-3

OEt

HO

OEt J

Ca-C0

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cleavage

cleavage

cleavage

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H O ^ O H

Γ

F i g u r e 6. (/?-0-4)-(/?-0-4) l i g n i n s u b s t r u c t u r e m o d e l t r i m e r 6 a n d i t s d e g r a d a t i o n p r o d u c t s b y l i g n i n peroxidase.

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CHO

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36.

UMEZAWA & HIGUCHI


513

Since we have been i n v e s t i g a t i n g the a r o m a t i c r i n g cleavage of β-0-4 l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y i n t a c t cells o f w h i t e - r o t f u n g i a n d l i g n i n peroxidase of P. chrysosporium as described above, we e x a m i n e d the a r o m a t i c r i n g cleavage a n d β-0-4 b o n d cleavage o f p o l y m e r i c l i g n i n b y l i g n i n peroxidase. W e s y n t h e s i z e d a c o p o l y m e r o f (β-0-4)-(β-β) lignin substructure model t r i m e r 6 a n d c o n i f e r y l a l c o h o l 7, w h i c h w i l l be referred t o as (β-0-4)-(β-β)D H P i n t h i s review ( F i g . 7). T h e (β-0-4)-(β-β)-ΌΕΡ was e t h y l a t e d w i t h d i a z o e t h a n e a n d the e t h y l a t i o n p r o d u c t 16 was s u b m i t t e d to gel filtration ( L H - 2 0 / D M F ) to remove low m o l e c u l a r weight f r a c t i o n s . T h e h i g h m o l e c u l a r weight f r a c t i o n o f the e t h y l a t e d (β-0-4)-(β-β)-ΌΕΡ 16 t h u s o b t a i n e d ( m o l e c u l a r weight > 2200, c a l i b r a t e d w i t h p o l y s t y r e n e , F i g . 8) was de g r a d e d b y l i g n i n peroxidase. T h e d e g r a d a t i o n p r o d u c t s were e x t r a c t e d w i t h e t h y l acetate, a c e t y l a t e d a n d p a r t i a l l y p u r i f i e d by t h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) . G a s c h r o m a t o g r a p h y mass spectroscopic ( G C - M S ) a n a l y s i s o f the T L C p u r i f i e d f r a c t i o n showed the f o r m a t i o n o f c y c l i c c a r b o n a t e s o f a r y l g l y c e r o l 8 a n d 9, f o r m a t e o f a r y l g l y c e r o l 11, a r y l g l y c e r o l 13, a n d o> c a r b o n y l - a r y l g l y c e r o l 14 ( F i g . 9) (32). Since the c o m p o u n d s were p r e v i o u s l y i d e n t i f i e d as a r o m a t i c r i n g cleavage p r o d u c t s a n d β-0-4 b o n d c l e a v age p r o d u c t s o f β-0-4 l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y the e n z y m e ( F i g s . 2 a n d 6), the results show t h a t l i g n i n peroxidase cleaves a r o m a t i c rings a n d β-0-4 bonds of the s y n t h e t i c l i g n i n ( D H P ) . T h u s , m o s t of the d e g r a d a t i v e reactions o f p o l y m e r i c l i g n i n s suggested p r e v i o u s l y b y the a n a l y s i s o f decayed l i g n i n i s o l a t e d f r o m decayed w o o d b y w h i t e - r o t f u n g i were c a t a l y z e d by l i g n i n peroxidase. A r o m a t i c R i n g Cleavage of M o n o m e r i c A r o m a t i c C o m p o u n d s by W h i t e - R o t Basidiomycetes M o n o m e r i c a r o m a t i c c o m p o u n d s s u c h as v a n i l l i c a c i d a n d s y r i n g i c a c i d are k n o w n t o be p r o d u c e d i n the d e g r a d a t i o n of p o l y m e r i c l i g n i n b y w h i t e - r o t basidiomycetes (13,14,33). It is s t i l l u n c e r t a i n to w h a t extent a r o m a t i c r i n g s o f p o l y m e r i c l i g n i n are cleaved b y l i g n i n peroxidase, as opposed to b e i n g degraded v i a s i d e c h a i n cleavages to produce the m o n o m e r i c C 6 - C 1 i n t e r m e d i a t e s s u c h as v a n i l l i c a c i d a n d s y r i n g i c a c i d . F u r t h e r m o r e , the fate o f the m o n o m e r i c d e g r a d a t i o n i n t e r m e d i a t e s is not f u l l y e l u c i d a t e d . A n d e r et ai (34) p r o p o s e d a d e g r a d a t i o n p a t h w a y o f v a n i l l i c a c i d v i a 1,2,4t r i h y d r o x y b e n z e n e b y i n t a c t cells o f Sporotrichum pulverulentum ( = P. chrysosporium). T h i s p a t h w a y involves d e c a r b o x y l a t i o n o f v a n i l l i c a c i d c a t a l y z e d b y v a n i l l a t e h y d r o x y l a s e to p r o d u c e m e t h o x y h y d r o q u i n o n e (35,36), followed b y d e m e t h y l a t i o n a n d subsequent a r o m a t i c r i n g cleavage o f the d e m e t h y l a t e d p r o d u c t , 1,2,4-trihydroxybenzene, c a t a l y z e d b y a d i o x y g e nase o f the fungus (37). O n the other h a n d , the role o f l i g n i n peroxidase i n the m e t a b o l i s m o f these a r o m a t i c m o n o m e r s r e m a i n s u n c e r t a i n , a l t h o u g h a n o n - p h e n o l i c a r o m a t i c m o n o m e r , v e r a t r y l a l c o h o l w h i c h is s y n t h e s i z e d de novo b y P. chrysosporium, is o x i d i z e d b y l i g n i n peroxidase to p r o d u c e m a i n l y v e r a t r a l d e h y d e a n d trace a m o u n t s of a r o m a t i c r i n g cleavage p r o d ucts (18,22).


Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989. >

3

CH CHN 2

OEt

J-LH-20/DMF

2

High molecular weight fraction

2

ÇHOH 0CH3 ÇH20H CHOH C H — 0 — C H ÇH-0-^p-CH-OH CHO-i ÇHOHOCH3 (^LocH3

F i g u r e 7. P r e p a r a t i o n of s y n t h e t i c l i g n i n 1 6 , e t h y l a t e d c o p o l y m e r o f c o n i f e r y l alcohol 7 a n d a r y l g l y c e r o l - / ? - s y r i n g a r e s i n o l ether 6. In 1 6 , the r e c t a n g u l a r enclosure represents a n assumed s t r u c t u r e of the m o i e t y der i v e d f r o m 7.

OCH.

v

Horseradish peroxidase

3

CH 0^ 2

CH OH -ÇH ÇHOH



36.

UMEZAWA & HIGUCHI


515

Void

-J

i*2

1

1

1

35

28

21

Ml—ι

ία

0

Elution volume (ml) F i g u r e 8. G e l f i l t r a t i o n o f e t h y l a t e d (/?-0-4)-(/?-/?)-DHP 16. S o l i d l i n e : E t h y l a t e d (/?-0-4)-(/?-/?)-DHP 16 after removal o f low m o l e c u l a r weight f r a c t i o n s . T h e c o l u m n w a s c a l i b r a t e d w i t h (/?-0-4)-(/?-/?) l i g n i n s u b s t r u c t u r e m o d e l t r i m e r 6 ( m o l e c u l a r weight 6 4 2 ) ; /3-0-4 l i g n i n m o d e l d i m e r 1 ( m o l e c u l a r weight 348) a n d polystyrenes o f m o l e c u l a r weight 9000, 4000 ( v o i d ) , 2200 ( i n d i c a t e d b y A ) . C o l u m n : Sephadex L H - 2 0 , 1.1 x 48 c m . E l u e n t : D M F , 13.5-14.4 m l / h r . D e t e c t o r : R e f r a c t i v e i n d e x detector R I - 2 ( J a p a n A n a l y t i cal I n d u s t r y C o . , L t d . ) .



F i g u r e 9. D e g r a d a t i o n p r o d u c t s of s y n t h e t i c l i g n i n ( D H P ) 1 6 b y l i g n i n peroxidase. 8 , 9 a n d 1 1 : A r o m a t i c r i n g cleavage p r o d u c t s ; 1 3 a n d 1 4 : /?0-4 b o n d cleavage p r o d u c t s .


36. UMEZAWA & HIGUCHI


517


Literature Cited 1. Chen, C.-L.; Chang, H.-m. In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic: Orlando, FL, 1985; pp. 53556. 2. Higuchi, T . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic: Orlando, F L , 1985; pp. 557-78. 3. Higuchi, T . Wood Res. 1986, 73, 58. 4. Umezawa, T.; Higuchi, T . FEBS Lett. 1985, 182, 257. 5. Umezawa, T.; Kawai, S.; Yokota, S.; Higuchi, T . Wood Res. 1986, 73, 8. 6. Umezawa, T.; Shimada, M.; Higuchi, T.; Kusai, K. FEBS Lett. 1986, 205, 287. 7. Umezawa, T.; Higuchi, T . FEBS Lett. 1986, 205, 293. 8. Umezawa, T.; Higuchi, T . Agric. Biol. Chem. 1987, 51, 2281. 9. Umezawa, T.; Higuchi, T . FEBS Lett. 1987, 218, 255. 10. Kirk, T . K.; Chang, H.-m. Holzforschung 1975, 29, 56. 11. von Ellwardt, P.-C.; Haider, K.; Ernst, L. Holzforschung 1981, 35, 103. 12. Chua, M . G. S.; Chen, C.-L.; Chang, H.-m.; Kirk, T . K. Holzforschung 1982, 36, 165. 13. Chen, C.-L.; Chang, H.-m.; Kirk, T . K. J. Wood Chem. Technol. 1983, 3, 35. 14. Tai, D.; Terazawa, M.; Chen, C.-L.; Chang, H.-m.; Kirk, T . K. In Re cent Advances in Lignin BiodegradationResearch;Higuchi, T.; Chang, H.-m.; Kirk, T . K., Eds.; Uni Publishers: Tokyo, 1983; pp. 44-63. 15. Haider, K.; Kern, H. W.; Ernst, L. Holzforschung 1985, 39. 16. Kawai, S.; Umezawa, T.; Higuchi, T . Appl. Environ. Microbiol. 1985, 50, 1505. 17. Yoshihara, K.; Umezawa, T.; Higuchi, T.; Nishiyama, M. Agric. Biol. Chem. 1988, 52, 2345. 18. Leisola, M . S. Α.; Schmidt, B.; Thanei-Wyss, U.; Fiechter, A. FEBS Lett. 1985, 189, 267. 19. Tien, M.; Kirk, T . K. Proc. Natl. Acad. Sci. USA 1984, 81, 2280. 20. Miki, K.; Renganathan, V.; Mayfield, M . B.; Gold, M . H. FEBS Lett. 1987, 210, 199. 21. Cain, R. B. In Lignin Biodegradation: Microbiology, Chemistry, and Potential Applications; Kirk, T . K.; Higuchi, T.; Chang, H.-m., Eds.; CRC Press: Boca Raton, FL, 1980; Vol. 1, pp. 21-60. 22. Shimada, M.; Hattori, T.; Umezawa, T.; Higuchi, T.; Uzura, K. FEBS Lett. 1987, 221, 327. 23. Haemmerli, S. D.; Schoemaker, H. E.; Schmidt, H. W. H.; Leisola, M . S. A. FEBS Lett. 1987, 220, 149. 24. Miki, K.; Kondo, R.; Renganathan, V.; Mayfield, M . B.; Gold, M . H. Biochem. 1988, 27, 4787. 25. Dodson, P. J.; Evans, C. S.; Harvey, P. J.; Palmer, J . M . FEMS Mi crobiol. Lett. 1987, 42, 17. 26. Kirk, T . K.; Tien, M.; Kersten, P. J.; Mozuch, M . D.; Kalyanaraman, B. Biochem. J. 1986, 236, 279.


518 27. 28. 29. 30.


31.

32. 33. 34. 35. 36. 37.

PLANT CELL WALL POLYMERS Miki, K.; Renganathan, V.; Gold, M . H. Biochem. 1986, 25, 4790. Umezawa, T.; Higuchi, T . Mokuzai Gakkaishi 1988, 34, 929. Tien, M.; Kirk, T . K. Science 1983, 221, 661. Haemmerli, S. D.; Leisola, M . S. Α.; Fiechter, A. FEMS Microbiol. Lett. 1986, 35, 33. Odier, E.; Mozuch, M.; Kalyanaraman, B.; Kirk, T . K. In Lignin En zymic and Microbial Degradation; Odier, E . , Ed.; INRA: Paris, 1987; p. 131. Umezawa, T.; Higuchi, T . FEBS Lett. 1989, 242, 325. Chen, C.-L.; Chang, H.-m.; Kirk, T . K. Holzforschung 1982, 36, 3. Ander, P.; Eriksson, K.-E.; Yu, H.-s. Arch. Microbiol. 1983, 136, 1. Buswell, J. Α.; Ander, P.; Pettersson, B.; Eriksson, K . - E . FEBS Lett. 1979, 103, 98. Yajima, Y.; Enoki, Α.; Mayfield, M . B.; Gold, M . H. Arch. Microbiol. 1979, 123, 319. Buswell, J. Α.; Eriksson, K.-E. FEBS Lett. 1979, 104, 258.

RECEIVED March 10,1989


Aromatic Ring Cleavage by Lignin Peroxidase - ACS Symposium

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