Mechanisms of Lignin Degradation by Lignin Peroxidase and Laccase

Chapter 35

Mechanisms of Lignin Degradation by Lignin Peroxidase and Laccase of White-Rot Fungi Takayoshi Higuchi Downloaded by PENNSYLVANIA STATE UNIV on August 29, 2013 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch035

Wood Research Institute, Kyoto University, Uji, Kyoto 611, Japan

The main cleavage mechanisms of side-chains and aro matic rings of lignin model compounds and synthetic lignin (DHP) by lignin peroxidase and laccase of white -rot fungi have been elucidated. Tracer studies using H-, C- and O-labeled arylglycerol-β-aryl ethers and diarylpropane-1,3-diols with O and H 18O indicated that side-chains and aromatic rings of these substrates were cleaved via aryl radical cation and phenoxy radi cal intermediates, in reactions mediated only by lignin peroxidase/HO and laccase/O. 2

13

18

18

2

2

2

2

2

The process of lignin biodégradation by microbes has evoked much interest in recent years (1-3). Several o f these studies have been d i r e c t e d t o w a r d s e s t a b l i s h i n g the exact c h e m i c a l m e c h a n i s m s i n v o l v e d i n l i g n i n sidec h a i n cleavage a n d a r o m a t i c r i n g - o p e n i n g reactions, w h i c h are c a t a l y z e d b y l i g n i n peroxidase (4,5) a n d laccase (6). A l m o s t a l l o f these studies n o r m a l l y employ, as l i g n i n - l i k e substrates, various d i m e r i c phenolic o r n o n phenolic m o d e l c o m p o u n d s w h i c h contain b o n d i n g p a t t e r n s c h a r a c t e r i s t i c of s u b s t r u c t u r e s k n o w n t o be present i n isolated l i g n i n p r e p a r a t i o n s . F o r e x a m p l e , u s i n g β A a n d β-ΟΆ m o d e l c o m p o u n d s , i t has been established t h a t b o t h l i g n i n peroxidase a n d laccase c a t a l y z e one electron o x i d a t i o n s (5-7). I n p a r t i c u l a r , l i g n i n peroxidase converts b o t h phenolic a n d n o n phenolic moieties t o their corresponding p h e n o x y r a d i c a l a n d a r y l r a d i c a l c a t i o n i n t e r m e d i a t e s , whereas laccase o n l y catalyzes p h e n o x y r a d i c a l for m a t i o n f r o m phenolic substrates; n o n p h e n o l i c c o m p o u n d s are n o t o x i d i z e d (8). T h e a r y l r a d i c a l cations (formed f r o m n o n p h e n o l i c m o d e l c o m p o u n d s by l i g n i n p e r o x i d a s e / ! ^ O 2 ) t h e n undergo n u c l e o p h i l i c a t t a c k (e.g., b y H 2 O , or h y d r o x y l groups o n adjacent functionalities) t o generate a r y l r a d i c a l i n termediates. S u c h reactive species, as w e l l as t h e p h e n o x y r a d i c a l s afore m e n t i o n e d , c a n t h e n react w i t h dioxygen r a d i c a l s o r a l t e r n a t i v e l y they c a n

0097-6156/89/0399-0482$06.00A) © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Downloaded by PENNSYLVANIA STATE UNIV on August 29, 2013 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch035

35.

HIGUCHI

483

Lignin Degradation by Peroxidase & Laccase

undergo free r a d i c a l c o u p l i n g reactions. T h u s t w o c o m p e t i n g reactions c a n o c c u r d u r i n g l i g n i n biodégradation: (1) r e a c t i o n w i t h d i o x y g e n r a d i c a l s res u l t i n g i n l i g n i n d e g r a d a t i o n a n d (2) free r a d i c a l c o u p l i n g l e a d i n g t o r e p o l y m e r i z a t i o n . T h e s e findings help e x p l a i n the current difficulties e x p e r i e n c e d i n efficiently d e g r a d i n g the l i g n i n p o l y m e r in vitro w i t h i s o l a t e d e n z y m e p r e p a r a t i o n s (1). A t t h i s j u n c t u r e , i t is p e r t i n e n t to briefly describe o u r c u r r e n t u n d e r s t a n d i n g of l i g n i n f o r m a t i o n i n x y l e m cell w a l l s (9). I n i t i a l l y , free r a d i c a l c o u p l i n g o f i n t e r m e d i a t e s , f o r m e d f r o m the c o r r e s p o n d i n g m o n o l i g n o l s v i a m e d i a t i o n o f cell w a l l peroxidases a n d H 2 O 2 , affords d i m e r i c q u i n o n e m e t h i d e s ; subsequent n u c l e o p h i l i c a t t a c k (e.g., b y H 2 O or adjacent h y d r o x y l groups) results i n r e a r o m a t i z a t i o n to afford d i l i g n o l s , s u c h as deh y d r o d i c o n i f e r y l a l c o h o l , d , l - p i n o r e s i n o l , g u a i a c y l g l y c e r o l - / ? - c o n i f e r y l ether, etc. T h e s e phenols c a n t h e n be reconverted i n t o t h e i r free r a d i c a l f o r m s , w h i c h t h e n ( i n a n analogous m a n n e r ) undergo c o u p l i n g a n d r e a r o m a t i z a t i o n to give the c o r r e s p o n d i n g oligolignols. R e p e t i t i o n o f these reactions results i n l i g n i n f o r m a t i o n ; t h i s process is often described as d e h y d r o g e n a s e p o l y m e r i z a t i o n ( F i g . 1). I n o u r o p i n i o n , the reactions i n v o l v e d i n l i g n i n biodégradation (as c a t a l y z e d b y l i g n i n peroxidase a n d laccase) c a n be c o n sidered as a n e x t e n s i o n of the reactions encountered i n l i g n i n f o r m a t i o n . W e propose t h i s i d e a since, as discussed l a t e r , i t appears t h a t d e g r a d a t i o n of the l i g n i n p o l y m e r involves s i m i l a r reactions. In t h i s c h a p t e r , some o f the m a i n reactions i n l i g n i n biodégradation (i.e., s i d e - c h a i n cleavage a n d a r o m a t i c r i n g - o p e n i n g ) are d e s c r i b e d , a n d the effects of b o t h laccase a n d l i g n i n peroxidase c o m p a r e d . T h e schemes p r o p o s e d are a l l based o n identified p r o d u c t s f r o m m o d e l c o m p o u n d s a n d s u p p o r t e d wherever possible b y a p p r o p r i a t e l a b e l l i n g s t u d i e s . F i n a l l y , the processes of l i g n i n biosynthesis a n d biodégradation are c o m p a r e d . Results and Discussion Side-Chain Cleavage Reactions of β-l and β-0-4 Lignin Model Compounds Catalyzed by Lignin Peroxidase. β-l Model Compounds. T h e s e can be separated i n t o two b r o a d c a t egories: n o n p h e n o l i c a n d p h e n o l i c . A s can be seen f r o m F i g u r e 2, the n o n p h e n o l i c β-l l i g n i n m o d e l c o m p o u n d 1 is first converted i n t o i t s a r y l r a d i c a l c a t i o n i n t e r m e d i a t e . T h i s t h e n undergoes C a - C / ? h o m o l y t i c c l e a v age to afford 3 , 4 , 5 - t r i m e t h o x y b e n z a l d e h y d e 2, a n d the d i o l 3, w i t h the l a t t e r t r a p p i n g oxygen as s h o w n b y a p p r o p r i a t e l a b e l l i n g e x p e r i m e n t s w i t h C>2 ( F i g . 2) (4,10). T h e g e n e r a l i t y of t h i s m e c h a n i s m was d e m o n s t r a t e d u s i n g a n u m b e r of methoxybenzenes a n d d i a r y l e t h a n e d i m e r s as s u b s t r a t e s (7,11). R e a c t i o n progress was m o n i t o r e d b y E S R s p e c t r o m e t r y (11); m e c h a n i s m s were f u r t h e r established by u s i n g m o d e l c o m p o u n d s specifically d e u t e r a t e d at C a a n d C/? as r e q u i r e d (12). W h i l e the c o r r e s p o n d i n g p h e n o l i c β-l m o d e l c o m p o u n d s are also s u b j e c t to C a - C / ? cleavage, other reactions can also occur (13). F i g u r e 3 s u m marizes the m a i n reactions i d e n t i f i e d to t h i s p o i n t . These i n c l u d e (i) C a - C / ? cleavage of 4 to afford s y r i n g a l d e h y d e 6 a n d the d i o l , 7, (ii) C a o x i d a t i o n a n d d e h y d r a t i o n t o give the ketone 5 a n d (iii) a l k y l - a r y l cleavage t o give the d e g r a d a t i o n p r o d u c t s 8 a n d 9 respectively (13). 18



Figure 1. Formation of guaiacyl lignin and lignin-carbohydrate complexes ( L C C ) via dehydrogenase polymerization of coniferyl alcohol.



35.

HIGUCHI


485

Figure 2. Side-chain cleavage of a nonphenolic β A model compound 1 by lignin peroxidase.



C a - Ο β CLEAVAGE

9

IALKYL-ARYL CLEAVAGE

F i g u r e 3. P o s s i b l e d e g r a d a t i o n p a t h w a y s o f the p h e n o l i c 0-\ m o d e l c o m p o u n d 4 b y l i g n i n peroxidase.

OCH.

4


S3

35.

HIGUCHI


487

β-0-4 Model Compounds. Investigations were c o n d u c t e d u s i n g b o t h n o n p h e n o l i c a n d phenolic l i g n i n m o d e l s u b s t r a t e s . S t u d i e s (12,14,15) w i t h the n o n p h e n o l i c d i m e r 10 revealed t h a t four m a i n r e a c t i o n p a t h w a y s were possible: (i) b e n z y l i c o x i d a t i o n ( C a ) to give the c o r r e s p o n d i n g ketone (not s h o w n ) ; (ii) f o r m a t i o n of the a r y l r a d i c a l c a t i o n i n t e r m e d i a t e as before, f o l lowed b y f r a g m e n t a t i o n to afford g u a i a c o l 11, the g l y c o l i c aldehyde 12 a n d 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 13 ( F i g . 4, p a t h w a y i i ) ; ( i i i ) a r y l r a d i c a l c a t i o n f o r m a t i o n , subsequent i n t e r m o l e c u l a r n u c l e o p h i l i c a t t a c k b y the 7 h y d r o x y l g r o u p a n d r e a r r a n g e m e n t , to give the isomeric d i o l 14 followed b y h o m o l y t i c cleavage to afford the aldehyde 15 a n d 2 - ( 2 - m e t h o x y p h e n o l ) ace t a l d e h y d e 16. These u n u s u a l reactions were confirmed b y l a b e l l i n g studies as s h o w n ( F i g . 4, p a t h w a y i i i ) , a n d (iv) d i s p l a c e m e n t a n d / o r a r o m a t i c r i n g d i s p l a c e m e n t t o y i e l d the t r i o l 19 a n d o-quinone 20 ( F i g . 4, p a t h w a y i v ) (3).


/

Studies (16) e m p l o y i n g p h e n o l i c β-0-4 m o d e l substrates revealed three m a j o r reactions, reminiscent of those p r e v i o u s l y described i n F i g u r e 3, i.e., (i) b e n z y l i c o x i d a t i o n , (ii) C a - C / ? cleavage to give either the c o r r e s p o n d i n g C a aldehyde or a c i d ; a n d ( i i i ) a l k y l - a r y l b o n d r u p t u r e to give the h y d r o q u i n o n e (cf. 8, F i g . 3) a n d the c o r r e s p o n d i n g g l y c e r a l d e h y d e - 2 - a r y l ether. Side Chain

Cleavage

of β-l

and β-0-4

Model Compounds

by

Laccase.

β-l Model Compounds. O n l y substrates w i t h p h e n o l i c f u n c t i o n a l i t i e s were e x a m i n e d , since n o n p h e n o l i c substrates were unaffected. T h u s , the two p h e n o l i c diols 4 a n d 21 were synthesized (6), a n d b o t h were i n d i v i d u a l l y exposed to laccase f r o m Coriolus versicolor (6). F o r d i o l 4, a l k y l a r y l cleavage gave 2 , 6 - d i m e t h o x y h y d r o q u i n o n e 8, the c o r r e s p o n d i n g b e n z o q u i n o n e 23 a n d the aldehyde 24 ( F i g . 5, p a t h w a y B ) ; C a o x i d a t i o n also afforded the ketone 22 ( p a t h w a y A ) . A d d i t i o n a l l y , other reactions were also observed ( F i g . 6), n a m e l y C a - C / ? cleavage to give s y r i n g a l d e h y d e 6, a n d the a r y l k e t o - a l c o h o l 25 ( F i g . 6, p a t h w a y A ) . O n the other h a n d , d i o l 21 o n l y u n d e r w e n t C a - C / ? cleavage, g e n e r a t i n g the s u b s t i t u t e d b e n z a l d e h y d e 27 a n d the p h e n y l g l y c o l 26 ( F i g . 6, p a t h w a y B ) . N o t e t h a t these m e c h a n i s m s were based u p o n a p p r o p r i a t e l a b e l l i n g studies u s i n g 0 (6). T h u s , reactions i n v o l v i n g p h e n o l i c substrates, a n d u s i n g b o t h l i g n i n peroxidase a n d laccase are s i m i l a r ; b o t h encompass C a - C / ? , a l k y l - a r y l b o n d cleavages a n d o x i d a t i v e reactions. 1

8

2

β-Ο-4 Model Compounds. I n t h i s i n v e s t i g a t i o n , the s u b s t r a t e used for i n c u b a t i o n w i t h laccase was the m o d e l c o m p o u n d , s y r i n g y l g l y c e r o l - / ? g u a i a c y l ether 28 (18). It was i n i t i a l l y r e p o r t e d t h a t t h i s u n d e r w e n t conver sion i n t o g l y c e r a l d e h y d e - 2 - g u a i a c y l ether a n d 2 , 6 - d i m e t h o x y b e n z o q u i n o n e (17). A d d i t i o n a l l y , the h y d r o x y l group of the h y d r o q u i n o n e was l a b e l l e d with 0 , w h e n the e x p e r i m e n t was c a r r i e d o u t i n the presence of H 2 0 . H o w e v e r , more comprehensive studies (18) have d e m o n s t r a t e d t h a t the d i m e r 28 also undergoes b e n z y l i c o x i d a t i o n to give the ketone 29, a n d Ca-C β cleavage to afford g u a i a c o l 11 a n d s y r i n g i c a c i d 30 ( F i g . 7). T h u s , once a g a i n , the reactions c a t a l y z e d b y laccase (for these p h e n o l i c substrates) show considerable resemblance to t h a t a l r e a d y noted for l i g n i n peroxidase. 1 8

1 8


POLYMERS


PLANT C E L L W A L L


HIGUCHI



35.


489


F i g u r e 5. P o s s i b l e m e c h a n i s m s for C a o x i d a t i o n ( A ) a n d a l k y l - a r y l cleavage ( B ) reactions of a phenolic β-l m o d e l c o m p o u n d 4 b y laccase.



F i g u r e 6. P o s s i b l e m e c h a n i s m s for Ca-C β cleavage o f p h e n o l i c β-l c o m p o u n d s 4 a n d 2 1 b y laccase.

model



492

PLANT C E L L W A L L POLYMERS

30

F i g u r e 7. S i d e - c h a i n cleavage o f a phenolic β-0-4 laccase.

model compound 28 by


35.

HIGUCHI

Aromatic Ring Cleavage of Nonphenolic Lignin Substructure Model Compounds and Veratryl Alcohol by Lignin Peroxidase. β-Ο-4 Model Compounds. T h r e e m o d e l c o m p o u n d s 31-33 were s y n thesized (19,20), a n d treated w i t h l i g n i n peroxidase as before. T h i s r e s u l t e d i n t h e i r conversion i n t o t h e a r y l g l y c e r o l c y c l i c c a r b o n a t e s 37-39, the O - f o r m a t e s 40,41, a n d t h e m e t h y l o x a l a t e dérivâtes 34-36. A f o u r t h m o d e l c o m p o u n d 42 was also s y n t h e s i z e d , a n d t h i s u n d e r w e n t d e g r a d a t i o n t o give t h e m e t h y l o x a l a t e d e r i v a t i v e 44 a n d cis-cis m u c o n a t e d e r i v a t i v e s 43 (19,20). L a b e l l i n g e x p e r i m e n t s , w i t h C>2 a n d H 2 0 , respectively, d e m o n s t r a t e d t h a t o n l y one o f t h e oxygens o n t h e c a r b o n y l groups o f t h e m e t h y l o x a l a t e 34 a n d m u c o n a t e s 43 were C>2 d e r i v e d , w i t h t h e other b e i n g f o r m e d f r o m H 2 0 ( F i g . 8) (19). T o account for t h e f o r m a t i o n o f 43 as i n i t i a l r i n g cleavage p r o d u c t s , t h e h y p o t h e t i c a l scheme s h o w n i n F i g u r e 9 is p r o p o s e d ; i n i t i a l f o r m a t i o n o f t h e a r y l r a d i c a l c a t i o n i n t e r m e d i a t e o c curs w h i c h c a n t h e n react w i t h H 2 O , O 2 a n d h y d r o g e n r a d i c a l s as s h o w n . T h e m e c h a n i s m s for t h e f o r m a t i o n o f o t h e r r i n g cleavage p r o d u c t s 34-41 v i a a r y l r a d i c a l c a t i o n i n t e r m e d i a t e s are s h o w n i n t h e c h a p t e r b y U m e z a w a and Higuchi. Veratryl Alcohol. L e i s o l a et ai (21,22) recently r e p o r t e d t h a t t r e a t m e n t o f v e r a t r y l a l c o h o l 45 w i t h l i g n i n peroxidase resulted m a i n l y i n t h e f o r m a t i o n o f v e r a t r y l aldehyde 49, the t w o 7 - l a c t o n e s 46 a n d 47 ( F i g . 10) a n d several other quinones n o t s h o w n . W e (23,24) have e s t a b l i s h e d t h a t t h e 6-lactone 48 was also f o r m e d . W h e n e x p e r i m e n t s were c o n d u c t e d i n t h e presence o f C>2 a n d H 2 0 , regiospecific i n c o r p o r a t i o n i n t o p r o d u c t s 46 a n d 47 was observed ( F i g . 11). T h i s regiospecificity d i d n o t o c c u r d u r i n g 6-lactone 48 f o r m a t i o n , a l t h o u g h t h e reasons for t h i s are n o t clear. [Note also t h a t w h e n v a n i l l y l a l c o h o l w a s used as s u b s t r a t e , t h e m a i n p r o d u c t s were the b i p h e n y l C5-C5 a d d u c t s , together w i t h s m a l l a m o u n t s o f i - l a c t o n e s (25).] 18


493


1 8

18

1 8

18

1 8

Aromatic Ring Cleavage of Phenolic β-0-4 Substructure Model Compounds by Laccase. W h e n v a n i l l y l a l c o h o l was used as a s u b s t r a t e , o n l y b i p h e n y l f o r m a t i o n ( C 5 - C 5 l i n k e d ) o c c u r r e d a n d n o evidence for t h e f o r m a t i o n o f a n y r i n g - o p e n e d p r o d u c t s w a s o b t a i n e d (26). H e n c e , we also e x a m i n e d t h e effect o f laccase o n t h e s t e r i c a l l y h i n d e r e d 4,6-di-2, a n d not H 0 , w a s i n c o r p o r a t e d i n t o t h i s p r o d u c t ( F i g . 13) (28). 18

2

1 8

Aromatic Ring Cleavage of Artificial (DHP) Lignin by Lignin Peroxidase. A s discussed, o u r p r e v i o u s studies established t h a t t h e a r o m a t i c r i n g s o f n o n p h e n o l i c / ? - 0 - 4 m o d e l c o m p o u n d s u n d e r w e n t o p e n i n g t o give cis,cism u c o n a t e derivatives as t h e i r i n i t i a l p r o d u c t s ( F i g . 9 ) . H o w e v e r , these studies p r o v i d e d n o i n f o r m a t i o n as t o whether higher oligomers, o r i n d e e d , the l i g n i n p o l y m e r itself, w o u l d undergo such reactions. S i n c e i t is generally v i e w e d t h a t coniferous l i g n i n has a l m o s t 4 0 % o f its linkages connected v i a / ? - 0 - 4 bonds, these aforementioned results s u g gest t h a t a l i g n i n p o l y m e r connected p r i m a r i l y v i a such linkages s h o u l d



A O

4

OEt

ι

.18, •: 0

Ε ι α

2

3

Ο

2

1 8

3

3

l

o

r

^

2

0

C

Eta.

Z

43

3

9

3

D: H.

H

2

3

OC

T

and/or

R'=H

0

36R=R'=CH CH

3

3SR=CH ,

34R=R'=H

OCH OEt

18 18„ 0 , 9: 0 of 0 , lo

Η,·

ROv^CH

ÂÔCH

7

38 R=CH

37R=H

3

< £ oOCH C OEt 39 3

OjLÔH

OCD OCH3

o-9

VÔCH, OEl

Q

0

44

0 "OCH, OEt

HO.

7

H0?0^V°

41R=CH

40R=H

F i g u r e 8. A r o m a t i c r i n g cleavage o f n o n p h e n o l i c β-0-4 m o d e l c o m p o u n d s 31-33 b y l i g n i n peroxidase.

Of H

2

3

R'=H

J

CH

33R=R'=CH CH

3

32R=CH ,

31R-R'=H

OEl

ROV- Ο

f


C D

3

35.

HIGUCHI

EtO-Ht^-v

EtOv

ΕίΟν,ΛοΛ^

EtO


495


O C Η·,

V*OCH OEl

OCH,

VÔCH,

3

OEt

J

OEt

J

°

C H

3

J

43 F i g u r e 9. M e c h a n i s m for the f o r m a t i o n o f a r y l g l y c e r o l cis-cis m u c o n a t e 4 3 b y l i g n i n peroxidase f r o m the n o n p h e n o l i c β-Ο-4 m o d e l c o m p o u n d 4 2 . ο

ο

^ O C H

3

0 47

F i g u r e 10. D e g r a d a t i o n o f v e r a t r y l a l c o h o l 4 5 b y l i g n i n peroxidase.


PLANT C E L L W A L L

POLYMERS


496



35.

HIGUCHI

497


i ^ r ^ i r

LACCASE -

A

OH

r

0

0

^

0

Ο

50

MH

+

m/z

269

51

AUTHENTIC

D-LABELED

ENZYMIC

COMPOUND

NON-ENZYMIC

AUTHENTIC

DEGRADATION

DEGRADATION

COMPOUND

PRODUCT

PRODUCT

' 4 3

m/z 269 272

V

3.5

Time (min.) F i g u r e 12. M a s s c h r o m a t o g r a m s b u t y l g u a i a c o l 50 b y laccase.

of d e g r a d a t i o n

product

51

of


4,6-di-t-

PLANT C E L L WALL POLYMERS


498

F i g u r e 13. P r o p o s e d m e c h a n i s m s for a r o m a t i c r i n g cleavage of 4 , 6 - d i - t b u t y l g u a i a c o l 50 by laccase.



35.

HIGUCHI

Lignin Dégradation by Peroxidase & Laccase

499

undergo s i m i l a r degradative reactions. L i g n i n is often represented b y v a r ious s y n t h e t i c d e h y d r o g e n a t i v e l y p o l y m e r i z e d ( D H P ) p r e p a r a t i o n s , p r o duced v i a the p e r o x i d a s e / H 2 0 2 i n d u c e d p o l y m e r i z a t i o n o f m o n o l i g n o l ( s ) (29). H o w e v e r , d e p e n d i n g u p o n the p o l y m e r i z a t i o n process e m p l o y e d ( Z u l a u f or Z u t r o p f ) the / ? - 0 - 4 linkage frequency can v a r y f r o m 10-40% (29). C o n s e q u e n t l y , we prepared a s y n t h e t i c D H P l i g n i n p o l y m e r f r o m c o n i f e r y l a l c o h o l v i a the Z u t r o p f m e t h o d , hence m a x i m i z i n g the / ? - 0 - 4 l i n k age frequency. F o l l o w i n g i n c u b a t i o n of t h i s s y n t h e t i c p o l y m e r , w i t h l i g n i n p e r o x i d a s e / H 2 0 2 , the degraded p r o d u c t s were s u b j e c t e d to gel p e r m e a t i o n c h r o m a t o g r a p h y u s i n g Sephadex L H 2 0 . T h e e l u t i o n profile revealed t h a t p a r t i a l d e p o l y m e r i z a t i o n h a d o c c u r r e d (30). G C - M S a n a l y s i s also revealed t h a t a s m a l l a m o u n t of v a n i l l i n was f o r m e d v i a C a - C / ? cleavage. S u r p r i s ingly, no a r o m a t i c r i n g cleavage p r o d u c t s were observed. T h u s another D H P , prepared f r o m a m i x t u r e o f 4 - e t h o x y - 3 - m e t h o x y p h e n y l g l y c e r o l - / ? - s y r i n g a r e s i n i n o l ether 51 a n d c o n i f e r y l a l c o h o l 52 was s y n thesized (31). T h i s p o l y m e r was o f interest as i t n o w c o n t a i n e d the m o r e r e a d i l y cleavable / ? - 0 - 4 s y r i n g y l linkages. F o l l o w i n g D H P f o r m a t i o n , the p o l y m e r s were e t h y l a t e d w i t h diazoethane. T h e r e s u l t i n g e t h y l a t e d p o l y mer p r o d u c t 53 was t h e n a p p l i e d to a Sephadex L H 2 0 c o l u m n , w i t h D M F as eluent. T h e higher m o l e c u l a r weight f r a c t i o n e l u t e d ( M W > 2200) was t h e n used as a substrate for l i g n i n p e r o x i d a s e / ^ O 2 t r e a t m e n t . F o l l o w i n g t r e a t m e n t w i t h l i g n i n p e r o x i d a s e / H 2 0 2 , the degraded D H P p o l y m e r so o b t a i n e d was a c e t y l a t e d a n d the r e s u l t i n g p r o d u c t s p a r t i a l l y p u r i f i e d b y t h i n layer c h r o m a t o g r a p h y a n d a n a l y z e d b y gas c h r o m a t o g r a p h y - m a s s spectroscopy ( G C - M S ) . F r o m t h i s m i x t u r e , the a r y l g l y c e r o l c y c l i c c a r b o n ates 37, 39, a n d formate 40 were i d e n t i f i e d , together w i t h 4 - e t h o x y - 3 m e t h o x y p h e n y l g l y c e r o l ( F i g . 14). These results therefore established t h a t l i g n i n p e r o x i d a s e / H 2 0 2 catalyzes s i d e - c h a i n cleavage a n d a r o m a t i c r i n g o p e n i n g reactions of e t h y l a t e d D H P l i g n i n p o l y m e r s , i n agreement w i t h the results p r e v i o u s l y o b t a i n e d f r o m the m o d e l s y s t e m s . Conclusions O v e r recent years, considerable progress has been m a d e i n e l u c i d a t i n g the b i o c h e m i c a l processes of l i g n i n f o r m a t i o n (biosynthesis) a n d l i g n i n biodégradation. T a b l e I s u m m a r i z e s , a n d compares, the m a i n reactions i n v o l v e d i n b o t h processes. I n o u r o p i n i o n , the o v e r a l l c h e m i c a l p r i n c i p l e s l e a d i n g to b o t h f o r m a t i o n a n d d e g r a d a t i o n are s i m i l a r . O u r investigations i n d i c a t e t h a t l i g n i n can be degraded i n t o s m a l l e r fragments v i a a r y l r a d i c a l c a t i o n a n d p h e n o x y r a d i c a l i n t e r m e d i a t e s , i n a reaction mediated by only lignin p e r o x i d a s e / ! ^ O 2 and laccase/02- H o w ever, i t has been s h o w n t h a t in vitro some of the l i g n i n is p o l y m e r i z e d , p r o b a b l y v i a c o u p l i n g reaction of p h e n o x y r a d i c a l s of p h e n o l i c moieties o f l i g n i n , d u r i n g t r e a t m e n t w i t h l i g n i n peroxidase (32) or laccase (33) i n aqueous s o l u t i o n . P r o t o l i g n i n , o n the other h a n d , w h i c h is present i n the p l a n t cell walls i n association w i t h polysaccharides, seems t o be degraded s m o o t h l y to lower m o l e c u l a r weight fractions. T h u s , the r e a c t i o n c o n d i t i o n s p r e v e n t i n g r e p o l y m e r i z a t i o n d u r i n g in vivo l i g n i n biodégradation need t o be identified.



F i g u r e 14. A r o m a t i c r i n g - o p e n i n g reactions of a D H P 53 b y l i g n i n p e r o x idase. ( T h e D H P p o l y m e r 53 was prepared f r o m 4 - e t h o x y - 3 - m e t h o x y p h e nylglycerol-/?-syringaresinol ether 51 a n d c o n i f e r y l a l c o h o l 52.)


35.

HIGUCHI


501

Table I. Comparison of Lignin Biosynthesis and Biodégradation Mechanisms Biodégradation

Biosynthesis Enzymatic Reaction:


1.

Cell wall peroxidase/H202 — • Formation of phenoxy radicals of monolignols

Non-enzymatic Reaction:

Enzymatic Reaction: 1.

Lignin peroxidase/H202 — • Formation of aryl radical cations of nonphenolic units

2.

Laccase/02 — • Formation of phenoxy radicals of phenolic units

Non-enzymatic Reaction:

1.

Coupling of phenoxy radicals to give dimeric quinone methides

1.

Homolytic or heterolytic cleavage of side-chains (Ca-C/?, alkyl-phenyl) and aromatic rings

2.

Dioxygen radical attack on carbon-centered radicals

2.

Dioxygen radical attack on carbon-centered radical intermediates

3.

Nucleophilic attack on quinone methides by H 2 O and R-OH to give dilignols and higher oligomers

3.

Nucleophilic attack on aryl cations and C a cations by H 0 and R-OH to give degradation products 2

A cknowledgment s This paper was prepared based on our recent work on lignin biodégradation in the Research Section of Lignin Chemistry. The author is indebted to Dr. M. Shimada, Dr. T . Umezawa, Messrs. S. Kawai, T . Habe, S. Yokota and T. Hattori in this section for their cooperation for this investigation. Literature Cited 1. 2. 3. 4. 5. 6.

Kirk, T . K.; Farrell, R. L. Ann. Rev. Microbiol. 1987, 41, 465-505. Higuchi, T . Wood Res. 1986, 73, 58-81. Buswell, J . Α.; Odier, E . CRC Rev. Biotechnol. 1987, 6, 1-60. Tien, M.; Kirk, T . K. Proc. Natl. Acad. Sci. USA 1984, 81, 2280-84. Umezawa, T.; Higuchi, T . FEBS Lett. 1988, 218, 255-60. Kawai, S.; Umezawa, T.; Higuchi, T . Arch. Biochem. Biophys. 1988, 262, 111-17. 7. Kersten, P. J.; Tien, M.; Kalyanarama, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 2609-12. 8. Kawai, S.; Umezawa, T.; Shimada, M.; Higuchi, T.; Koide, K.; Nishida, T.; Morohoshi, N.; Haraguchi, T . Mokuzai Gakkaishi 1987, 33, 792-97. 9. Higuchi, T . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T . , Ed.; Academic Press: Orlando, F L , 1985; Ch. 7.



502

PLANT CELL WALL POLYMERS

10. Kuwahara, M.; Glenn, J . K.; Morgan, Μ. Α.; Gold, M . H. FEBS Lett. 1984, 169, 247-50. 11. Hammel, Κ. E.; Tien, M.; Kalyanarama, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 8348-53. 12. Habe, T.; Shimada, M.; Umezawa, T.; Higuchi, T . Agric. Biol. Chem. 1985, 49, 3505-10. 13. Yokota, S.; Umezawa, T.; Kawai, S.; Higuchi, T . Abst. 38th Mtg. Japan Wood Res. Soc. 1988, Asahikawa, Japan. 14. Umezawa, T.; Higuchi, T . FEBS Lett. 1985, 192, 147-50. 15. Umezawa, T.; Higuchi, T . FEMS Microbiol. Lett. 1985, 26, 123-26. 16. Yokota, S.; Umezawa, T.; Higuchi, T . , unpublished. 17. Higuchi, T . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic Press: Orlando, FL, 1985; Ch. 20. 18. Kawai, S.; Umezawa, T.; Higuchi, T., unpublished. 19. Umezawa, T.; Higuchi, T . FEBS Lett. 1986, 205, 293-98. 20. Umezawa, T.; Higuchi, T . Agric. Biol. Chem. 1987, 51, 2281-84. 21. Leisola, M . S. Α.; Schmidt, B.; Thanei-Wyss, U.; Fiechter, A. FEBS Lett. 1985, 189, 267-90. 22. Leisola, M. S. Α.; Haemmerli, S. D.; Smit, J. D. G.; Troller, J.; Waldner, R.; Schoemaker, Η. E.; Schmidt, H. In Lignin Enzymic and Microbial Degradation, INRA Ed.; INRA: Paris, 1987; pp. 81-86. 23. Shimada, M.; Hattori, T.; Umezawa, T.; Higuchi, T.; Uzura, K. FEBS Lett. 1987, 221, 327-31. 24. Hattori, T.; Shimada, M.; Umezawa, T.; Higuchi, T.; Uzura, K. Agric. Biol. Chem. 1988, 52, 879-80. 25. Hattori, T., et al., unpublished. 26. Kawai, S.; Umezawa, T.; Higuchi, T. Abst. 38th Ann. Mtg. Japan Wood Res. Soc. 1988, Asahikawa, Japan. 27. Gierer, J.; Imsgard, F. Acta Chem. Scand. 1977, 31, 546-50. 28. Kawai, S.; Umezawa, T.; Shimada, M.; Higuchi, T . FEBS Lett. 1988, 236, 309-11. 29. Sarkanen, Κ. V. In Lignins: Occurrence, Formation, Structure and Re actions; Sarkanen, Κ. V.; Ludwig, C. H., Eds.; Wiley-Interscience: New York, 1971; Ch. 4. 30. Umezawa, T.; Higuchi, T . Abst. 38th Ann. Mtg. Japan Wood Res. Soc. 1988, Asahikawa, Japan. 31. Umezawa, T.; Higuchi, T . FEBS Lett. 1989, 242, 325-29. 32. Haemmerli, S. D.; Leisola, M . S. Α.; Fiechter, A. FEMS Microbiol. Lett. 1986, 35, 33-36. 33. Ishihara, T.; Miyazaki, M . Mokuzai Gakkaishi 1972, 18, 416-19. RECEIVED May 19, 1989


Mechanisms of Lignin Degradation by Lignin Peroxidase and Laccase

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