Biomimetic Studies in Lignin Degradation - American Chemical Society

most cases in degrading β-l, β-0-4, and β-b model compounds. ... The oxidations of anisyl alcohol, in the presence of veratryl alcohol or 1,4- ...
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Chapter 37

Biomimetic Studies in Lignin Degradation Futong Cui and David Dolphin Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Y6, Canada

A redox stable, water-soluble iron porphyrin has been used as a model ligninase. The reactions of lignin model compounds catalyzed by this biomimetic system were found to be dependent on pH and the solvent being used. Model studies showed that veratryl alcohol could mediate the oxidation of a polymeric lignin model compound under certain conditions but could not mediate the oxidation of small molecule model compounds. L i g n i n is the second most a b u n d a n t renewable o r g a n i c c o m p o u n d o n e a r t h . It composes about 15-25% o f the l a n d - p r o d u c e d biomass. A considerable effort has been m a d e t o u n d e r s t a n d l i g n i n biodégradation d u r i n g the last 20 or 30 years, a n d since the discovery o f l i g n i n d e g r a d i n g enzymes (ligninases) i n 1983 (1,2), progress has been m a d e i n u n d e r s t a n d i n g the m e c h a n i s m s o f l i g n i n biodégradation. M o d e l e n z y m e studies are i m p o r t a n t for b o t h m e c h a n i s t i c e l u c i d a t i o n a n d for p r a c t i c a l a p p l i c a t i o n s . T h i s is p a r t i c u l a r l y true for t h e non-specific l i g n i n d e g r a d i n g enzymes. L i g n i n a s e is synthesized b y P. chrysosponum o n l y under c e r t a i n phases o f g r o w t h a n d is difficult t o o b t a i n i n q u a n t i t y . It is a powerful o x i d a n t w h i c h i n i t i a t e s l i g n i n d e g r a d a t i o n b y one electron o x i d a t i o n s , a n d a m a j o r role o f the ligninase p r o t e i n is t o s t e r i c a l l y p r o t e c t the o x i d i z e d heme prosthetic g r o u p . It seems u n l i k e l y t h a t ligninase w i l l c o n t a i n a n " a c t i v e s i t e " to specifically b i n d the diverse s t r u c t u r a l components of l i g n i n . Instead, electron transfer (either direct or m e d i a t e d ) w i l l take place between t h e enzyme a n d p o l y m e r i c l i g n i n . A s t e r i c a l l y protected, water-soluble s y n t h e t i c i r o n p o r p h y r i n c o u l d p r o v i d e a r e a d i l y available b i o m i m e t i c c a t a l y s t for b o t h basic research a n d p o t e n t i a l i n d u s t r i a l a p p l i c a t i o n s . S u c h a s y n t h e t i c h e m i n m i g h t be s u p e r i o r to t h e e n z y m e , i n t h a t b e i n g a s m a l l molecule i t c o u l d interact, w i t h the p o l y m e r i c l i g n i n molecule more r e a d i l y t h a n c a n ligninase. S h i m a d a et al. (3) first f o u n d t h a t a s y n t h e t i c p o r p h y r i n i r o n mesot e t r a p h e n y l p o r p h y r i n ( F e T P P (I)) c a t a l y z e d C - C b o n d cleavage o f β-l type 0097-6156/89/0399-0519$06.00/0 © 1989 American Chemical Society

520

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l i g n i n m o d e l c o m p o u n d s , a n d further e x p e r i m e n t s (4) showed t h a t the re­ a c t i o n h a d an o p t i m a l p H of 3.0 a n d was s t i m u l a t e d b y the presence of i m ­ idazole. Isotope l a b e l i n g experiments (5) showed t h a t the i r o n p o r p h y r i n c a t a l y z e d reactions followed the same m e c h a n i s t i c routes as those of the ligninase c a t a l y z e d reactions (6). P r o t o h e m i n ( I I ) was also r e p o r t e d to be able to c a t a l y z e r i n g cleavage reactions of v e r a t r y l a l c o h o l (7). Shi­ m a d a ei al (8) recently r e p o r t e d t h a t p r o t o h e m i n m i m i c k e d ligninase i n most cases i n d e g r a d i n g β-l, β-0-4, a n d β-b m o d e l c o m p o u n d s . T h e s i m ­ ple i r o n p o r p h y r i n s (1,11), however, suffer a great disadvantage i n t h a t they are r a p i d l y destroyed b y o x i d a n t s . S t e r i c a l l y p r o t e c t e d w a t e r - s o l u b l e m e t a l l o p o r p h y r i n s have been used i n our l a b o r a t o r y as ligninase m i m i c s a n d we discuss here some observations o n our most p r o m i s i n g c a t a l y s t i r o n m e s o - t e t r a - ( 2 , 6 - d i c h l o r o - 3 - s u l f o n a t o p h e n y l ) p o r p h y r i n chloride ( T D C S P P F e C l (III)). Experimental T h e o x i d a t i o n of v e r a t r y l a l c o h o l was carried out under air at r o o m t e m p e r ­ a t u r e . T h e r e a c t i o n m i x t u r e contained 30 //moles of v e r a t r y l a l c o h o l , 0.05 //moles of T D C S P P F e C l , 30 //moles of ???-chloroperbenzoic acid ( ? ? i C P B A ) m a d e u p to 6 m l u s i n g phosphate buffer or as otherwise i n d i c a t e d i n T a b l e I. 4-methoxyacetophenone (30 /imoles) was added as a n i n t e r n a l s t a n d a r d . T h e r e a c t i o n was s t o p p e d after 2 hours by p a r t i t i o n i n g the m i x t u r e be­ tween m e t h y l e n e chloride a n d s a t u r a t e d s o d i u m b i c a r b o n a t e s o l u t i o n . T h e aqueous layer was t w i c e e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e a n d the ex­ t r a c t s c o m b i n e d . T h e p r o d u c t s were a n a l y z e d b y G C after a c e t y l a t i o n w i t h excess 1:1 acetic a n h y d r i d e / p y r i d i n e for 24 hours at r o o m t e m p e r a t u r e . T h e o x i d a t i o n s of a n i s y l a l c o h o l , i n the presence of v e r a t r y l a l c o h o l or 1,4d i m e t h o x y b e n z e n e , were p e r f o r m e d as i n d i c a t e d i n T a b l e III a n d I V i n 6 m l of phosphate buffer ( p H 3.0). O t h e r c o n d i t i o n s were the same as for the o x i d a t i o n of v e r a t r y l a l c o h o l described above. T D C S P P F e C l r e m a i n ­ i n g after the reaction was e s t i m a t e d f r o m its Soret b a n d a b s o r p t i o n before a n d after the r e a c t i o n . F o r the d e c o l o r i z a t i o n of P o l y B-411 ( I V ) by T D C ­ S P P F e C l a n d m C P B A , 25 //moles of m C P B A were added to 25 m l 0.05% P o l y B - 4 1 1 c o n t a i n i n g 0.01 //moles T D C S P P F e C l , 25 //moles of manganese sulfate a n d 1.5 mmoles of l a c t i c a c i d buffered at p H 4.5. T h e d e c o l o r i z a t i o n of P o l y B - 4 1 1 was followed b y the decrease i n a b s o r p t i o n at 596 n m . F o r the e l e c t r o c h e m i c a l d e c o l o r i z a t i o n of P o l y B-411 i n the presence of v e r a t r y l a l c o h o l , a t w o - c o m p a r t m e n t cell was used. A glassy c a r b o n p l a t e was used as the anode, a p l a t i n u m p l a t e as the a u x i l i a r y electrode, a n d a silver wire as the reference electrode. T h e p o t e n t i a l was controlled at 0.900 V . P o l y B - 4 1 1 (50 m l , 0.005%) i n p H 3 buffer was added to the anode c o m p a r t m e n t a n d p H 3 buffer was added to the cathode c o m p a r t m e n t to the same level. T h e d e c o l o r i z a t i o n of P o l y B-411 was followed by the change i n absorbance at 596 n m a n d the s i m u l t a n e o u s o x i d a t i o n of v e r a t r y l alcohol was followed at 310 n m . T h e same electrochemical a p p a r a t u s was used for the decol­ o r i z a t i o n of P o l y B-411 adsorbed o n t o filter p a p e r . T e t r a b u t y l a m m o n i u m p e r c h l o r a t e ( T B A P ) was used as s u p p o r t i n g electrolyte when methylene chloride was the solvent.

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522

PLANT C E L L W A L L P O L Y M E R S

Results S h i m a d a et ai have c a r r i e d out most o f t h e i r studies o n m o d e l ligninases i n o r g a n i c solvents (3-5,8). However, reactions i n o r g a n i c a n d aqueous so­ l u t i o n s m a y take different routes a n d D o r d i c k et ai (9) have r e p o r t e d t h a t horseradish peroxidase a n d other peroxidases are able t o d e p o l y m e r i z e b o t h n a t u r a l a n d s y n t h e t i c l i g n i n s i n organic solvent b u t are u n a b l e t o do so i n aqueous s o l u t i o n , a l t h o u g h these results have recently been reassessed (10). W e have s t u d i e d the solvent effect for T D C S P P F e C l c a t a l y z e d reactions u s i n g the s i m p l e s t l i g n i n m o d e l c o m p o u n d , v e r a t r y l a l c o h o l . T a b l e I shows t h a t under the same concentrations o f o x i d a n t a n d c a t a l y s t the y i e l d o f v e r a t r a l d e h y d e was higher i n aqueous s o l u t i o n t h a n i n o r g a n i c solvent. It was i n t e r e s t i n g t o note t h a t a n a d d i t i o n a l p r o d u c t was f o r m e d at a b o u t the same y i e l d as v e r a t r a l d e h y d e w h e n m e t h a n o l was the solvent. A l t h o u g h the s t r u c t u r e o f t h i s c o m p o u n d has not yet been e l u c i d a t e d , t h i s result suggests t h a t solvent can also change the p r o d u c t d i s t r i b u t i o n . T h i s was also d r a m a t i c a l l y d e m o n s t r a t e d i n the f o l l o w i n g e x a m p l e . T h e β-0-4 link­ age is one of the most a b u n d a n t s u b s t r u c t u r e s of l i g n i n . I n spruce l i g n i n the β-Ο-4 b o n d was e s t i m a t e d t o comprise 4 8 % o f the linkages connect­ i n g the p h e n y l p r o p a n o i d u n i t s (11). W h i l e F e T P P (I) (8) cannot degrade 4 - 0 - e t h y l g u a i a c y l g l y c e r o l - / ? - g u a i a c y l ether i n o r g a n i c solvents b y the same p a t h w a y as the e n z y m i c reactions (12), our i n i t i a l results show t h a t T D C ­ S P P F e C l degraded t h i s c o m p o u n d i n water to give, a m o n g other p r o d u c t 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 a n d g u a i a c o l ( F i g . 1). T a b l e I. 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 b y T D C S P P F e C l a n d m C P B A i n various solvents Solvent 6 m l p H 2 phosphate buffer 6 ml D M F 6 m l methanol α

b

% Y i e l d of V e r a t r a l d e h y d e 49.6 12.0 11.0

0

6

Based on veratryl alcohol. A second p r o d u c t was o b t a i n e d .

T h e o p t i m a l p H for T D C S P P F e C l c a t a l y s i s was e x a m i n e d for the p r o ­ d u c t i o n o f v e r a t r a l d e h y d e f r o m v e r a t r y l a l c o h o l . T h e y i e l d was highest at the lowest p H used ( T a b l e II). It can also be seen f r o m T a b l e II t h a t T D C ­ S P P F e C l was f a i r l y stable over a w i d e range o f p H . T h e p H o f the m e d i u m is c r u c i a l for the n a t u r a l d e g r a d a t i o n o f l i g n i n . C a t i o n r a d i c a l s are i n v o l v e d i n b o t h the biosynthesis a n d biodégradation o f l i g n i n . T h e biosynthesis occurs at a r o u n d p H 7 w h i c h favors r a d i c a l c o u p l i n g o f the neutral p h e n o late r a d i c a l w h i l e biodégradation occurs under a c i d i c c o n d i t i o n w h i c h favors c a t i o n r a d i c a l i n d u c e d C - C b o n d cleavage. A l t h o u g h no a t t e m p t was m a d e to o p t i m i z e the t u r n o v e r o f the c a t a l y s t , T a b l e II shows t h a t T D C S P P F e C l was stable under the extreme

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o x i d i z i n g c o n d i t i o n s , i.e., w i t h a c a t a l y s t to o x i d a n t r a t i o of 1:600. O t h e r s i m p l e h e m i n s are r a p i d l y degraded under these c o n d i t i o n s . T a b l e I I . O x i d a t i o n of v e r a t r y l a l c o h o l b y T D C S P P F e C l a n d m C P B A i n aqueous s o l u t i o n as a f u n c t i o n of p H

a

PH

% Yield of Veratraldehyde"

% T D C S P P F e C l Left After Reaction

2 4 6 8 10

49.6 11.4 5.8 3.7 9.9

98 98 97 74 96

Y i e l d based o n v e r a t r y l a l c o h o l .

P a l m e r et ai (13) p r o p o s e d t h a t ligninase degrades l i g n i n b y single electron o x i d a t i o n s a n d t h a t the c a t i o n r a d i c a l s of s m a l l a r o m a t i c molecules c o u l d serve as diffusible redox m e d i a t o r s d u r i n g l i g n i n d e g r a d a t i o n . V e r a t r y l a l c o h o l was f o u n d to s t i m u l a t e the o x i d a t i o n of a n i s y l c o m p o u n d s (14) by ligninase a n d H a r v e y et ai suggested t h a t the c a t i o n r a d i c a l of v e r a t r y l a l c o h o l f u n c t i o n e d as a redox m e d i a t o r i n t h i s r e a c t i o n . Since the c a t i o n r a d i c a l of v e r a t r y l a l c o h o l has not so far been detected (15), the m e d i a t i n g role o f v e r a t r y l a l c o h o l is s t i l l a n open q u e s t i o n . T h e o x i d a t i o n of a n i s y l a l c o h o l by T D C S P P F e C l a n d m C P B A was c a r r i e d o u t i n the presence of v a r y i n g a m o u n t s of v e r a t r y l a l c o h o l . T h e results i n T a b l e III show t h a t the presence of v e r a t r y l a l c o h o l i n h i b i t e d the o x i d a t i o n of a n i s y l a l c o h o l . These observations are r e a d i l y r a t i o n a l i z e d o n the basis of the o x i d a t i o n p o t e n t i a l s of the two c o m p o u n d s . V e r a t r y l a l c o h o l , w h i c h has a lower o x i d a t i o n p o t e n t i a l t h a n a n i s y l a l c o h o l , (1.52 V vs. 1.76 V as d e t e r m i n e d by c y c l i c v o l t a m e t r y i n a c e t o n i t r i l e ) , is m o r e easily o x i d i z e d t h u s decreasing the y i e l d of a n i s y l a l c o h o l b y c o m p e t i n g w i t h i t for the o x i d a n t ; the same w i l l be true i n the n a t u r a l systems. T h e m e d i a t i n g role of a w e l l k n o w n single-electron transfer agent, 1,4-dimethoxybenzene, was tested for c o m p a r i s o n . It can be seen f r o m T a b l e I V t h a t 1,4-dimethoxybenzene c a n n o t s t i m u l a t e the o x i d a t i o n of a n i s y l a l c o h o l either. T h e r e f o r e , the i n creased y i e l d of a n i s y l a l c o h o l o x i d a t i o n by ligninase c a n n o t be a t t r i b u t e d d i r e c t l y to the m e d i a t i n g role of v e r a t r y l a l c o h o l . However the recent w o r k of H a r v e y et ai (17) provides a g o o d e x p l a n a t i o n of t h e i r e a r l y results (14) a n d confirms the s p e c u l a t i o n of K i r k et ai (16). V e r a t r y l a l c o h o l can protect ligninase f r o m i n a c t i v a t i o n by p r e v e n t i n g the f o r m a t i o n of ligninase c o m p o u n d III (17) ( F i g u r e 2). Indeed, the low y i e l d of v e r a t r a l d e h y d e at h i g h p H , c o u p l e d to the s t a b i l i t y of the h e m i n ( T a b l e I I , p H 10) was due to the facile f o r m a t i o n of the c o m p o u n d III a n a l o g of T D C S P P F e C l w h i c h is r e l a t i v e l y l o n g l i v e d at h i g h p H . O u r i n a b i l i t y to e s t a b l i s h a m e d i a t i n g role for v e r a t r y l a l c o h o l i n the o x i d a t i o n of a n i s y l a l c o h o l does not exclude the p o s s i b i l i t y t h a t i t f u n c t i o n s as a m e d i a t o r i n l i g n i n d e g r a d a t i o n . T h e m e d i a t i n g role of v e r a t r y l a l c o h o l m i g h t be difficult to observe for s m a l l m o l e c u l e

PLANT

524

C E L L WALL POLYMERS

CHO

OH

OEt

F i g u r e 1. C o m p a r i s o n of the reactions of 4 - O - e t h y l g u a i a c y l g l y c e r o l - / ? g u a i a c y l ether c a t a l y z e d b y F e T P P / t - B u O O H / C H C l , T D C S P P F e C l / m C P B A / H 2 0 a n d l i g n i n a s e / h y d r o g e n peroxide. 3

0

Resting E n z y m e

Ligningx

Lignin

C o m p o u n d II

H 0 2

2

Compound m

F i g u r e 2. T h e c a t a l y t i c cycles of ligninase.

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i n t e r a c t i o n s w i t h the low m o l e c u l a r weight m o d e l c o m p o u n d s . Indeed, as d e s c r i b e d below, the results of e l e c t r o c h e m i c a l studies w i t h a p o l y m e r i c m o d e l c o m p o u n d s h o w n i n F i g u r e 3 suggest s u c h a m e d i a t i n g role. T a b l e I I I . O x i d a t i o n of a n i s y l a l c o h o l b y T D C S P P F e C l i n aqueous s o l u t i o n i n the presence of v e r a t r y l a l c o h o l ( p H 3) Substrates Anisyl Alcohol (//moles) 30 30 30 30 30 a

Veratryl Alcohol (pmoles) 30 15 3 0.3 0

% y i e l d of Anisaldehyde"

% T D C S P P F e C l Left After Reaction > > > > >

1.3 4.2 14.7 20.8 25.0

90 90 90 90 90

Y i e l d based o n a n i s y l a l c o h o l .

T a b l e I V . O x i d a t i o n of A n i s y l A l c o h o l by T D C S P P F e C l a n d m C P B A i n A q u e o u s S o l u t i o n i n the Presence of 1,4-dimethoxybenzene ( p H 3) Substrates Anisyl Alcohol (/xmoles) 30 30 30 30 30 a

1,4-dimethoxy Benzene (/zmoles)

% Y i e l d of Anisaldehyde"

30 15 3 0.3 0

1.3 3.3 17.5 20.4 25.0

% T D C S P P F e C l Left After Reaction > > > > >

90 90 90 90 90

Y i e l d based o n a n i s y l a l c o h o l .

P o l y B - 4 1 1 , a w a t e r - s o l u b l e , blue dye ( I V ) has been used as a l i g n i n m o d e l b y G l e n n et ai (18). T h e dye was deposited onto a piece of filter p a p e r , w h i c h was t h e n a t t a c h e d d i r e c t l y to a glassy c a r b o n anode. T h e a n o l y t e c o n t a i n e d 10 m M v e r a t r y l a l c o h o l a n d 0.1 M t e t r a b u t y l a m m o n i u m p e r c h l o r a t e ( T B A P ) i n methylene chloride. T h e c a t h o l y t e c o n t a i n e d o n l y 0.1 M T B A P i n m e t h y l e n e chloride. A f t e r s i x hours c o n t r o l l e d p o t e n t i a l o x i d a t i o n at 1.2 V , the blue color of filter p a p e r o n the side f a c i n g the anode t u r n e d to r e d d i s h b r o w n . C o m p l e t e o x i d a t i o n of P o l y B - 4 1 1 i n aqueous s o l u t i o n by T D C S P P F e C l a n d m C P B A gave the same color. T h e filter paper was not decolorized i n a c o n t r o l e x p e r i m e n t l a c k i n g v e r a t r y l a l c o h o l . A s P o l y B - 4 1 1 was not able to m a k e direct contact w i t h the electrode (since

PLANT C E L L W A L L P O L Y M E R S

526

A596 0.540 0.520-f 0.500 0.480

2"

0.460

3

0.440 H 0.420

-i

10

1

1

1

1

20

30

40

50

*

60

Time (min) F i g u r e 3. E l e c t r o c h e m i c a l o x i d a t i o n of P o l y B-411 i n the presence of v e r a t r y l a l c o h o l at various concentrations ( m M ) . 1, 0; 2, 0.1; 3, 1.0; 4, 10. A596 0.70 Η 0.60 0.500.400.30 0.20

Time (min) F i g u r e 4. D e c o l o r i z a t i o n o f P o l y B - 4 1 1 b y T D C S P P F e C l a n d ? n C P B A i n the presence o f manganese sulfate a n d l a c t i c a c i d (curve 3). C u r v e s 1 a n d 2 show c o n t r o l e x p e r i m e n t s l a c k i n g T D C S P P F e C l a n d manganese sulfate, respectively.

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it is insoluble in methylene chloride) in the above experiment, veratryl alcohol (or one of its oxidation products) must have mediated its oxidation. TDCSPPFeCl can also mimic the function of the Mn(II)-dependent peroxidase isolated from ligninolytic cultures of P. chrysosporium (19,20). The mCPBA-oxidized TDCSPPFeCl can oxidize Mn(II) to Mn(III) which in turn oxidizes Poly B-411 in the presence of various α-hydroxy carboxylic acids. Figure 4 shows that TDCSPPFeCl can rapidly decolorize Poly B411 in the presence of manganese sulfate, lactic acid, and mCPBA. Control experiments lacking either manganese sulfate or TDCSPPFeCl caused inef­ ficient and very slow decolorization of Poly B-411 (Fig. 4). It has been sug­ gested (23) that one function of manganese is that of a diffusible mediator for lignin degradation. The above observations support this mediating role and at the same time emphasize the role of the porphyrin (enzyme). Hy­ drogen peroxide, a two electron oxidant, cannot oxidize Mn(II) to Mn(III) (a one electron process), but at the same time manganese ions unlike iron do not initiate Fenton-like chemistry with peroxide. Peroxide can oxidize a ferric porphyrin to the compound I oxidation state (O = Fe(IV)Por ) and this can bring about the one electron oxidation of Mn(II) leaving Compound II (O = Fe(IV)Por). Compound II can then oxidize a second equivalent of Mn(II). Indeed this latter step is probably critically important in preventing Compound III formation in the manganese dependent peroxidase. +#

Conclusions We have shown that TDCSPPFeCl ( Π Ι ) is so far the most stable and efficient catalyst among the iron porphyrins used as model ligninases. All the known reactions catalyzed by this porphyrin mimic the ligninases quite well. TDCSPPFeCl can be used in both aqueous and polar organic solvent (such as methanol, DMF) so that solvent effects of lignin degradation can be studied. The catalyst is stable over a wide range of pH so the reactions at different pH can be compared. Under carefully controlled conditions veratryl alcohol can act as a redox mediator but from the experiments described above, we expect that such a role must be of only minor importance in nature. The ability of veratryl alcohol (and Mn(II)) to reduce Compound II (preventing the formation of compound III) is probably of far more importance for the ligninases. Acknowledgment This work was supported by the Natural Sciences and Engineering Research Council of Canada. Literature Cited 1. Tien, M.; Kirk, T . K. Science 1983, 221, 661-63. 2. Glenn, J. K.; Morgan, Μ. Α.; Mayfield, M . B.; Kuwahara, M.; Gold, M. H. Biochem. Biophys. Res. Commun. 1983, 114, 1077-83. 3. Shimada, M . ; Habe, T.; Umezawa, T.; Higuchi, T.; Okamoto, T . Biochem. Biophys. Res. Commun. 1984, 122, 1247-52.

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4. Habe, T.; Shimada, M.; Higuchi, T . Mokuzai Gakkaishi 1985, 31, 54-55. 5. Habe, T.; Shimada, M . ; Okamoto, T.; Panijpan, B.; Higuchi, T . J. Chem. Soc., Chem. Commun. 1985, 1323-24. 6. Tien, M.; Kirk, T . K. Proc. Natl. Acad. Sci. USA 1984, 81, 2280-4. 7. Shimada, M . ; Hattori, T.; Umezawa, T.; Higuchi, T.; Okamoto, T . Proc. Intl. Symp. on Lignin Enzymic and Microbial Degradation; Paris, France, April 23-24, 1987; p. 151. 8. Shimada, M.; Habe, T.; Higuchi, T.; Okamoto, T.; Panijpan, B.; Holz­ forschung 1987, 41, 277-85. 9. Dordick, J. S.; Marletta, Μ. Α.; Klibanov, A. M . Proc. Natl. Acad. Sci. USA 1986, 83, 6255-57. 10. Lewis, N. G.; Razal, R. Α.; Yamamoto, E . Proc. Natl. Acad. Sci. USA 1987, 84, 7925-27. 11. Adler, E . Wood Sci. Technol. 1977, 11, 169-218. 12. Kirk, T . K.; Tien, M.; Kersten, P. J.; Mozuch, M. D. Biochem. J. 1986, 236, 279-87. 13. Schoemaker, Η. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J . M . FEBS Lett. 1985, 183, 7-12. 14. Harvey, P. J.; Schoemaker, Η. E.; Palmer, J. M . FEBS Lett. 1986, 195, 242-46. 15. Tien, M.; Kirk, T . K.; Bull, C.; Fee, J . A. J. Biol. Chem. 1986, 261, 1687-93. 16. Kirk, T . K.; Farrell, R. L. Ann. Rev. Microbiol. 1987, 41, 465-505. 17. Harvey, P. J.; Schoemaker, Η. E.; Palmer, J . M.; Proc. Intl. Symp. on Lignin Enzymic and Microbial Degradation; Paris, France, April 23-24, 1987; p. 145. 18. Glenn,J. K.; Gold, M . H. Appl. Environ. Microbiol. 1983, 45, 1741-47. 19. Kuwahara, M.; Glenn, J . K.; Morgan, Μ. Α.; Gold, M . H. FEBS Lett. 1984, 169, 247-50. 20. Paszczynski, Α.; Huynh, Van-Ba; Crawford, R. FEMS Microbiol. Lett. 1985, 29, 37-41. 21. Glenn, J. K.; Gold. M . H. Arch. Biochem. Biophys. 1985, 242, 329-41. 22. Paszczynski, Α.; Huynh, Van-Ba; Crawford, R. Arch. Biochem. Bio­ phys. 1986, 244, 750-65. 23. Glenn, J . K.; Akileswaran, L.; Gold, M . H. Arch. Biochem. Biophys. 1986, 251, 688-96. RECEIVED March 17, 1989