Enzymatic Modification of Lignin for Technical Use ... - ACS Publications

Chapter 27

Enzymatic Modification of Lignin for Technical Use

Downloaded by NORTH CAROLINA STATE UNIV on April 13, 2013 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0397.ch027

Strategies and Results A. Hüttermann, O. Milstein, B. Nicklas, J. Trojanowski, A. Haars, and A . Kharazipour Forstbotanisches Institut der Universität Göttingen, Büsgenweg 2, D-3400 Göttingen, Federal Republic of Germany Extracellular phenoloxidases from white-rot fungi are promising tools for the biotechnological conversion of technical by-product lignins into valuable polymers. A short review of lignin-degrading enzymes of white-rot basidiomycetes is given as well as two examples for the potential application of laccases in lignin transforming processes. (1) In nature, enzymes react generally in aqueous solution. However, the bulk of technical by-product lignins (e.g., kraft lignin) is water insoluble, causing reaction rates of phenol-oxidases to be slow. This problem was addressed by the development of a process which uses immobilized phenoloxidases in an organic-aqueous system. (2) The same class of enzymes can efficiently be used for the biologically catalyzed bonding of particle boards. Adhesive cure is based on the oxidative polymerization of lignin using phenoloxidases as radical donors. This lignin-based "bio-adhesive" can be applied under conventional pressing conditions. The resulting particle boards meet German performance standards without emission of harmful vapors. A n added advantage is the total utilization of lignin from spent pulp liquor. T h e concept o f using the n a t u r a l p o l y p h e n o l l i g n i n as a feedstock for p o l y mer p r o d u c t i o n or as a n adhesive i n w o o d composites has encouraged n u merous scientific endeavors (1), t h e basic rationale for w h i c h concerns r e p l a c i n g expensive p e t r o c h e m i c a l resins w i t h this c o m p a r a t i v e l y low-cost renewable r a w m a t e r i a l . A l t h o u g h l i g n i n occurs as a p o l y m e r w i t h several a t t r a c t i v e s t r u c t u r a l features i n t h e plant cell w a l l , h a v i n g m a c r o m o l e c u lar architecture a n d m a n y types o f reactive f u n c t i o n a l groups, t h e l i g n i n available f r o m cellulose p r o d u c t i o n is m u c h less a t t r a c t i v e for use i n p o l y mers. T h e h a r s h reaction c o n d i t i o n s o f the p u l p i n g process t r a n s f o r m l i g n i n 0097-6156/89A)397-0361$06.00A) © 1989 American Chemical Society

In Lignin; Glasser, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


362

LIGNIN: PROPERTIES AND MATERIALS

i n t o a r a t h e r inert m a t e r i a l . T h e content of phenolic groups, for instance, is d r a s t i c a l l y changed b y a l l c o m m e r c i a l l y a p p l i e d p u l p i n g processes, a n d the n u m b e r of a l k y l a r y l ether bonds declines. T h u s , several i m p o r t a n t properties are u n f a v o r a b l y altered i n the c o m m e r c i a l l y available l i g n i n s as c o m p a r e d to the o r i g i n a l p l a n t m a t e r i a l . T h e properties of t o d a y ' s c o m m e r c i a l l y available l i g n i n s can be s u m m a r i z e d as follows (2): L i g n i n s f r o m the sulfite process ( l i g n i n sulfonates) are e x t r e m e l y p o l a r o w i n g t o the presence of sulfonate groups, a n d they are h i g h l y water soluble. K r a f t l i g n i n s , w h i c h are d e r i v e d f r o m the k r a f t (or s u l fate) process, have low p o l a r i t y a n d are water i n s o l u b l e . T h e s e c o m p o u n d s are therefore rather u n a t t r a c t i v e as feedstocks for p o l y m e r p r o d u c t i o n , a n d l i t t l e progress has been m a d e i n t h i s d i r e c t i o n so far. I n a d d i t i o n , the l i g n i n c o m i n g f r o m the organosolv process is water i n s o l u b l e a n d i t s m o l e c u l a r weight is p o s s i b l y too s m a l l to be s u i t a b l e as a p r e p o l y m e r for plastics production. A strategy for the e n z y m a t i c m o d i f i c a t i o n of l i g n i n for its t e c h n i c a l use s h o u l d t h u s concentrate o n the f o l l o w i n g goal: to p r o d u c e a h o m o g e neous, pure l i g n i n p r e p a r a t i o n of reasonably h i g h m o l e c u l a r weight w i t h h i g h r e a c t i v i t y p r o v i d e d b y reactive f u n c t i o n a l groups. Extracellular E n z y m e s of Lignin-Transforming F u n g i T h e f o l l o w i n g e n z y m a t i c a c t i v i t i e s have been characterized w h i c h are able to change n a t i v e l i g n i n : • ligninase, i.e., l i g n i n peroxidase resp. v e r a t r y l peroxidase, a n e n z y m e isolated a n d characterized b y K i r k a n d his c o l l a b o r a t o r s (3) (for a n u p d a t e d review see ref. 4-5). T h i s e n z y m e is considered t o be the m a i n l i g n o l y t i c s y s t e m i n w h i t e - r o t f u n g i . It is d e t e r m i n e d v i a its a b i l i t y to catalyze 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 . • laccase, i.e., p o l y p h e n o l oxidase ( E . C . I . 1 0 . 3 . 2 . ) . T h i s e n z y m e is c o m m o n i n m a n y m i c r o o r g a n i s m s , a n d i t is r e p o r t e d to have m a n y different p h y s i o l o g i c a l functions (6). It has been k n o w n for a l o n g t i m e t h a t l a c case is able to p o l y m e r i z e phenols. Its role i n l i g n i n d e g r a d a t i o n has first been discussed b y A n d e r a n d E r i k s s o n (7). • poly-blue-oxidase. T h i s e n z y m e was first described b y G l e n n a n d G o l d (8); i t oxidizes the l i g n i n m o d e l c o m p o u n d p o l y - b l u e w h i c h is a p o l y m e r i c dye. A l l three enzymes have the advantage of b e i n g subject to convenient spectroscopic assays. T h e most i m p o r t a n t gross changes i n the l i g n i n molecule w h i c h enzymes c a n c a t a l y z e , a n d w h i c h are i m p o r t a n t for possible i n d u s t r i a l uses, are the f o l l o w i n g : • solubilization, • demethylation, • changes i n phenolic a n d a l i p h a t i c h y d r o x y l contents, a n d • changes i n m o l e c u l a r weight d i s t r i b u t i o n . I n our l a b o r a t o r y , we have screened various m i c r o o r g a n i s m s for s o l u b i l i z a t i o n a n d d e m e t h y l a t i o n a c t i v i t y i n a d d i t i o n to t e s t i n g for the presence



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H U T T E R M A N N FT AL.

Enzymatic Modification for Technical Use

363

of ligninase, laccase a n d p o l y - b l u e - o x i d a s e . D a t a o n changes i n the m o l e c u l a r weight d i s t r i b u t i o n have been given elsewhere ( 9 , 1 0 ) . A l t h o u g h the b i o c h e m i c a l work o n l i g n i n d e g r a d a t i o n has focused m a i n l y o n a single ( a l t h o u g h r a t h e r suitable) o r g a n i s m , the w h i t e rot fungus Phanerochaeie chrysosporium (5), we decided to analyze the l i g n i n t r a n s f o r m i n g c a p a c i t y of f u n g i c o m i n g f r o m different niches i n the ecosystem. T h e s e f u n g i r e p resent a w i d e ecological range, a n d they take i n t o c o n s i d e r a t i o n the m a n y different c o n d i t i o n s under w h i c h f u n g i degrade w o o d resp. l i g n i n . T h e results are s u m m a r i z e d i n T a b l e I. T h e d a t a i n d i c a t e t h a t , w i t h i n t h i s v a r i e t y of different species, no c o r r e l a t i o n exists between the a c t i v i t i e s of the different enzymes a n d d e m e t h y l a t i o n or s o l u b i l i z a t i o n . S u r p r i s i n g l y , no c o r r e l a t i o n , either, was observed between the a b i l i t y to s o l u b i l i z e o r g a n o solv l i g n i n a n d the a c t i v i t y o f the l i g n i n peroxidase. B y c o n t r a s t , a r e l a t i o n was f o u n d between l i g n i n s o l u b i l i z a t i o n a n d p o l y - b l u e oxidase a c t i v i t y ( T a ble I). S t a t i s t i c a l a n a l y s i s of the d a t a g i v e n i n T a b l e I revealed a c o r r e l a t i o n coefficient of r = 0.82, w h i c h indicates a significant r e l a t i o n between these two p a r a m e t e r s . W e therefore consider t h i s enzyme as a t o o l for g e t t i n g l i g n i n i n t o aqueous s o l u t i o n . P a r t of our f u t u r e work w i l l concentrate o n this specific e n z y m a t i c a c t i v i t y . Laccase from White-Rot Fungi. A l t h o u g h there is no s t r i n g e n t c o r r e l a t i o n between laccase a c t i v i t y a n d l i g n i n d e c o m p o s i t i o n (e.g., T a b l e I), a p r o m i nent role of t h i s enzyme i n l i g n i n d e g r a d a t i o n has been discussed b y several a u t h o r s ( 4 , 7 , 1 1 ) . T h e presence of laccase has also been s h o w n to result i n p o l y m e r i z a t i o n b o t h in vivo ( 1 2 , 1 3 ) a n d in vitro ( 1 4 , 1 5 ) . Laccase acts o n phenolics v i a a non-specific o x i d a t i o n w h i c h generates q u i n o i d i n t e r m e diates. T h i s results i n the f o r m a t i o n of reactive intermediates w h i c h m a y subsequently p o l y m e r i z e (16-18). It is therefore reasonable to expect t h a t the low m o l e c u l a r weight substances c o m i n g f r o m e n z y m a t i c a l l y degraded l i g n i n are r e a d i l y r e p o l y m e r i z e d , a n d p o l y m e r i z a t i o n m a y be d o m i n a t i n g over d e p o l y m e r i z a t i o n processes d u r i n g l i g n i n t r a n s f o r m a t i o n in vitro (19). I n a d d i t i o n t o p o l y m e r i z a t i o n , w h i c h a m o u n t s to a c r o s s l i n k i n g o f l i g n i n v i a o x i d a t i v e c o u p l i n g , t h i s group of enzymes also catalyzes another i m p o r t a n t r e a c t i o n i n l i g n i n : It h y d r o x y l a t e s p h e n o l i c s u b s t r a t e s , t h e r e b y i n t r o d u c i n g phenolic h y d r o x y l groups w h i c h serve as new reactive sites on the molecule. These c a t a l y t i c properties make the e n z y m e a n i n t e r e s t i n g c a n d i d a t e for a v a r i e t y of possible uses i n l i g n i n b i o t r a n s f o r m a t i o n . A l t h o u g h the role of the enzyme d u r i n g l i g n i n d e g r a d a t i o n in vivo does not seem to have been e l u c i d a t e d so far, there are several i n d i c a t i o n s t h a t t h i s a c t i v i t y is i m p o r t a n t for the f u n g i w h i c h generate the e n z y m e . W e have s t u d i e d t h i s u s i n g the w h i t e - r o t fungus Heterobasidion annosum, w h i c h causes root a n d b u t t rot i n conifers. I n these i n v e s t i g a t i o n s we f o u n d the f o l l o w i n g : • A l t h o u g h H. annosum has a n e x t r e m e l y h i g h v a r i a t i o n i n the i s o e n z y m e p a t t e r n o f other enzymes, i t p r o d u c e d o n l y a single b a n d i n the isoelectric focusing e x p e r i m e n t of the laccase p r e p a r a t i o n s i s o l a t e d f r o m more t h a n 60 different s t r a i n s (20). • A l t h o u g h H. annosum is r e a d i l y m u t a g e n i z e d by a v a r i e t y of agents, i t



364

T a b i c I. Peroxidase a n d O x i d a s e P r o d u c t i o n b y Selected W o o d - I n h a b i t i n g F u n g i , a n d their C a p a c i t y to D e m e t h y l a t e a n d S o l u b i l i z e L i g n i n U n i t s χ 1000/1 m g M y c e l i u m


Trametes versicolor Polyporus pinsitus Phallus impudicus Oudemansiella radicata Bjerkandera adusia Pleurotus florida F6 Pleurotus florida PP Polyporus platensis Ustulina deusta Polyporus varius Xylaria polymorpha Phlebia radiata Polyporus brumalis Merulius tremellosus Daedaleopsis confragosa 1

3

4

5

Solubi lization'

2.9 5.6

29.5 28.0

52.1 65.1

14.0

0.0

27.1

13.3

2.2

5.1

1.4

16.0

20.3

7.6

8.6

0.0

16.8

19.5

1.7

26.5

1.7

14.6

35.3

1.7

20.6

3.9

14.0

45.3

1.6 1.0 3.2

24.0 5.6 7.1

0.9 0.8 0.0

14.0 14.0 12.0

60.0 19.2 16.9

1.7 3.9

0.9 25.0

0.3 4.7

12.0 11.0

15.8 56.8

1.5

23.4

1.8

10.6

56.1

2.8

27.0

1.2

8.0

58.9

3.4

13.4

0.0

5.0

31.2

Laccase

1.6 1.7

20.8 33.3

2.5

1

4

3

1 U n i t = increase of A /mm/l m l m e d i u m using veratryl alcohol and H 0 at p H 3. 1 U n i t = decrease of the adsorbance r a t i o A^s/A^/ ^ m e d i u m after 24 h i n c u b a t i o n u s i n g 0 . 0 1 % P o l y B l u e . 1 U n i t = change of A / m i n / m l m e d i u m using 0.01% T M B . F i g u r e s i n d i c a t e a c c u m u l a t i v e release of C 0 using C H 0 - o r g a n o solv-lignin. F i g u r e s i n d i c a t e C - w a t e r solubles i n c u l t u r e i n % of i n i t i a l Cactivity using C-/?-organosolv lignin. 31Q

2

2

Demethyl ation

Poly-Blue Oxidase

Ligninperoxidase

Fungus

2

1

6 4 5

1

4

2

1 4

3

1 4

1 4

14

was i m p o s s i b l e to detect laccase-free m u t a n t s i n the m a n y thousands of m u t a g e n i z e d clones w h i c h were inspected (21). • T h e s o p h i s t i c a t e d r e g u l a t i o n of the synthesis a n d secretion of the en-


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z y m e after i n d u c t i o n w i t h a s u i t a b l e p h e n o l as revealed b y d e n s i t y l a b e l l i n g studies (6) indicates t h a t t h i s enzyme is under s t r i n g e n t m e t a b o l i c c o n t r o l o f the o r g a n i s m . Laccase Activity and Properties of the Immobilized Enzyme in Organic Solvents. It has recently been s h o w n t h a t a n u m b e r o f enzymes, w h e n i m m o b i l i z e d , c a n express their a c t i v i t y i n a r e a c t i o n m e d i u m i n w h i c h the b u l k o f water has been replaced b y organic solvents (22-26). F u r t h e r m o r e , K l i b a n o v a n d co-workers (27) have s h o w n t h a t l i g n i n c o u l d be d e p o l y m e r ized w i t h horseradish peroxidase i n organic m e d i a , a n o b s e r v a t i o n however w h i c h is s t i l l s u r r o u n d e d b y controversy (28). Because o f these findings, a n d i n v i e w o f the fact t h a t the b u l k o f the c o m m e r c i a l l y available l i g n i n s are w a t e r - i n s o l u b l e , we have recently c o n c e n t r a t e d o n h o w t o use the e n z y m e i n solvents i n w h i c h organosolv a n d kraft l i g n i n s w o u l d be soluble, t o o . For o u r studies we used laccase f r o m the basidiomycete Trametes versicolor, w h i c h was p u r i f i e d u s i n g D E A E - S e p h a d e x A - 5 0 c h r o m a t o g r a p h y . W h e n a l y o p h i l i z e d powder o f the purified laccase was a d d e d t o a s o l u t i o n o f d i m e t h o x y p h e n o l ( D M P ) or s y r i n g a l d a z i n e i n w a t e r - s a t u r a t e d e t h y l a c e t a t e , the color of the substrates t u r n e d r a p i d l y yellow or r e d - v i o l e t , i n d i c a t i n g t h a t t h e u s u a l l y observed laccase reaction takes place indeed under those c o n d i t i o n s . However, the above described enzyme reaction s y s t e m has several disadvantages a n d inconveniences, s i m i l a r t o those described for the horseradish peroxidase i n organic solvents (26): Since the e n z y m e does n o t dissolve i n the organic solvent, i t forms particles w h i c h c l u m p together a n d stick t o the walls o f the reaction vessel. M o r e o v e r , the o v e r a l l r e a c t i o n is slow a n d i r r e p r o d u c i b l e , since o n l y the active sites at t h e surface o f the c l u m p s are available for catalysis a n d the size o f the i n d i v i d u a l c l u m p s varies considerably. N o c o r r e l a t i o n c o u l d therefore be established between the a m o u n t o f enzyme a n d the extent o f its r e a c t i o n . These p r o b l e m s were overcome b y i m m o b i l i z i n g the e n z y m e . Since the u s u a l m e t h o d s for i m m o b i l i z a t i o n of laccase d i d n o t w o r k , we a d o p t e d a new m e t h o d , details o f w h i c h w i l l be described elsewhere (29). R e a s o n a b l e measurements were possible w i t h this technique. T y p i c a l p a t t e r n s o f l a c case a c t i v i t y c o u l d be m o n i t o r e d v i a the changes of absorbance o f 2 , 6 D M P a n d s y r i n g a l d a z i n e . W h e n the r e a c t i o n took place i n o r g a n i c solvents, the a b s o r p t i o n s p e c t r a o f the p r o d u c t s were s i m i l a r t o those o b t a i n e d for the same r e a c t i o n i n buffer. F u r t h e r m o r e , the c a t a l y t i c a c t i o n o f the T. versicolor laccase followed M i c h a e l i s - M e n t e n - k i n e t i c s i n m o s t o f the o r g a n i c solvents w h i c h were tested (see T a b l e II for specific e x a m p l e s ) . E x p r e s s i o n o f the c a t a l y t i c c a p a c i t y o f the i m m o b i l i z e d laccase w a s also observed i n more t h a n a dozen different solvents, p r o v i d e d t h a t they were either s a t u r a t e d w i t h water o r , i n the case o f solvents m i s c i b l e w i t h w a t e r , s m a l l a m o u n t s o f water h a d been added ( T a b l e I I I ) . N o e n z y m a t i c r e a c t i o n was observed when the solvents tested were free o f w a t e r . N o c o r r e l a t i o n was f o u n d between the a c t i v i t y o f the i m m o b i l i z e d laccase a n d the h y d r o p h o b i c i t y o f the solvent i n w h i c h the reaction took place. T h e rate of laccase r e a c t i o n i n ethylacetate was o n l y twice t h a t i n toluene, despite the fact t h a t w a t e r - s a t u r a t e d ethylacetate contains 50 times more water t h a n



366

T a b l e II. K i n e t i c P a r a m e t e r s of O x i d a t i o n of 2,6 D M P a n d S y r i n g a l d a z i n e w i t h Laccase i n O r g a n i c Solvents

Substrate 2,6 D M P

Incubation Media

Km mM

Reaction Rate

E t h y l acetate Acetonitrile Acetone Tetrahydrofuran

0.52 1.10 0.65 1.06

11.6Δ468 n m · m i n " - m g E " 12.6Δ468 n m · m i n " - m g E " 21.3Δ468 n m · m i n " - m g E " 3.4Δ468 n m · m i n " " - m g E

E t h y l acetate Acetonitrile Acetone Tetrahydrofuran

0.15 0.08 0.08 0.31

216 μπιοί 282 μπιοί 206 μπιοί 36 μπιοί ·

a

6


6

Syringaldazine

a

6

6

a

6

6

1

1

1

1

1

1

• min - mg E " • m i n- · m g E " • m i n- - m g E " m i n "- · mg E "

-

1

_ 1

1

1

1

1

1

1

1

1

E t h y lacet ate was p r e s a t u r a t e d w i t h water. C o n t a i n i n g 7% ( v / v ) w a t e r .

w a t e r - s a t u r a t e d toluene (30). Toluene, o n the other h a n d , reacted ten t i m e s as fast as m e t h a n o l . T h e r e a c t i o n d i d not t a k e place i n either 3.5% aqueous d i m e t h y l s u l f o x i d e or 3.5% aqueous d i m e t h y l f o r m a m i d e . T h u s , i m m o b i l i z e d laccase seems to be tolerant of a m u c h w i d e r range of solvents i n w h i c h i t can f u n c t i o n t h a n was reported for peroxidase (26). T h i s m a y be due t o the fact t h a t different methods of i m m o b i l i z a t i o n have been e m p l o y e d . A d d i t i o n of water always enhanced the efficiency of i m m o b i l i z e d l a c case. T h e o p t i m a l a m o u n t s of water were s u r p r i s i n g l y l o w : rates of s y r i n g a l d a z i n e o x i d a t i o n c o m p a r a b l e to those observed i n buffer were observed i n 6 5 % aqueous a c e t o n i t r i l e , 5 0 % aqueous acetone, 5 0 % aqueous dioxane, a n d others. I m m o b i l i z e d laccase is s u r p r i s i n g l y stable w h e n stored i n o r g a n i c solvents. S t o r e d i n n-hexane at 3 0 ° C , i t was stable for more t h a n eight days w i t h less t h a n 1 0 % loss of i n i t i a l a c t i v i t y , whereas at the same c o n d i tions the s y s t e m lost about 9 0 % of its a c t i v i t y w h e n stored i n buffer. T h e a d d i t i o n of water to the pure solvents h a d a d e t r i m e n t a l effect on s t a b i l i t y . I n s u m m a r y , the d a t a i n d i c a t e t h a t i m m o b i l i z e d laccase retains m u c h of i t s a c t i v i t y i n o r g a n i c solvents. T h i s m a y have i m p o r t a n t technological implications. Application of Laccase as a Radical Donor in Adhesives for Particle Boards. T h e properties of laccase w i t h regard to l i g n i n render it a c a n d i d a t e for a p p l i c a t i o n i n t e c h n i c a l processes. A two-component adhesive was f o r m u l a t e d w i t h l i g n i n as the phenolic component a n d laccase as r a d i c a l donor. T h e process is described i n more d e t a i l elsewhere (1). It is evident t h a t the a p p l i c a t i o n of a b i o l o g i c a l c a t a l y s t , the e n z y m e , i n a t e c h n i c a l process ( i n t h i s case p a r t i c l e b o a r d p r o d u c t i o n ) d e m a n d s as conditiones sine qua non the f o l l o w i n g parameters: i . T h e e n z y m e has to be p r o d u c e d o n inexpensive substrates i n large quantities; i i . It m u s t be a p p l i c a b l e i n crude f o r m w i t h o u t p r i o r p u r i f i c a t i o n ; i i i . It has to be reasonably stable at r o o m t e m p e r a t u r e ;


27.

HUTTERMANN ET A L


367

T a b l e I I I . T h e R a t e s of L a c c a s e - C a t a l y z e d O x i d a t i o n of S y r i n g a l d a z i n e i n Different O r g a n i c Solvents Reaction Rate / / m o l e / m i n / m g enzyme

Solvent E t h y l acetate Toluene Benzene Ether Isooctane n-Hexane Cyclohexane Chloroform Dichlorethane Acetonitrile Acetone Ethanol 1.4-Dioxane Tetrahydrofuran Methanol D i m e t h y l sulfoxide Ν ,N-Dimethylformamide a

0

a


a

a

a

a

0

a

6

6

6

6

6

6

6

6

216 104 81 75 68 62 48 15 8 116 80 28 16 11 10 0 0

a N o r e a c t i o n was observed i n the a n h y d r o u s solvents; the solvents were

p r e s a t u r a t e d w i t h d i s t i l l e d water at r o o m t e m p e r a t u r e . b R e a c t i o n took place i n solvent following a d d i t i o n of 3.5% ( v / v ) of d i s tilled water. i v . Since p a r t i c l e boards are pressed at h i g h t e m p e r a t u r e s , the e n z y m e must be heat tolerant; v . B o a r d pressing has to follow current state-of-art, w i t h press t i m e s as short as possible; a n d v i . T h e price of the final p r o d u c t has to be c o m p e t i t i v e w i t h c o n v e n t i o n a l p e t r o c h e m i c a l resins. W e have succeeded w i t h the development of a n e n z y m e - c a t a l y z e d a d hesive s y s t e m o n l i g n i n basis t h a t meets a l l the requirements s t a t e d above. T h i s s y s t e m is based o n a basidiomycete (Trametes versicolor), grown in a fermenter o n spent sulfite l i q u o r a n d a m i n o p h e n o l as a d d i t i o n a l e n z y m e i n d u c i n g agent. A m o n g m a n y other basidiomycetes, t h i s fungus was cho sen because of its h i g h e x t r a c e l l u l a r laccase a c t i v i t y w h i c h p r o d u c e d best adhesion results. T h e c u l t u r e b r o t h o b t a i n e d after 4 days of c u l t i v a t i o n is concentrated b y e v a p o r a t i o n or u l t r a f i l t r a t i o n , a n d the r e s u l t i n g concen t r a t e d , crude e n z y m e s o l u t i o n is a p p l i e d d i r e c t l y i n the b i n d i n g s y s t e m . A sterile storage of the enzyme concentrate at r o o m t e m p e r a t u r e is possible for at least one m o n t h . C o m p a r e d t o the c h e m i c a l l y cured adhesive systems i n c o m m o n use today, t h i s e n z y m a t i c b i n d e r has c e r t a i n advantages: 1. It is based o n the t o t a l u t i l i z a t i o n of waste l i g n i n , first as b i n d i n g




368

c o m p o n e n t i n the adhesive a n d second as n u t r i e n t source for e n z y m e production. 2. Because of the h i g h c a t a l y t i c a c t i v i t y of the e n z y m e , the b i n d i n g p r o cess can be p e r f o r m e d under m i l d c o n d i t i o n s w i t h o u t a p p l i c a t i o n of large a m o u n t s of h a r m f u l chemicals. 3. I n contrast to p a r t i c l e boards b o n d e d w i t h s y n t h e t i c resins, the " b i o b o n d e d " boards do not e m i t any h a r m f u l vapors as do, for example, formaldehyde-bonded boards. T h e p r o d u c t i o n of the " b i o b o n d e d " p a r t i c l e boards has been described recently i n d e t a i l ( 1 , 3 1 ) . A n a l y t i c a l results b y 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 a n d u l t r a c e n t r i f u g a t i o n have been presented i n s u p p o r t of a n in vivo a n d in vitro p o l y m e r i z a t i o n of l i g n i n sulfonates. Besides p o l y m e r i z a t i o n , the l i g n i n molecule was f o u n d to be m o d i f i e d b y c a r b o x y l a t i o n , a n d t h i s was s h o w n b y difference spectroscopy (32). T h e f o l l o w i n g is a short s u m m a r y of p e r t i n e n t results. M i l l e d w o o d l i g n i n was m i x e d w i t h the crude e n z y m e s o l u t i o n of Trametes versicolor e x t r a c e l l u l a r phenoloxidases p r o d u c e d o n spent sulfite l i q u o r i n a r a t i o of a p p r o x i m a t e l y 2 : 1 . T h i s c o m p r i s e d the m a i n p a r t of the twoc o m p o n e n t "bio-adhesive." I n d u s t r i a l particles were b o n d e d w i t h 1 5 % b i o adhesive under c o n v e n t i o n a l pressing c o n d i t i o n s to have 19 m m p a r t i c l e boards (40 x 50 cm) of the properties described i n T a b l e I V . T h e b o n d i n g r e a c t i o n (crosslinking) took place i n aqueous s o l u t i o n at r o o m t e m p e r a t u r e . If c o n v e n t i o n a l pressing technology is a p p l i e d , the t e m p e r a t u r e s h o u l d be elevated i n order to m a i n t a i n water e v a p o r a t i o n w i t h i n a reasonable press time. T a b l e I V . T e c h n o l o g i c a l a n d M e c h a n i c a l P r o p e r t i e s of " B i o b o n d e d " P a r t i c l e B o a r d s : Effect of L i g n i n T y p e , E n z y m e A c t i v i t y a n d P r e s s i n g T i m e

Lignin Type

Press Time (min

Density (kg/m )

Versai Tensile Strength (N/mm )

1 -f enzyme 2 + enzyme 2 -f enzyme

5 5 3

774 775 745

0.47 0.42 0.44

5.4% 5.4% 6.7%

5 3

734 700

0.18 0.37

40.1% 10.0%

3

2

Thickness Swelling** After 2h

24h 24.0% 19.9% 29.3%

Controls: 1 — enzyme* Urea formaldehyde (66%)

Enzymatic Modification of Lignin for Technical Use ... - ACS Publications

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