Structure and Properties of the LigninâCarbohydrate Complex

Chapter 2

Structure and Properties of the Lignin-Carbohydrate Complex Polymer as an Amphipathic Substance T . Koshijima , T . Watanabe , and F . Y a k u 1

1

1

2

Wood Research Institute, Kyoto University, Gokasho, U j i , Kyoto 611,

Downloaded by UNIV OF PITTSBURGH on September 23, 2013 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0397.ch002

Japan 2

Government Industrial Research Institute, Osaka 563, Japan

It has been f o u n d t h a t l i g n i n - c a r b o h y d r a t e complexes (LCC's) consist o f sugar chains a n d r e l a t i v e l y s m a l l l i g n i n fragments a t t a c h e d as p e n d a n t side chains; they have number-average m o l e c u l a r weights of ca. 6000-8000. T h e linkage between sugar a n d l i g n i n was d e t e r m i n e d t o be m a i n l y o f the b e n z y l ether t y p e by a n e w l y developed m e t h o d u s i n g DDQ o x i d a t i o n . Some of the LCC's exh i b i t a s t r o n g tendency to f o r m micelles or aggregates in aqueous s o l u t i o n due t o h y d r o p h o b i c a n d also electrostatic interactions. Since B j o r k m a n first proposed the existence o f l i g n i n - c a r b o h y d r a t e c o m plexes ( L C C ' s ) as species i n c a p a b l e of s e p a r a t i o n i n t o the respective c o m ponents b y selective c h e m i c a l t r e a t m e n t s or s p e c i a l p u r i f i c a t i o n techniques, e x p e r i m e n t a l results s u p p o r t i n g the presence o f L C C ' s i n w o o d have been r e p o r t e d b y M e r e w e t h e r (1), K o s h i j i m a (2), Wegener (3), a n d Y a k u (4). T h e r e a f t e r , a lot of experiments designed to isolate fractions c o n t a i n i n g L C C ' s f r o m p a r t l y degraded w o o d preserving the n a t i v e c o n f i g u r a t i o n t o different extents have been c o n d u c t e d . A m o n g t h e m , B j o r k m a n (5), B r o w n e l l (6), K o s h i j i m a (7), Y a k u (8,9), E r i k s s o n (10) a n d W a t a n a b e (11) have ext r a c t e d L C C ' s f r o m finely d i v i d e d w o o d powder f r o m w h i c h m i l l e d w o o d l i g n i n h a d been e x t r a c t e d previously. B y c o n t r a s t , L u n d q u i s t (12), A z u m a (13), M u k o y o s h i (14), T a k a h a s h i (15) a n d K a t o (16) have separated the L C C ' s c o n t a i n e d i n a m i l l e d w o o d l i g n i n f r a c t i o n e x t r a c t e d w i t h 8 0 % aqueous dioxane f r o m v e r y fine w o o d powder. M e a n w h i l e , F r e u d e n b e r g (17) was the first person w h o d e m o n s t r a t e d the f o r m a t i o n o f a n a d d i t i o n c o m p o u n d f r o m a q u i n o n e m e t h i d e a n d s u crose d u r i n g e n z y m a t i c d e h y d r o g e n a t i o n o f c o n i f e r y l a l c o h o l i n a concent r a t e d sucrose s o l u t i o n . T h e r e a f t e r , T a n a k a (18) observed the f o r m a t i o n of a b e n z y l ester between the q u i n o n e m e t h i d e o f a d i l i g n o l a n d a u r o n i c 0097-6156/89/0397-0011$06.00A> © 1989 American Chemical Society

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

LIGNIN: PROPERTIES AND MATERIALS


12

a c i d . K a t a y a m a (19) isolated a c o m p o u n d i n w h i c h glucose is l i n k e d t o c o n i f e r y l a l c o h o l t h r o u g h a b e n z y l ether b o n d . A n a l o g o u s c o m p o u n d s were also o b t a i n e d b y Feckel (20). Since those e x p e r i m e n t s were c a r r i e d o u t i n the presence of 20 t o 500 t i m e s the a m o u n t of c o n i f e r y l a l c o h o l i n r e l a t i o n to sugar, those results i n d i c a t e d o n l y the p o s s i b i l i t y o f c h e m i c a l l i n k a g e f o r m a t i o n between l i g n i n precursors a n d sugars. N o t e w o r t h y results have been o b t a i n e d f r o m electron m i c r o s c o p i c o b servations of L C C ' s b y Fengel (21) a n d K o s i k o v a (22). W h e n c o m p a r i n g these t w o schematic i l l u s t r a t i o n s , coiled a n d w o u n d fibrils of p o l y s a c c h a rides are b u r i e d i n the l i g n i n m a t r i x i n b o t h cases, b u t the p o l y s a c c h a r i d e fibril i n FengePs scheme is m u l t i p l y connected t o a b i g l i g n i n p a r t i c l e . I n K o s i k o v a ' s scheme, a fibril is a t t a c h e d to a r e l a t i v e l y s m a l l l i g n i n p a r t i c l e at o n l y one p o s i t i o n . D e s p i t e so m a n y investigations of L C C ' s , no c h e m i c a l p r o o f has been advanced so far a b o u t the k i n d of linkage c o n n e c t i n g the sugar t o the l i g n i n moiety. A l s o , no d i s t i n c t m a c r o m o l e c u l a r c o n f i r m a t i o n has yet been p r o v i d e d for the s c h e m a t i c m o l e c u l a r forms t h a t have been proposed o n the basis of electron microscopic observations. I n t h i s p a p e r , the molecular shape a n d micelle or aggregate f o r m a t i o n of L C C molecules, the n a t u r e of the s u g a r - l i g n i n linkage, a n d the m o l e c u l a r weights of L C C ' s w i l l be d o c u m e n t e d o n c h e m i c a l or p h y s i c o c h e m i c a l grounds. New Methods Oligomers

for

Extraction of L C C ' s

and Isolation of

L C C

T h e use of h i g h - b o i l i n g p o i n t solvents, such as d i m e t h y l f o r m a m i d e or d i m e t h y l s u l f o x i d e used b y B j o r k m a n for e x t r a c t i o n of L C C ' s f r o m spruce w o o d , m a y lead to d e n a t u r a t i o n or p a r t i a l d e g r a d a t i o n o f L C C ' s i n the course of solvent r e m o v a l or c o n c e n t r a t i o n . W e have developed the f o l l o w i n g two m e t h o d s for easily o b t a i n i n g L C C ' s . O n e involves i s o l a t i o n o f L C C ' s f r o m a n aqueous 8 0 % dioxane e x t r a c t o b t a i n e d f r o m finely d i v i d e d w o o d p o w d e r . A f t e r s e p a r a t i n g the p r e c i p i t a t e of m i l l e d w o o d l i g n i n f r o m the aqueous s o l u t i o n r e m a i n i n g after dioxane r e m o v a l , the L C C ' s i n the s u p e r n a t a n t are t a k e n out a n d purified b y u s i n g a p y r i d i n e - a c e t i c a c i d w a t e r - c h l o r o f o r m solvent s y s t e m t o remove sugar-free l i g n i n . T h e L C C - W t h u s o b t a i n e d is analogous t o B j o r k m a n L C C i n respect t o c h e m i c a l c o m p o s i t i o n a n d m o l e c u l a r weight, b u t differs i n y i e l d w h i c h ranges f r o m 0.75 to 1.1% of w o o d (13). T h e second way consists o f hot-water e x t r a c t i o n o f finely d i v i d e d w o o d p o w d e r , p r e v i o u s l y e x t r a c t e d w i t h aqueous dioxane for r e m o v i n g m i l l e d w o o d l i g n i n . T h e r e s u l t a n t L C C - W E is o b t a i n e d i n 9.3-10.0% y i e l d a n d does not differ f r o m B j o r k m a n L C C i n r e l a t i o n to c h e m i c a l c o m p o s i t i o n a n d m o l e c u l a r weight (11) (see T a b l e I). T h i s fact is v e r y significant i n d e m o n s t r a t i n g t h a t L C C ' s c o n t a i n i n g u p t o 1 8 % l i g n i n are able t o be ext r a c t e d w i t h hot w a t e r alone f r o m w o o d powder. I n order t o estimate the frequency of l i g n i n - s u g a r linkages i n p i n e L C C ' s , L C C oligomers were a t t e m p t e d t o be prepared f r o m L C C - W E of p i n e w o o d . T h e greatest p r o b l e m s to be solved were the s e p a r a t i o n o f


KOSHIJIMA ETAL.

2.

Complex Polymer as an Amphipathic Substance

13

T a b l e I. C h e m i c a l C o m p o s i t i o n a n d P r o p e r t i e s o f P i n e L C C - W Lignin-Carbohydrate Complexes Components

LCC-WE


R e c o v e r y (%) C a r b o h y d r a t e content N e u t r a l sugar Uronic acid L i g n i n content (%) A c e t y l content Mi? S(S) M M w

n

a

6

c

(%)

C-l-A

C-l-M 43.4*

9.3°

C-l-R 2.1*

48.7*

(%) 80.0 4.2 17.9

95.5 N.D. 3.7

76.0 6.4 26.6

41.5 1.9 43.6

3.3 -15.5° N.D.

7.6 -28.2° 0.9

N.D. -11.4° 0.8

N.D. -8.0° N.D.

1.2 x 1 0 7.6 x 1 0

1.2 x 1 0 7.5 x 1 0

1.1 x 1 0 6.7 x 1 0

C

C

4

3

e

e

4

3

C

4

3

N.D. N.D.

C

C

V a l u e s are expressed as weight percentages o f the w o o d m e a l e x t r a c t e d w i t h 8 0 % aqueous dioxane. V a l u e s are expressed as weight percentages o f L C C - W E . Not determined.

L C C oligomers f r o m oligosaccharides a n d the p r e v e n t i o n o f reaggregation o f L C C oligomers t h r o u g h h y d r o p h o b i c i n t e r a c t i o n s . C h r o m a t o g r a p h y o f a n e n z y m e - d e g r a d e d C - l - A f r a c t i o n was successful o n a T o y o p e a r l H W - 4 0 S c o l u m n w h i c h h a d been a l r e a d y d e m o n s t r a t e d t o adsorb o n l y l i g n i n - c o n t a i n i n g oligosaccharides (23). T h e m i x t u r e o f L C C oligomers a n d oligosaccharides was p r e p a r e d b y h y d r o l y z i n g the f r a c t i o n C - l - A t h a t h a d been f r a c t i o n a t e d f r o m L C C - W E w i t h purified C e l l u l o s i n A C ( p r o t e i n 93.7%) a n d t h e n w i t h a m i x t u r e o f purified Meicelase ( p r o t e i n 92.2%) a n d C e l l u l o s i n A C at 40° C for 72 h o u r s . T h e e n z y m e - d e g r a d e d p r o d u c t s were i n t r o d u c e d o n t o the T o y o p e a r l H W - 4 0 S c o l u m n , w h i c h was e l u t e d w i t h water t o remove the oligosaccharide components ( A - E S W ) a n d subsequently w i t h 5 0 % aqueous d i o x a n e t o recover the adsorbed L C C oligomers ( A - E S D ) , w h i c h c o n t a i n e d n o c o n t a m i n a t i n g l i g n i n or oligosaccharide (see ref. 23 a n d T a b l e II). Estimation of Lignin-Sugar Linkages i n L C C ' s In 1982, O i k a w a et al. r e p o r t e d t h a t 2,3-dichloro-5,6-dicyanobenzoquinone ( D D Q ) reacts w i t h 4 - m e t h o x y b e n z y l ether t o give the a l c o h o l q u a n t i t a t i v e l y (24). P r o v i d e d t h a t D D Q a t t a c k s specifically at the p - a l k o x y b e n z y l ether l i n k a g e , direct evidence c a n be o b t a i n e d for the occurrence o f b e n z y l i c l i g n i n - c a r b o h y d r a t e linkages w h i c h m a y be present i n L C C molecules. N i n e m o d e l c o m p o u n d analogs of L C C ' s were synthesized a n d s u b j e c t e d to r e a c t i o n w i t h D D Q at 5 0 ° C for 1 h r or 40° C for 24 hrs i n 5 0 % d i o x a n e s o l u t i o n (25). F u r t h e r , m o d e l c o m p o u n d s ( I - I V ) were also s y n t h e s i z e d t o check the effect o f s u b s t i t u e n t s at the para p o s i t i o n s o f the g u a i a c y l moieties u p o n the release o f sugar residues b y D D Q o x i d a t i o n ( F i g . 1). A t w o - h o u r r e a c t i o n o f the m o d e l c o m p o u n d s w i t h D D Q at the b o i l i n g t e m p e r a t u r e




F i g u r e 1. S y n t h e s i z e d m o d e l

compounds.


K O S H U I M A ET AL.

2.


15

T a b l e I I . N e u t r a l S u g a r C o m p o s i t i o n of L C C S u b f r act ions Lignin-Carbohydrate Complexes


Components

C-l-A

A-ESD

C a r b o h y d r a t e content, %

76.0

8.3

Carbohydrate composition": L-Arabinose D-Xylose D-Mannose D-Galactose D-Glucose

7.3 49.5 27.0 7.2 9.1

7.5 17.6 48.7 7.7 18.6

a

V a l u e s are expressed as m o l e percentages o f the t o t a l n e u t r a l sugars.

o f the d i c h l o r o m e t h a n e - w a t e r (18:1) m i x t u r e e m p l o y e d was o p t i m u m (26). T h e L C C models 3 - m e t h o x y - 4 - h y d r o x y (I) a n d 3 - m e t h o x y - 4 - b e n z y l o x y b e n z y l ether ( I V ) were o x i d a t i v e l y decomposed b y D D Q , w h i l e the p - a c e t o x y L C C m o d e l c o m p o u n d II was inert to the o x i d a t i o n process because o f the electron w i t h d r a w i n g i n d u c t i v e effect of the acetoxy g r o u p , as r e p o r t e d b y B e c k e r . F u r t h e r m o r e , a l l o f the /?-ether linkages i n the L C C models I - I V were stable d u r i n g the D D Q o x i d a t i o n . However, the L C C m o d e l c o m p o u n d III h a v i n g the 3 , 4 - d i m e t h o x y p h e n y l m o i e t y was stable d u r i n g D D Q o x i d a t i o n under these c o n d i t i o n s . P r o b a b l y , t h i s is because the o x i d a t i o n p o t e n t i a l of the L C C m o d e l III is not low enough t o f o r m a charge t r a n s fer c o m p l e x w i t h D D Q . In fact, w h e n the para p o s i t i o n was s u b s t i t u t e d w i t h the more electron d o n a t i n g b e n z y l o x y g r o u p , q u a n t i t a t i v e o x i d a t i v e cleavage was observed i n the L C C m o d e l I V . M o s t o f the p a m - s u b s t i t u e n t s i n l i g n i n s are far more electron d o n a t i n g t h a n the m e t h o x y l g r o u p a n d , i n m a n y cases, t h a n the b e n z y l o x y group i n their effects. T h u s , i t m a y be c o n c l u d e d t h a t D D Q effectively decomposes n o n p h e n o l i c b e n z y l ether linkages i n l i g n i n s . G l y c o s i d i c b o n d s between sugar residues, however, were i n e r t to D D Q o x i d a t i o n (24,25). T o identify w h i c h sugar h y d r o x y Is p a r t i c i p a t e i n b e n z y l ether linkages t o l i g n i n , W a t a n a b e a n d K o s h i j i m a (23) developed a new m e t h o d w h i c h i n volves a c e t y l a t i o n o f L C C ' s or L C C oligomers followed b y D D Q o x i d a t i o n a n d P r e h m ' s m e t h y l a t i o n . Before a p p l i c a t i o n o f t h i s m e t h o d t o L C C ' s , i t was confirmed b y u s i n g 1,2,3,4-tetraacetyl- a n d 1 , 2 , 3 , 6 - t e t r a a c e t y l - D glucose t h a t D D Q does not cause a c e t y l group r e m o v a l f r o m or m i g r a t i o n i n the sugar a n d t h a t P r e h m ' s m e t h y l a t i o n does not i n d u c e a c e t y l m i g r a t i o n . A c c o r d i n g to t h i s m e t h o d (Scheme 1), the L C C oligomers A - E S D were s u b j e c t e d to a n a l y s i s of the t y p e of linkage present a n d the c o r r e s p o n d i n g m o n o m e t h y l a t e d sugars are s u m m a r i z e d i n T a b l e I I I . T h e sites o f b e n z y l ether linkages t o the sugar moieties of the L C C ' s are p r e d o m i n a n t l y at C - 6 i n hexoses a n d m o s t l y at C - 3 a n d somewhat less so at C - 2 i n pentoses. T h e sugars l i b e r a t e d by t h i s r e a c t i o n are the ones located at the a - c a r b o n s of l i g n i n p h e n y l p r o p a n e u n i t s etherified at the p - h y d r o x y l p o s i t i o n . M o s t o f


16


the n o n - p h e n o l i c b e n z y l ether linkages are easily b r o k e n b y the a c t i o n o f D D Q due t o the enhanced e l e c t r o n - d o n a t i n g properties o f para s u b s t i t u t e d p h e n y l p r o p a n e u n i t s . H o w e v e r , phenolic h y d r o x y l s are a c e t y l a t e d d u r i n g the first step o f t h i s m e t h o d i n p h e n o l i c b e n z y l ethers a n d the enhanced electron w i t h d r a w i n g properties o f the a c e t o x y groups m a k e cleavage o f the b e n z y l ether l i n k e d sugar residues (as s h o w n i n Scheme 1) d i f f i c u l t . T a b l e I I I . M e t h y l E t h e r s o f M o n o s a c c h a r i d e s f r o m the H y d r o l y z a t e o f Methylated L C C Oligomers ( A - E S D )


M e t h y l a t e d Sugars

a

Mol %

a

2- 0 - m e t h y l - D - x y l o s e 3- 0 - m e t h y l - D - x y l o s e T o t a l xylose

9.2 24.2 33.4

2- O - m e t h y l - D - m a n n o s e 6- O m e t h y l - D - m a n n o s e T o t a l mannose

2.8 41.6 44.4

2- 0 - m e t h y l - D - g l u c o s e 6- O - m e t h y l - D - g l u c o s e T o t a l glucose

2.0 20.3 22.3

B a s e d o n t o t a l m e t h y l a t e d sugar c o m p o n e n t s i d e n t i f i e d .

Molecular Shape of L C C ' s T h e m o l e c u l a r sizes o f L C C ' s e x t r a c t e d f r o m finely d i v i d e d w o o d p o w d e r o f 10 t o 30 n a n o m e t e r d i a m e t e r p a r t i c l e size m a y be s o m e w h a t s m a l l e r t h a n o r i g i n a l l y the case o w i n g t o vigorous m e c h a n i c a l a c t i o n d u r i n g the course o f m i l l i n g . H o w e v e r , t h i s a c t i o n affects b o t h l i g n i n a n d p o l y s a c c h a r i d e molecules evenly (2), a n d so i t does not seem to be the case t h a t o n l y specific linkages are cleaved b y m i l l i n g . F i r s t , f r a c t i o n a t i o n of the L C C ' s e x t r a c t e d f r o m p i n e (Pinus densiflora) w o o d was a t t e m p t e d a c c o r d i n g t o the m e t h o d o f B j o r k m a n (5) i n t o three f r a c t i o n s b y means o f a d s o r p t i o n c h r o m a t o g r a p h y w i t h a D E A E S e p h a d e x c o l u m n . I n the case o f pine L C C ' s , the f r a c t i o n C - l - M e l u t i n g first w i t h w a t e r was composed o f n e u t r a l L C C ' s (fraction C - l - M - 1 , 5 % y i e l d ) consisti n g o f mannose, glucose, arabinose, xylose a n d l i g n i n , as well as lignin-free a c e t y l g l u c o m a n n a n ( 4 5 % y i e l d ) . Subsequent e l u t i o n w i t h one m o l a r a m m o n i u m carbonate solution liberated acidic L C C ' s (fraction C - l - A , 25-30% y i e l d ) w h i c h c o n s t i t u t e d the m a j o r p o r t i o n o f the p i n e L C C ' s c o n s i s t i n g o f 1 2 - 1 3 % l i g n i n , a b o u t 6% u r o n i c a c i d a n d 7 6 % n e u t r a l sugars. T h e last e l u t i o n ( f r a c t i o n C - l - R , 2 - 5 % y i e l d ) was effected b y u s i n g 10 m o l a r acetic a c i d , w h i c h released a f r a c t i o n c o n t a i n i n g a r o u n d 4 4 % l i g n i n a n d 4 3 % sugars. T h e f o l l o w i n g e x p e r i m e n t s were c o n d u c t e d u s i n g f r a c t i o n C - l - A , w h i c h was t h o u g h t t o be representative of the L C C ' s (17). T h e a c i d i c L C C f r a c t i o n , C - l - A , was p a r t i a l l y h y d r o l y z e d w i t h a cellulase p r e p a r a t i o n , C e l l u l o s i n A C , a n d changes i n the l i g n i n a n d sugar d i s t r i b u t i o n s were a n a l y z e d


2.

KOSHIJIMA ETAL.


CH OH 2


—

O-CH

WOAc —0—CH fc-0

D-Glucose 4

I

Hydrolysis

Scheme 1. E s t i m a t i o n of linkage sites i n l i g n i n - c a r b o h y d r a t e moieities.


17


18


b y means of Sephadex c o l u m n c h r o m a t o g r a p h y . F i g u r e 1, where the l i g n i n c o m p o n e n t is m a r k e d b y a d o t t e d l i n e , shows t h a t not o n l y the p o l y s a c c h a r i d e b u t also even the l i g n i n component was b r o k e n d o w n to s m a l l e r molecules b y the a c t i o n of the cellulase p r e p a r a t i o n . T h i s was q u i t e u n e x p e c t e d , since the C e l l u l o s i n A C has been confirmed t o have intense carb o x y met hylcellulase, hemicellulase a n d /?-glucosidase as w e l l as avicelase a c t i v i t i e s b u t no ligninase or peroxidase a c t i v i t i e s . F i g u r e 2 a shows the p r o file of f r a c t i o n C - l - A f r o m a Sephadex G - 1 5 c o l u m n . T h e m a j o r f r a c t i o n i n F i g u r e 2 A was secured, p a r t i a l l y h y d r o l y z e d w i t h C e l l u l o s i n A C a n d rechrom a t o g r a p h e d ( F i g . 2b). W h e n peaks I a n d I I i n F i g u r e 2b were i s o l a t e d a n d a g a i n h y d r o l y z e d w i t h the same cellulase p r e p a r a t i o n , the l i g n i n c o m ponents a g a i n separated i n t o four fractions i n the lower m o l e c u l a r weight region of the profile as s h o w n i n F i g u r e 2c (4). T h i s p h e n o m e n o n is easily u n d e r s t a n d a b l e b y a s s u m i n g the l i g n i n component of the L C C ' s t o consist of low m o l e c u l a r weight p e n d a n t - l i k e fragments a t t a c h e d to a sugar c h a i n ( F i g . 3). I n other words, C e l l u l o s i n A C c o u l d break d o w n o n l y the i n t e r c o n n e c t i n g sugar c h a i n between l i g n i n moieties, w i t h the result t h a t the l i g n i n components a p p e a r e d t o be cleaved by the cellulases a n d separated i n t o several peaks c o r r e s p o n d i n g t o different m o l e c u l a r sizes o n the g e l - f i l t r a t i o n c h r o m a t o g r a m s (4). T h i s v i e w was s u p p o r t e d b y the v i s c o s i m e t r i c b e h a v i o r of the n e u t r a l L C C ( C - l - M - 1 ) s o l u t i o n i n water (9). Micelle or Aggregate F o r m a t i o n a n d H y d r o p h o b i c Interactions of L C C Molecules in Aqueous Solution W h e n h y d r o p h i l i c a n d h y d r o p h o b i c groups are present i n a single molecule a n d t h e i r r a t i o a n d d i s t r i b u t i o n over the molecule are s u i t a b l e , the molecules associate w i t h each other a n d f o r m micelles or aggregates i n aqueous s o l u t i o n . A s L C C molecules have b o t h h y d r o p h o b i c l i g n i n a n d h y d r o p h i l i c sugar moieties, i t is reasonable t o consider t h a t they s h o u l d associate t o f o r m a g gregates or micelles. F i g u r e 4 displays the r e l a t i o n s h i p between e l e c t r i c a l c o n d u c t i v i t y of the L C C s o l u t i o n a n d c o n c e n t r a t i o n , w h i c h changes s h a r p l y at the p o i n t where the micelles or aggregates are f o r m e d , c o n f i r m i n g indeed t h a t s u c h entities do occur. T h e c r i t i c a l micelle c o n c e n t r a t i o n (c.m.c.) was 0 . 0 3 5 % i n t h i s case (8). M e a n w h i l e , p i n a c y a n o l c h l o r i d e , a k i n d of p i g m e n t , has two v i s i b l e a b s o r p t i o n m a x i m a , the a - b a n d ( A 6 0 5 n m ) a n d /?-band ( A 5 5 0 nm). These \ ' s shift to longer wavelengths w i t h increasing m o l a r e x t i n c t i o n coefficient as the L C C c o n c e n t r a t i o n increases. F i g u r e 5 i l l u s t r a t e s p l o t s of the m o l a r e x t i n c t i o n coefficients of the L C C - p i n a c y a n o l c h l o r i d e s o l u t i o n against the c o n c e n t r a t i o n of C - l - A - 1 , the purified a c i d i c L C C f r a c t i o n . T h e d i s c o n t i n u i t y (corresponding to the c.m.c.) i n F i g u r e 5 confirms t h a t m i c e l l e f o r m a t i o n occurs. F i g u r e 5 shows the same c.m.c. value, 0.035%, as F i g u r e 4, i n d i c a t i n g t h a t m i c e l l e or aggregate f o r m a t i o n takes place i n L C C s o l u t i o n s (8). F i g u r e 6 provides another instance d e m o n s t r a t i n g m i celle f o r m a t i o n or aggregation of L C C ' s i n s o l u t i o n as a result of c a t i o n i c d e t e r g e n t - L C C i n t e r a c t i o n s . T h e L C C ' s used here were isolated together w i t h m i l l e d w o o d l i g n i n f r o m finely d i v i d e d p i n e w o o d w i t h a n aqueous d i o x m a r

m a r

m a x



Figure 2. Gel-filtration patterns of enzyme-hydrolyzates of L C C ' s (fraction C - l - A ) on Sephadex G-15 column. (Reprinted with permission from réf. 4. Copyright 1976 Walter de Gruyter & Co.)


so

h*

J

s-

î

8

f

f

η

20



|

: Attack

points

of

carbohydrolase

F i g u r e 3. D e g r a d a t i o n p a t t e r n o f L C C molecule b y carbohydrolases.



2.

KOSHIJIMA ET AL.


Concentration, %

F i g u r e 4. P l o t of c o n d u c t i v i t y a g a i n s t LCC ( f r a c t i o n C-l-A-1) c o n c e n t r a t i o n . ( R e p r i n t e d w i t h p e r m i s s i o n from r e f . 8, C o p y r i g h t 1979 Walter de G r u y t e r & Co.)


21


22


Concentration

of C - l - A - 1 , %

F i g u r e 5. P l o t of molar e x t i n c t i o n c o e f f i c i e n t a g a i n s t LCC ( f r a c t i o n C-l-A-1) c o n c e n t r a t i o n . ( R e p r i n t e d w i t h p e r m i s s i o n from r e f . 8. C o p y r i g h t 1979 Walter de Gruyt e r & Co.)


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

1

volume,

1

ml

1

1

T

Figure 6. Gel-filtration o f pine L C C - W with the addition of a variety of detergents on Sepharose 4 B column. (Reprinted with permission from ref. 27. Copyright 1981.)

o oo

AAA • • •

without detergent c a t i o n i c detergent anionic detergent non-ionic detergent amphionic detergent

T


24


ane s o l u t i o n ; the f r a c t i o n is denoted b y L C C - W i n t h i s c h a p t e r . L C C - W was f r a c t i o n a t e d i n t o three f r a c t i o n s , n a m e l y W - l , W - 2 a n d W - 3 , t h r o u g h a Sepharose 4 B c o l u m n (13). W - l is the one e l u t e d at the v o i d v o l u m e , W - 2 (M = 2 x 10 ) c o n t a i n e d 5 1 % l i g n i n a n d 3 8 % n e u t r a l sugar moieties, a n d W - 3 ( M = 3 x 10 ) i n c l u d e d 2 1 % l i g n i n a n d 7 3 % sugars. 5

n


n

3

T h e a d d i t i o n o f 0 . 1 % o f a v a r i e t y o f detergents t o the eluents i n g e l filtration c h r o m a t o g r a p h y u s i n g a Sepharose 4 B c o l u m n resulted i n the decrease or disappearance o f the higher m o l e c u l a r weight f r a c t i o n W - 2 , w h i l e the lower molecular weight f r a c t i o n W - 3 r e m a i n e d at its i n i t i a l p o s i t i o n i n the c h r o m a t o g r a m . W - 3 a p p e a r e d i n the c h r o m a t o g r a m alone w h e n c a t i o n i c or a m p h i o n i c detergents were used as a d d i t i v e s (27). A s the f r a c t i o n W - 2 h a d a number-average m o l e c u l a r weight i n the region o f 1 0 a n d the r e m a i n i n g W - 3 is o f the order o f 1 0 , W - 2 corresponds to micelles or aggregates o f pine L C C ' s w h i c h are decomposed b y i n t e r a c t i o n s w i t h c a t i o n i c detergents. F u r t h e r , i t became evident f r o m t h i s e x p e r i m e n t t h a t electrostatic i n t e r a c t i o n s c o n t r i b u t e as d r i v i n g forces for m i c e l l e or aggregate f o r m a t i o n i n a d d i t i o n to h y d r o p h o b i c interactions (27). 5

3

T h e L C C molecules e x h i b i t i n g a tendency to f o r m micelles or aggregates are t h o u g h t to be confined t o those w i t h l i g n i n rsugar r a t i o s a r o u n d 1:1. T h i s is e x e m p l i f i e d b y f r a c t i o n W - 2 here. O n the other h a n d , L C C molecules c o n t a i n i n g a larger a m o u n t o f c a r b o h y d r a t e a n d less l i g n i n w o u l d l a c k the a b i l i t y to f o r m micelles as i n the case of those L C C molecules i n f r a c t i o n W - 3 w h i c h c o n t a i n e d 7 0 % sugar a n d 2 0 % l i g n i n o n average ( F i g . 7 b ) . F i g ure 7 depicts the s t r u c t u r a l d e t e r m i n a n t s of the L C C molecules a n d t h e i r micelles or aggregates proposed o n the basis o f the s u m m a r i z e d r e s u l t s . T h e L C C molecules proposed here f u r n i s h a c h e m i c a l a n d p h y s i c o c h e m i c a l basis to the images o b t a i n e d b y K o s i k o v a a n d Fengel f r o m elect r o n m i c r o s c o p i c observations. A s s h o w n i n F i g u r e 7, some o f the sugar c h a i n t e r m i n a l s are e x p e c t e d to l i n k g l y c o s i d i c a l l y t o l i g n i n moieties since sugar-free l i g n i n fragments have been isolated b y e n z y m a t i c d e g r a d a t i o n o f C - l - A u s i n g C e l l u l o s i n A C (4). H o w e v e r , there is no direct evidence c o n c e r n i n g the occurrence of g l y c o s i d i c linkages, so t h i s p r o b l e m r e m a i n s a n open q u e s t i o n . It is expected f r o m the m o l e c u l a r shapes of L C C ' s t h a t s t r o n g h y d r o p h o b i c i n t e r a c t i o n s due to the l i g n i n moieties act between the L C C molecules i n aqueous s o l u t i o n . W h e n the c h r o m a t o g r a p h y o f L C C ' s was carried o u t w i t h h y d r o p h o b i c s t a t i o n a r y phases u s i n g c o l u m n s o f p h e n y l a n d o c t y l Sepharose, 8 0 . 4 % a n d 7 7 . 6 % of the charged L C C ' s were a d s o r b e d o n t o the c o l u m n , w h i c h were b e i n g eluted w i t h a solvent m i x t u r e c o m p o s e d o f a n i n c r e a s i n g c o n c e n t r a t i o n o f e t h y l cellosolve (0, 15, 30, 45 a n d 50%) a n d decreasing c o n c e n t r a t i o n of a m m o n i u m sulfate (0.8, 0.6, 0.4, 0.2 a n d 0 . 0 M ) , respectively, adjusted t o p H 6.8. T h e c h e m i c a l c o m p o s i t i o n s o f the five components hereby o b t a i n e d i n d i c a t e d t h a t the fractions w i t h higher l i g n i n content are e l u t e d at the higher c o n c e n t r a t i o n o f e t h y l cellosolve ( T a ble I V ; ref. 15). E v e n t h o u g h one p h e n y l g r o u p is a p p r o x i m a t e l y equivalent t o a threec a r b o n a l k y l c h a i n i n h y d r o p h o b i c i t y , p h e n y l Sepharose adsorbs a larger



2.

KOSHIJIMA ETAL.

a

e


F r . W-2

b.

Lignin

W-3

fragment

Carbohydrate

moiety

glycosidically Carbohydrate to

Fr.

25

lignin

to

linking

lignin

moiety

non-

fragment

linking

glycosidically

fragment

Figure 7. Structural determinants of L C C molecules and their micelle (or aggregate) formation. (Reprinted with permission from ref. 27. Copyright 1981.)



(X)

0

3

(%)

(X)

13.9 37.4 13.9 20.5 14.3 12.7

7.8

17 .5

34.0

P-III

19.0 22.6 28.8 19.3 10.4

22.6

P-II

.4 .5 .8 .0 .3

23 24 25 15 11

8 .0

40,.5

22, 5 15. 2 29. 2 19..1 11..0

P -I

LCC- •W

29.7

17.0 22.3 28.6 23.0 9.2

43.5

P-IV

30. 6 17. 8 18.,5 10.,2 23.,4 12.,7

32 .3

8. 8

0-•I

.4 .7 .5 .0 .4

13 13 32 18 22

50 .5

P -V

D e t a i l s of the procedure are given i n the Experimental S e c t i o n . ^Percentage of the dry weight of each l i g n i n - c a r b o h y d r a t e complex. P e r c e n t a g e of the t o t a l n e u t r a l sugar. ^Percentage of the e l u t e d l i g n i n - c a r b o h y d r a t e complex. Source: Reprinted w i t h permission from r e f . 15. Copyright 1982 Elsevier.

a

Recovery**

Neutral sugar L-Arabinose D-Xylose D-Mannose D-Galactose D-Glucose

L i g n i n content*

Component

Phenyl-Sepharose

25.3

24.0 15.6 32.2 21.3 7.9 10.9

15.4 22.7 18.0 23.1 20.7 14.6

15.0 12.4 27.7 32.1 12.6 36.5

60.5

44.9

38.8

28.4

14.7 16.0 31.5 25.3 12.5

0-V

0-IV

0-III

O-II

,0c tvl-Sepharose

Table IV. P r o p e r t i e s of F r a c t i o n s Obtained from Hydrophobic Chromatography of Pine LCC's


2.

KOSHIJIMA ETAL.


27

a m o u n t o f L C C ' s t h a n o c t y l Sepharose, i n d i c a t i n g t h a t 7T-7T i n t e r a c t i o n s due t o the l i g n i n a r o m a t i c r i n g moieties o f the L C C ' s act i n a d d i t i o n to h y d r o p h o b i c i n t e r a c t i o n s i n v o l v i n g the c a r b o n a t o m s o f l i g n i n side chains. T h e number-average m o l e c u l a r weights were 6 x 1 0 for p i n e L C C ' s , and 6 x 1 0 a n d 2 x 1 0 for the species (present i n a 1:1 r a t i o ) c o m p r i s i n g beech L C C ' s (28). T h e beech L C C molecules seem t o consist o f very few b u t bigger l i g n i n moieties c o m p a r e d w i t h those of pine L C C ' s . T h e progress o f l i g n i n biosynthesis i n the presence o f oligo- or p o l y s a c charides i n the p r i m a r y cell w a l l w o u l d necessarily engender the f o r m a t i o n o f s u g a r - l i n k e d l i g n i n s or l i g n i n - i n t e r c o n n e c t i n g sugar c h a i n s , p r o v i d e d t h a t l i g n i n biosynthesis involves q u i n o n e m e t h i d e i n t e r m e d i a t e s . T h e r e s u l t i n g p r o d u c t s are a l l l i g n i n - c a r b o h y d r a t e complexes i n a b r o a d sense, b u t the L C C ' s w h i c h c a n be e x t r a c t e d by the u s u a l m e t h o d s w i l l be l i m i t e d t o those w i t h less t h a n a 5 0 % l i g n i n content. A p a r t o f the c h e m i c a l s t r u c t u r e s o f the i n t e r u n i t linkages i n p i n e L C C ' s is presented i n F i g u r e 8. T h e role o f L C C ' s i n l i v i n g p l a n t tissues is p r e s u m e d t o be r e l a t e d t o the p r e v e n t i o n o f water-soluble hemicelluloses f r o m d i s s o l v i n g o u t o f the cell w a l l b y the f o r m a t i o n o f micelles or aggregates t h a t i m m o b i l i z e sugar c h a i n s , a n d the s o l u b i l i z a t i o n o f w a t e r - i n s o l u b l e m a t e r i a l s u c h as l i g n i n , thereby e n a b l i n g i t to move t o a n y place i n the c e l l . 3


3

5

F i g u r e 8. S t r u c t u r a l features o f the i n t e r u n i t linkages i n the c a r b o h y d r a t e moieties i n p i n e L C C ' s .


lignin-

28



Literature Cited 1. Merewether, J . W. T . Holzforschung 1957, 11, 65-80. 2. Koshijima, T.; Taniguchi, T.; Tanaka, R. Holzforschung 1972, 26, 21117. 3. Wegener, G . Ph.D. Thesis, Universitat München, 1975. 4. Yaku, F.; Yamada, Y.; Koshijima, T . Holzforschung 1976, 30, 148-56. 5. Björkman, A . Svensk Papperstidn. 1957, 60, 243-51. 6. Brownell, H. H. Tappi 1971, 54, 66-71. 7. Koshijima, T.; Yaku, F.; Tanaka, R. Appl. Polym. Symp. 1976, 28, 1025-39. 8. Yaku, F.; Tsuji, S.; Koshijima, T . Holzforschung 1979, 33, 54-9. 9. Yaku, F.; Tanaka, R.; Koshijima, T . Holzforschung 1981, 35, 177-81. 10. Eriksson, O.; Lindgren, B. O. Svensk Papperstidn. 1977, 80, 59-63. 11. Watanabe, T.; Azuma, J.; Koshijima, T . Mokuzai Gakkaishi 1987, 33, 798-803. 12. Lundquist, K.; Simonson, R.; Tingsvik, K. Svensk Papperstidn. 1980, 83, 452-4. 13. Azuma, J.; Takahashi, N.; Koshijima, T . Carbohydr. Res. 1981, 93, 91-104. 14. Mukoyoshi, S.; Azuma, J.; Koshijima, T . Holzforschung 1981, 35, 23340. 15. Takahashi, N.; Azuma, J.; Koshijima, T . Carbohydr. Res. 1982, 107, 161-8. 16. Kato, A.; Azuma, J.; Koshijima, T . Chem. Lett. 1983, 137-40. 17. Freudenberg, K.; Harkin, J . M . Chem. Ber. 1960, 93, 2814-9. 18. Tanaka, K.; Nakatsubo, F.; Higuchi, T . Mokuzai Gakkaishi 1979, 25, 653-9. 19. Katayama, Y.; Morohoshi, N.; Haraguchi, T . Mokuzai Gakkaishi 1980, 26, 358-62. 20. Feckel, J . Ph.D. Thesis, Universitat München, 1982. 21. Fengel, D. Svensk Papperstidn. 1976, 79, 24-8. 22. Kosíková, B.; Zakutna, L.; Joniak, D. Holzforschung 1978, 32, 15-8. 23. Watanabe, T.; Kaizu, S.; Koshijima, T . Chem. Lett. 1986, 1871-4. 24. Oikawa, Y.; Yoshida, T.; Yonemitsu, O. Tet. Lett. 1982, 23, 885-8; Yonemitsu, O. Yuki-Goseikagaku 1985, 43, 691-6. 25. Koshijima, T.; Watanabe, T.; Azuma, J . Chem. Lett. 1984, 1737-40. 26. Watanabe, T.; Koshijima, T . Mokuzai Gakkaishi 1989, 35, in press. 27. Koshijima, T.; Azuma, J.; Takahashi, N. The Ekman Days 1981, I, 67. 28. Takahashi, N.; Koshijima, T . Wood Sci. Technol. 1988, 22, 177-89. RECEIVED May 29,1989


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