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UBC Theses and Dissertations

A biochemical investigation of wood cellulose Stevenson, George William 1956

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A BIOCHEMICAL INVESTIGATION OF WOOD CELLULOSE by GEORGE WILLIAM STEVENSON B.A., U n i v e r s i t y o f B r i t i s h Columbia,  1952  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department of Biochemistry We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard.  THE UNIVERSITY OF BRITISH COLUMBIA September, 1956  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the requirements f o r an advanced degree a t the  University  of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may of my  be granted by the  Department or by h i s r e p r e s e n t a t i v e .  stood that  I t i s under-  copying or p u b l i c a t i o n of t h i s t h e s i s f o r  f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department of  Biochemistry  The U n i v e r s i t y of B r i t i s h Vancouver Canada. Date  Head  Columbia,  September 28,  1956  written  ABSTRACT  (11)  A method i e d e s c r i b e d f o r t h e p r e p a r a t i o n o f an induced x y l a n a s e from Chaetomlum globosum. micro-organism  This  soil  was f i r s t grown i n s y n t h e t i c l i q u i d media  c o n t a i n i n g x y l a n as t h e s o l e carbon source.  After i n -  c u b a t i o n a t room temperature f o r a s u i t a b l e t i m e , t h e m y c e l l a were h a r v e s t e d , c e n t r i f u g e d and exposed t o h i g h frequency  v i b r a t i o n s w h i c h r u p t u r e d t h e c e l l w a l l s . The  r e s u l t i n g c l e a r enzyme s o l u t i o n was shown t o be o f h y d r o l y s i n g x y l a n from wheat s t r a w . product  capable  The main end  o f h y d r o l y s i s d e t e c t e d by means o f paper chromato-  graphy was x y l o s e . The a c t i o n o f t h i s induced enzyme p r e p a r a t i o n on wood c e l l u l o s e was s t u d i e d . the x y l a n was removed w i t h o u t mannose o r g l u c o s e In  A considerable f r a c t i o n of s i g n i f i c a n t l y changing the  content.  c o n n e c t i o n w i t h t h e enzyme s t u d i e s , a method  i s given f o r the q u a n t i t a t i v e determination of polysaccharides i n wood-cellulose preparations.  D i r e c t photo-  m e t r i c a n a l y s e s o f t h e wood h y d r o l y s a t e s p o t s on paper chromatograms were made w i t h an adapted Beckman s p e c t r o photometer.  An a n a l y s i s o f s e l e c t e d commercial p u l p s i s  g i v e n t o g e t h e r w i t h a comparison o f t h e method w i t h c o n v e n t i o n a l pentosan a n a l y s e s .  (v) ACKNOWLEDGEMENT My s i n c e r e t h a n k s a r e extended t o Dr. W.J. P o l g l a s e f o r h i s guidance and i n t e r e s t i n t h i s s t u d y .  Appreciation  i s a l s o extended t o t h e N a t i o n a l R e s e a r c h C o u n c i l f o r t h e i r f i n a n c i a l support o f t h i s work. The  author i s i n d e b t e d  t o t h e f o l l o w i n g companies  f o r s u p p l y i n g commercial wood p u l p samples: E. I . Du Pont De Nemours & Company I n c . , Benger L a b o r a t o r y , Waynesboro, V i r g i n i a . Weyerhaeuser Timber Company, P u l p D i v i s i o n , R e s e a r c h Dept., E v e r e t t , Washington. West V i r g i n i a P u l p and Paper Company, Luke, M a r y l a n d . Brown Company, B e r l i n , New Hampshire. A b i t i b l Power & Paper Company, L i m i t e d , C e n t r a l R e s e a r c h & Development D i v i s i o n , S a u l t S t e . M a r i e , O n t a r i o , Canada. Wheat s t r a w x y l a n was k i n d l y e u p p l i e d by Dr. R. L. W h i s t l e r , Purdue U n i v e r s i t y , L a f a y e t t e , I n d i a n a .  The  c u l t u r e s o f (3. globosum Kunze were g e n e r o u s l y p r o v i d e d by Dr. H. Sj^rensen, Dept. o f B a c t e r i o l o g y , S t a t e of P l a n t C u l t u r e , Lyngby, Denmark. of S o i l S c i e n c e ,  Laboratory  Dr. D. G. L a i r d , Dept.  U n i v e r s i t y o f B.C. a l s o a i d e d t h i s work  by s u p p l y i n g a d d i t i o n a l m i c r o - o r g a n i s m s as w e l l as g i v i n g helpful  suggestions.  (iii) TABLE OF CONTENTS Page INTRODUCTION  1  HISTORICAL  3  A.  The Q u a n t i t a t i v e D e t e r m i n a t i o n Polysaccharides I.  o f Wood  3  The s t r u c t u r e o f t h e more predominant 3  p o l y s a c c h a r i d e s i n wood II. III. B.  The q u a n t i t a t i v e d e t e r m i n a t i o n o f mannan . .  6  The q u a n t i t a t i v e d e t e r m i n a t i o n o f x y l a n  8  Hemlcellulases  10 13  EXPERIMENTAL A.  The Q u a n t i t a t i v e D e t e r m i n a t i o n  o f Mannan and 13  Xylan I. II. III.  IV.  . .  H y d r o l y s i s o f wood c e l l u l o s e  13  Sugar s t a n d a r d s  1^  Chromatography P l a t e I Photograph o f a 72 hour paper chromatogram  16  Photometric  17  analysis  P l a t e I I A d i a g r a m o f t h e c a r r i a g e and removable paper s t r i p h o l d e r adapted f o r use w i t h a Beckman Model <T3 spectrophotometer B.  Enzyme S t u d i e s  18  I.  18  Substrate  P l a t e I I I Graph showing t h e l i n e a r r e l a t i o n o f mannose and x y l o s e c o n c e n t r a t i o n s to the o p t i c a l density of t h e i r chromatogrammed s p o t s  (iv)  TABLE OF CONTENTS(cont'd.) Page II. III. IV.  Micro-organisms  19  Enzyme p r e p a r a t i o n  21  The d e t e r m i n a t i o n  o f enzyme a c t i v i t y  ...  22  RESULTS  24  DISCUSSION  26  SUMMARY  30a  BIBLIOGRAPHY  31  1  INTRODUCTION  The purpose  of t h i s study was t o i n v e s t i g a t e  the a c t i o n o f enzymes on the p o l y s a c c h a r i d e s a s s o c i a t e d w i t h wood c e l l u l o s e . was  In p a r t i c u l a r , t h i s  investigation  concerned w i t h x y l a n and mannan which, next t o  c e l l u l o s e , are the most abundant c a r b o h y d r a t e s o f wood. Commercial wood p u l p s were s e l e c t e d f o r study i n t h i s work.  I n i t i a l l y , the p r o j e c t was  s e t up t o determine  the  e f f e c t o f i n d u c e d x y l a n a s e s on t h e x y l a n c o n t e n t o f v a r i o u s wood p u l p s . The methods used were concerned w i t h two o f t h i s study:  aspects  the q u a n t i t a t i v e carbohydrate a n a l y s i s of  v a r i o u s wood p u l p s and the p r e p a r a t i o n o f s u i t a b l e enzymes. F i r s t , i n o r d e r t o e s t i m a t e changes i n the wood c a r b o h y d r a t e c o n t e n t b e f o r e and a f t e r enzyme a c t i o n , i t was n e c e s s a r y t o d e v e l o p a s u i t a b l e q u a n t i t a t i v e method o f a n a l y s i s .  The most s a t i s f a c t o r y method was  found t o be a p h o t o m e t r i c e s t i m a t i o n o f t h e h y d r o l y s e d samples s p o t t e d on paper chromatograms.  Considerable  time d u r i n g the e x p e r i m e n t a l work was g i v e n t o t h e development o f t h i e method and i t s a p p l i c a t i o n t o v a r i o u s p r e p a r a t i o n s o f wood c e l l u l o s e . Second, i n o r d e r t o o b t a i n the d e s i r e d enzyme a c t i v i t y t h e s e l e c t e d micro-organisms  were grown i n  l i q u i d media c o n t a i n i n g a p a r t i c u l a r p o l y s a c c h a r i d e as  2  the s o l e carbon s o u r c e .  I n t h i s way, enzymes were I n -  duced w h i c h were c a p a b l e o f m e t a b o l i s i n g t h e p a r t i c u l a r polysaccharide. This i n v e s t i g a t i o n i s o f value to both  funda-  mental u n d e r s t a n d i n g and i n d u s t r i a l a p p l i c a t i o n i n t h e cellulose field.  A t p r e s e n t , no c h e m i c a l methods a r e  a v a i l a b l e f o r p r e p a r i n g undegraded wood c e l l u l o s e , from c l o s e l y a s s o c i a t e d p o l y s a c c h a r i d e s .  free  I f specific  enzymes c o u l d be used t o a c h i e v e t h i s o b j e c t i v e , i t would p r o v i d e a b e t t e r s t a r t i n g m a t e r i a l f o r s t u d i e s on cellulose.  In considering possible Industrial  applica-  t i o n s ( e s p e c i a l l y i n t h e p u l p and paper i n d u s t r y ) , p u l p s from w h i c h s p e c i f i c p o l y s a c c h a r i d e s c o u l d be removed s y s t e m a t i c a l l y without degradation of the c e l l u l o s e s h o u l d prove t o be c o m m e r c i a l l y s u p e r i o r i n many r e s p e c t s . Furthermore,  t h e s p e c i f i c removal  o f wood p o l y s a c c h a r i d e s  would enable a r e l i a b l e e v a l u a t i o n o f t h e i r  contribution  to the p r o p e r t i e s o f the o r i g i n a l wood-cellulose preparation.  HISTORICAL  A.  The Q u a n t i t a t i v e D e t e r m i n a t i o n I.  3  o f Wood P o l y s a c c h a r i d e s  The s t r u c t u r e o f t h e more predominant p o l y s a c c h a r i d e s i n wood A b r i e f summary o f t h e s t r u c t u r e o f t h e more  i m p o r t a n t wood p o l y s a c c h a r i d e s o r p o l y o s e s I s i m p o r t a n t t o an u n d e r s t a n d i n g d e t e r m i n i n g them.  o f t h e a n a l y t i c a l methods used f o r C e l l u l o s e , by f a r t h e most abundant  wood p o l y s a c c h a r i d e , w i l l be d i s c u s s e d f i r s t . concept  The p r e s e n t  o f the m o l e c u l a r s t r u c t u r e o f c e l l u l o s e (as  o b t a i n e d from c o t t o n l i n t e r e ) i s a l i n e a r , unbranched polymer o f D-glucose a n h y d r i d e u n i t s , l i n k e d by 4- > 1, B - D - g l u c o s i d i c bonds ( l ) .  In the native state,  cellulose  i s composed o f a network o f these l i n e a r c h a i n s w h i c h v a r y c o n s i d e r a b l y i n t h e number o f component monomeric unite.  The i n d i v i d u a l l i n e a r m o l e c u l e s  thus form a  polymer-homologous s e r i e s w i t h i n any s i n g l e preparation.  cellulose  Superimposed on t h e s e m o l e c u l a r d i f f e r e n c e s  are v a r i a t i o n s i n t h e arrangement o f t h e c h a i n s w h i c h produce s t r u c t u r e s c h a r a c t e r i z e d by d i f f e r e n t o r d e r s o f l a t t i c e p e r f e c t i o n and degrees o f o r i e n t a t i o n .  There  are formed, t h e r e f o r e , g r a d a t i o n s between random m o l e c u l a r arrangement I n amorphous r e g i o n s and n e a r l y p e r f e c t three-dimensional c r y s t a l l i t e formation i n w e l l portions.  ordered  k I n wood c e l l u l o s e p r e p a r a t i o n s t h e r e i s not the c h e m i c a l homogeneity found i n c o t t o n c e l l u l o s e .  This  f a c t i s p a r t l y due t o t h e Incomplete removal o f such substances  as l i g n i n , a s h , wood e x t r a c t i v e s , and  e s p e c i a l l y t h e c l o s e l y a s s o c i a t e d p o l y o s e s , x y l a n and mannan.  Furthermore, w o o d - c e l l u l o s e p r e p a r a t i o n s have  a higher carboxyl-group  content than l i n t e r s  cellulose.  These groups a r e b e l i e v e d t o e x i s t as p o l y u r o n i d e s o r as u r o n i c anhydride u n i t s i n the c e l l u l o s e molecule or associated with  it.  Among t h e more i m p o r t a n t minor p o l y s a c c h a r i d e s i n wood c e l l u l o s e i s mannan.  I t s molecular s t r u c t u r e  a p p a r e n t l y v a r i e s c o n s i d e r a b l y w i t h t h e source  (2,3,4).  The main p o r t i o n o f t h e p o l y s a c c h a r i d e c o n s i s t s o f Dmannopyranose a n h y d r i d e u n i t s l i n k e d k -v 1 by B-D g l y c o s i d i c bonds.  I n a d d i t i o n , most o f t h e mannans  s t u d i e d c o n t a i n v a r y i n g amounts o f g l u c o s e , g a l a c t o s e o r arabinose residues.  Owing t o t h e l i m i t e d  understanding  o f the e x a c t s t r u c t u r e o f mannans i n wood, a r e l i a b l e a n a l y s i s o f t h i s p o l y o s e i s v e r y d i f f i c u l t and i s g e n e r a l l y c o n f i n e d t o t h e d e t e r m i n a t i o n o f t h e amount o f anhydromannose u n i t s p r e s e n t . The r e m a i n i n g p o l y s a c c h a r i d e o f I n t e r e s t i n t h i s d i s c u s s i o n i e t h e pentosan, x y l a n .  This polyose i s  composed m a i n l y o f anhydroxylose  I n esparto  units.  grass,  5 the x y l a n molecule  i s a l i n e a r s t r u c t u r e c o n t a i n i n g 70  t o 80 u n i t s o f anhydro-D-xylopyranose J o i n e d by k B-D l i n k a g e .  Branching  from t h i s c h a i n i s a s i n g l e  D-xylose r e s i d u e J o i n e d by a 3  1  <K-union.  Other  sources o f x y l a n show t h e presence o f L - a r a b i n o s e , g l u c u r o n l c or methylhexuronic 6,7).  1,  D-  u n i t s i n t h e polymer ( 5 ,  Uronic acid residues are also detectable.  O'Dwyer (8) has shown t h a t many x y l a n s o f wood a r e heterogeneous p o l y s a c c h a r i d e s .  Analyses f o r xylan i n  wood-cellulose p r e p a r a t i o n s are consequently t o t h e d e t e r m i n a t i o n o f anhydroxylose  restricted  units.  The p o l y s a c c h a r i d e s mentioned do n o t e x i s t i n d i s c r e t e l a y e r s o r segments i n p l a n t t i s s u e s .  On t h e  c o n t r a r y , much o f t h e d i f f i c u l t y i n a n a l y z i n g and s t u d y i n g t h e i r i n d i v i d u a l s t r u c t u r e i s due t o an i n t i m a t e a s s o c i a t i o n o f a l l t h r e e - an a s s o c i a t i o n w h i c h has so f a r d e f i e d a l l c h e m i c a l means o f complete s e p a r a t i o n and has g i v e n r i s e t o t h e o r i e s o f c h e m i c a l b o n d i n g between the p o l y s a c c h a r i d e s  (9.10)<.  S e v e r a l terms based on a s s o c i a t i o n r a t h e r than c h e m i c a l s t r u c t u r e a r e used t o r e f e r t o t h e p o l y s a c c h a r i d e s in plant tissue.  A l l of the carbohydrates  ively oalled holocellulose.  are c o l l e c t -  The n o n - c e l l u l o s i c p o l y -  s a c c h a r i d e s i n t h e h o l o c e l l u l o s e a r e c l a s s e d as hemicelluloses.  These u n o r i e n t e d components form a c o n t i n u o u s  i n t e r p e n e t r a t i n g system w i t h t h e c e l l u l o s e s t r u c t u r e . Most o f t h e h e m i c e l l u l o s e s a r e removed by e x t r a c t i o n w i t h dilute alkalies.  The h e m i c e l l u l o s e f r a c t i o n i s f u r t h e r  s u b d i v i d e d i n t o t h e amorphous p o l y u r o n i d e  hemicelluloses  w h i c h i n c l u d e methoxyhexuronic a c i d s as w e l l as x y l o s e , a r a b l n o s e , g l u c o s e and g a l a c t o s e r e s i d u e s , and c e l l u l o s a n s which c o n s i s t o f r e l a t i v e l y s i m p l e hexosans o r pentosans more o r l e s s o r i e n t e d i n t h e m i c e l l a r c e l l u l o s e s t r u c t u r e The c e l l u l o s a n s a r e r e l a t i v e l y s h o r t - c h a i n  polysacchar-  i d e s whose pyranose s t r u c t u r e s occupy t h e same space as the a n h y d r o g l u c o s e u n i t s and t h e r e f o r e p e r m i t t h e i r c l o s e packing i n t o the o r i e n t e d c e l l u l o s e f a b r i c . l a t e r a l forces which are present also r e t a i n the c e l l u l o s a n s .  The same  i n t h e c e l l u l o s e would  I n commercial wood-  c e l l u l o s e p r e p a r a t i o n s , x y l a n and mannan a r e t h e most important c e l l u l o s a n s . II.  The q u a n t i t a t i v e d e t e r m i n a t i o n o f mannan From t h e p r e c e d i n g  c o n s i d e r a t i o n s i t i s easy  to appreciate the d i f f i c u l t i e s o f analysing the polyoses of wood i n a q u a n t i t a t i v e f a s h i o n .  A t t e n t i o n w i l l be  g i v e n t o t h e a n a l y t i c a l methods used f o r d e t e r m i n i n g two o f t h e s e p o l y s a c c h a r i d e s - mannan and x y l a n .  only  As  p r e v i o u s l y mentioned, methods c u r r e n t l y a v a i l a b l e do n o t a c t u a l l y d e t e r m i n e t h e mannan o r x y l a n as polymers b u t  7 r a t h e r d e t e r m i n e them I n d i r e c t l y by t h e amount o f anhydromannoee o r a n h y d r o x y l o s e u n i t e found p r e s e n t . Reviews o f t h e l i t e r a t u r e  (11,12,13)  give  t h r e e g e n e r a l methods f o r e s t i m a t i n g mannan i n wood o r wood-cellulose preparations.  The method o f Schorger  (Ik),  w h i c h was m o d i f i e d  by Wise, R a t l i f f  and Browning  (15).  c o n s i s t s o f an a c i d h y d r o l y s i s o f t h e mannan t o  mannose, n e u t r a l i z a t i o n and subsequent p r e c i p i t a t i o n o f the mannose as t h e p h e n y l h y d r a z o n e . reported  D i l u t e acids are  t o give incomplete h y d r o l y s i s but stronger  concentrations  cause d e s t r u c t i o n o f t h e mannose.  acid Further-  more, when mannan i s p r e s e n t i n s m a l l amounts (1% o r l e s s ) other phenylhydrazones which c o - p r e c i p i t a t e w i t h the mannose phenylhydrazone i n t e r f e r e i n t h e a n a l y s i s . amounts o f x y l o s e a l s o i n t e r f e r e by l o w e r i n g  Large  t h e mannan  values. A recent  method ( 1 6 ) e s t i m a t e s mannan by t h e  r a t e d i f f e r e n c e o f l e a d t e t r a a c e t a t e consumption.  The  c i s - g l y c o l g r o u p i n g s i n mannan a r e more r a p i d l y o x i d i z e d than t h e t r a n s - g l y c o l g r o u p i n g s i n c e l l u l o s e .  Con-  s t r u c t i o n o f a curve showing time v e r s u s l e a d  tetra-  acetate  consumption p e r m i t s e x t r a p o l a t i o n o f t h e f i n a l  s t r a i g h t - l i n e p o r t i o n o f t h e curve t o z e r o t i m e f o r an e s t i m a t e o f t h e mannan p r e s e n t . The use o f q u a n t i t a t i v e paper chromatography  8 i a c u r r e n t l y t h e most s a t i s f a c t o r y method f o r d e t e r m i n i n g n o t o n l y mannan b u t a l s o x y l a n ( l ? ) .  Wise (18)  used t h i s method f o r d e t e c t i n g x y l o s e and mannose i n wood p r e p a r a t i o n s . G-ustafssbn sugar  L a t e r , Sundman, S a a r n i o and  (19) adapted t h e method f o r q u a n t i t a t i v e wood  determinations.  III.  The q u a n t i t a t i v e d e t e r m i n a t i o n o f x y l a n Before the i n t r o d u c t i o n of chromatographic  methods t h e d e t e r m i n a t i o n o f x y l a n was most f r u s t r a t i n g . U s u a l l y , x y l a n e s t i m a t i o n s were based on " c o r r e c t e d " pentosan d e t e r m i n a t i o n s w h i c h were, t h e m s e l v e s , p r e v i o u s l y corrected.  Pentosans were c o n v e r t e d by a c i d h y d r o l y s i s  t o p e n t o s e s and then t o f u r f u r a l .  The f u r f u r a l was d i s -  t i l l e d and d e t e r m i n e d by c o l o r i m e t r i c , v o l u m e t r i c o r g r a v i m e t r i c methods ( 2 0 ) . U r o n i c a c i d s , p o l y u r o n i d e s , hexose u n i t s , 6-deoxyhexose, formaldehyde, acetone and l e v u l i n i c a c i d a l l y i e l d f u r f u r a l or methyl f u r f u r a l d e r i v a t i v e s w h i c h a r e d e t e r m i n e d as f u r f u r a l . The g r e a t v a l u e o f paper chromatography l i e s i n i t s a b i l i t y t o s e p a r a t e and i d e n t i f y microgram q u a n t i t i e s o f x y l o s e and mannose i n complex m i x t u r e s o f carbohydrates  q u i c k l y , s i m p l y and a c c u r a t e l y .  Before  chromatographing, t h e p o l y s a c c h a r i d e s a r e h y d r o l y s e d u s u a l l y by a c i d s o l u t i o n s (21,22).  However, c a t i o n  9  exchange r e s i n s have a l s o been used f o r t h i s (23).  purpose  To reduce t h e sugar d e c o m p o s i t i o n t o a minimum,  Saeman and co-workers  (2*0 recommend a two hour  initial  d i s s o l v i n g time i n c o n c e n t r a t e d a c i d a t 30° f o l l o w e d by a u t o c l a v l n g f o r one hour at 15 p . s . i . and n e u t r a l i z a t i o n  After hydrolysis  ( u s u a l l y w i t h barium c a r b o n a t e or  a n i o n exchange r e s i n s ) the sugar s o l u t i o n s a r e t r a t e d and then chromatographed.  concen-  Several excellent  r e v i e w s o f methods and t e c h n i q u e s i n paper  chromatography  have been p u b l i s h e d ( 2 5 , 2 6 , 2 7 ) .  sugar  A f t e r the  s o l u t i o n s have been chromatographed they can be  developed  and e s t i m a t e d d i r e c t l y on the paper o r , t h e y can be e l u t e d from the sheet and d e t e r m i n e d by any one o f s e v e r a l c o n v e n t i o n a l methods.  E l u t i o n t e c h n i q u e s can  g i v e r i s e t o sugar l o s s e s t h r o u g h m a n i p u l a t i o n or decomposition during e x t r a c t i o n .  P r e - t r e a t m e n t o f the  paper i s o f t e n n e c e s s a r y t o a v o i d e l u t i n g substances.  Interfering  The more r a p i d method i s t o d e v e l o p t h e  sugars as c o l o r e d s p o t s and t o determine t h e i r c o n c e n t r a t i o n by comparison w i t h s t a n d a r d s w h i c h a r e r u n s i m u l t a n e o u s l y on the same sheet.  V i s u a l comparisons  are  o f t e n s a t i s f a c t o r y but p h o t o m e t r i c methods a r e more p r e c i s e (28,29,30).  The p h o t o m e t r i c d e t e r m i n a t i o n s are  based on t h e r e l a t i o n s h i p o f t h e spot a r e a , l e n g t h o r c o l o r d e n s i t y t o sugar c o n c e n t r a t i o n . Unknown sugar  10  c o n c e n t r a t i o n s a r e e v a l u a t e d from a s t a n d a r d curve i s p l o t t e d from d e n s i t y r e a d i n g s o f t h e sugar  which  standards  r u n on t h e same chromatogram as t h e unknowns.  B.  Hemlcellulases U n t i l r e c e n t l y , l i t t l e was known about t h e  specificity cytases.  o f t h e enzymes known as h e m c e l l u l a s e s or  The names mannanase, x y l a n a s e , arabanase, e t c .  used i n t h e l i t e r a t u r e were a p p l i e d t o enzymes w h i o h h y d r o l y s e d the d i f f e r e n t p o l y s a c c h a r i d e s .  However, most  of t h e s t u d i e s were c a r r i e d out w i t h crude enzyme p r e p a r a t i o n s and r e s u l t s were o f t e n d i f f i c u l t t o I n t e r p r e t . Furthermore,  t h e l a c k o f knowledge o f t h e e x a c t  structure  of the v a r i o u s p o l y s a c c h a r i d e s u b s t r a t e s and t h e d i f f i c u l t y o f q u a n t i t a t i v e l y d e t e r m i n i n g them h i n d e r e d p r o g r e s s i n t h e s e enzyme i n v e s t i g a t i o n s . I n 1933, G-rassman, S t a d l e r and Bender (31) were a b l e t o remove x y l a n a s e a c t i v i t y  from e x t r a c t s o f  A s p e r g i l l u s oryzae (Luizym) by a d s o r p t i o n on c h a r c o a l , l e a v i n g the c e l l u l a s e s t i l l a c t i v e .  These f i n d i n g s i n d i -  c a t e d t h a t c e l l u l a s e and x y l a n a s e were d i f f e r e n t enzymes but no i n v e s t i g a t i o n o f t h e mode o f a c t i o n o f t h e x y l a n a s e was  undertaken.  Sorensen ( 3 2 ) , i n s t u d i e s on t h e s p e c i f i c i t y and p r o d u c t s o f a c t i o n o f x y l a n a s e on wheat s t r a w x y l a n ,  11 has attempted t o g i v e more d e f i n i t e i n f o r m a t i o n about t h i s cytase.  The p a r t i c u l a r x y l a n a s e from Chaetomium  globosum kunze showed optimum a c t i v i t y at pH 6 . 5 and 3 7 ° . No s i g n i f i c a n t d i f f e r e n c e was found i n t h e r a t e o f enzymatic h y d r o l y s i s between x y l a n s from wheat s t r a w and wood.  I t was f u r t h e r demonstrated t h a t t h i s x y l a n a s e i s  a d a p t i v e and d i f f e r e n t from mannanase, c e l l u l a s e o r diastase.  Mannanase from t h e same m i c r o - o r g a n i s m was  a l s o shown t o be a d a p t i v e . The main p r o d u c t s o f h y d r o l y s i s by t h i s x y l a n a s e was x y l o s e .  Although heating or s t o r i n g of the  enzyme caused a l o s s o f t h e a b i l i t y t o produce x y l o s e from x y l a n , t h e power t o produce i n t e r m e d i a t e o l i g o s a c c h a r i d e s was not a f f e c t e d . Subsequent s t u d i e s by Sorensen ( 3 3 ) s u p p o r t e d the t h e o r y t h a t x y l a n a s e i s composed o f a t l e a s t two enzymes.  One enzyme behaves as an exoenzyme, c a p a b l e o f  randomly s p l i t t i n g o f f low m o l e c u l a r weight saccharides. splitting  oligo-  The o t h e r , an endoenzyme, i s c a p a b l e o f  xyloblose. Yundt ( 3 4 ) , i n e x p e r i m e n t s w i t h c r y s t a l l i n e  x y l a n and an enzyme p r e p a r a t i o n from A s p e r g i l l u s n l g e r . showed t h a t an I n i t i a l l y r a p i d h y d r o l y s i s o f t h e x y l a n was f o l l o w e d by a much slower r e a c t i o n .  The e x t e n t o f  enzymatic h y d r o l y s i s was d e t e r m i n e d by t h e c y a n i d e consumption o f t h e a l d o s e end groups.  12 I t was a l s o shown t h a t r e t r o g r a d a t i o n o f t h e p o l y s a c c h a r i d e was a p h y s i c a l i n f l u e n c e on enzyme a c t i o n r a t h e r than an i n d i c a t i o n o f c h e m i c a l  inhomogenelty.  EXPERIMENTAL  A.  13  The Q u a n t i t a t i v e D e t e r m i n a t i o n o f Mannan and X y l a n I.  H y d r o l y s i s o f wood c e l l u l o s e P r e p a r a t o r y t o h y d r o l y s i s , an a i r - d r i e d sample  o f wood c e l l u l o s e ( u s u a l l y c o n t a i n i n g about 1 0 $ m o i s t u r e ) was b e a t e n t o a f i n e , f l u f f y p u l p i n an o s t e r i z e r .  For  m o i s t u r e d e t e r m i n a t i o n , a t e n gram sample o f t h e a l r d r i e d p u l p was weighed out a t t h i s t i m e , o v e n - d r i e d a t 110°  f o r 8 hours and weighed a g a i n .  The two-step method  o f a c i d h y d r o l y s i s o f wood c e l l u l o s e d e v e l o p e d by L. E. Wise and co-workers  ( 1 5 ) was s u b s e q u e n t l y f o l l o w e d .  A  one gram sample o f t h e f l u f f e d p u l p was t r a n s f e r r e d t o a 250 m l . round-bottom f l a s k and d i s s o l v e d a t 20° i n 10 mis. o f 7 2 $ s u l f u r i c a c i d ( s p . g r . 1 . 6 4 ) . hours was s u f f i c i e n t f o r complete polysaccharides.  Usually 2 to 3  s o l u t i o n o f t h e wood  Next, t h e m i x t u r e was d i l u t e d w i t h  140 m i s . o f water, and t h e h y d r o l y s i s completed by h e a t i n g under r e f l u x on a b o i l i n g water b a t h f o r 4 h o u r s . The h y d r o l y s a t e was n e u t r a l i z e d w i t h barium u s i n g congo r e d paper as an i n d i c a t o r .  carbonate,  I t was found t h a t  a p p r o x i m a t e l y 230 grams o f barium carbonate were n e c e s s a r y f o r n e u t r a l i z a t i o n of the a c i d i c s o l u t i o n .  The n e u t r a l  s o l u t i o n was f i l t e r e d I n a Buchner f u n n e l (Coors 2A) u s i n g No. 44 Whatman f i l t e r paper.  Pressure applied t o  14 the t o p o f t h e f i l t e r cake a i d e d f i l t r a t i o n  considerably.  R e f i l t e r i n g t h e s o l u t i o n t o o b t a i n a c l e a r f i l t r a t e was found unnecessary filtering  i f p r o p e r c a r e was g i v e n t o t h e f i r s t  operation.  A 75 ml. a l i q u o t o f t h e c l e a r  f i l t r a t e was c o n c e n t r a t e d on a 40° water b a t h under r e duced p r e s s u r e t o a volume o f 1 o r 2 m i s .  The r e s u l t i n g  syrupy s o l u t i o n , t o g e t h e r w i t h any p r e c i p i t a t e d barium carbonate  o r s u l f a t e , was t r a n s f e r r e d by means o f a  micropipette to a 3 ml. f r i t t e d glass f i l t e r funnel of medium p o r o s i t y . volumetric flask.  The f i l t r a t e was c o l l e c t e d i n a 5 m l . As an a d d i t i o n a l p r e c a u t i o n i n  p r e v e n t i n g any p r e c i p i t a t e from p a s s i n g i n t o t h e f i l t r a t e , a s m a l l d i s c o f No. 44 Whatman f i l t e r paper, c u t out w i t h a s u i t a b l e c o r k b o r e r , was p l a c e d over t h e f r i t t e d g l a s s surface o f the f u n n e l .  The f l a s k w h i c h c o n t a i n e d t h e  c o n c e n t r a t e d sugar s o l u t i o n was s u c c e s s i v e l y washed w i t h s m a l l amounts o f d i s t i l l e d w a t e r .  A l l o f t h e s e washings  were f i l t e r e d i n t o t h e v o l u m e t r i c f l a s k .  Water was  added t o t h e f i l t r a t e t o b r i n g t h e volume o f t h e f i l t r a t e to  e x a c t l y 5 mis.  T h i s r e s u l t i n g s o l u t i o n , w h i c h was  s u b s e q u e n t l y a n a l y s e d by paper chromatography, now r e p r e s e n t e d a 10$ s o l u t i o n o f sugars from t h e h y d r o l y s e d wood-cellulose II.  Sugar  sample. standarde  By e x p e r i e n c e i t was found t h a t (on paper  15  chromatograms) s t a n d a r d s o l u t i o n s , each c o n t a i n i n g onlyone pure sugar, d i d n o t g i v e t r u l y comparable q u a n t i t a t i v e r e s u l t s w i t h m i x t u r e s o f sugars i n w o o d - c e l l u l o s e hydrolysates.  Consequently,  i n t h e s e experiments  each s t a n d a r d s o l u t i o n used  was a c t u a l l y composed o f t h r e e pure  sugars i n a r a t i o c l o s e l y r e s e m b l i n g t h e i r p r o p o r t i o n i n the w o o d - c e l l u l o s e h y d r o l y s a t e s t u d i e d .  These t h r e e most  predominant sugars were g l u c o s e , mannose and x y l o s e . However, as p r e v i o u s l y mentioned, o n l y t h e mannose and x y l o s e c o n t e n t s were o f concern i n t h i s s t u d y . A l l o b s e r v a t i o n s were based on h y d r o l y s e d  wood-cellulose  p r e p a r a t i o n s r e p r e s e n t i n g 10$ sugar s o l u t i o n s from t h e o r i g i n a l p u l p samples.  Therefore, the standard  s o l u t i o n s were p r o p o r t i o n a l l y p r e p a r e d  sugar  so as t o be  comparable w i t h t h e s e d i l u t e d w o o d - c e l l u l o s e h y d r o l y s a t e s . The r e l a t i v e p r o p o r t i o n o f g l u c o s e i n woodc e l l u l o s e samples was about 90$.  T h e r e f o r e , t h e amount  o f g l u c o s e i n a l l o f t h e s t a n d a r d s was f i x e d a t 9$. I n softwood  p u l p s t h a t were s t u d i e d t h e x y l o s e c o n t e n t  v a r i e d from 1 t o 6 p e r cent whereas t h e mannose v a r i e d from 1 t o 12 p e r c e n t .  content  Sugar s t a n d a r d s were  p r e p a r e d c o v e r i n g t h e s e approximate r a n g e s .  Benzoic  acid  (0.25 gm./lOO mis.) was added as an a n t i s e p t i c t o t h e standard s o l u t i o n s .  When n e c e s s a r y , a p p r o p r i a t e d i l u -  t i o n s were made f o r sugars w i t h c o n c e n t r a t i o n s o u t s i d e these  ranges.  16  % Xylose  % Mannose  % Glucose  1  0.1  0.2  9.0  2  0.2  0.4  9.0  3  0.3  0.6  9.0  4  0.4  0.8  9.0  5  0.6  1.1  9.0  Standard  solution  III.  Chromatography The h y d r o l y s e d w o o d - c e l l u l o s e s o l u t i o n s  p r e p a r e d by t h e p r e c e d i n g method were chromatographed on l a r g e s h e e t s o f No. 1 Whatman f i l t e r paper  (18£  M  A l o n g t h e s t a r t l i n e , a s e r i e s o f s t a n d a r d sugar  x 22^"). solutions  was a l t e r n a t e l y s p o t t e d w i t h a p r e p a r e d h y d r o l y s a t e (see f i g u r e 2 ) . The s p o t s were spaced a t 4 cm.  intervals.  The amount o f chromatographed s o l u t i o n was about 2 N o r s u f f i c i e n t t o produce a spot 10 mm.  i n diameter. A l l  a p p l i c a t i o n s o f t h e s t a n d a r d and unknown s o l u t i o n s were made w i t h t h e same m i c r o p i p e t t e . A f t e r each  application  the p i p e t t e was r i n s e d by s u c t i o n w i t h d i s t i l l e d  water  and then w i t h a c e t o n e . The s o l v e n t system r e p o r t e d by G u s t a f s s o n (21) was found t o be most s a t i s f a c t o r y f o r t h i s chromatog r a p h i c method. distilled.  n - B u t a n o l was s a t u r a t e d w i t h water and  P h t h a l l c a c i d (1.66  gr./lOO m i s . s o l v e n t ) ,  w h i c h i s a c t u a l l y a component o f t h e d e v e l o p e r , was then  ^  5  •  Fig.  u  •  2  l  4  I I  U  l  U  5  • #  2  2  I f  U  2  1  I *  Photograph o f a chromatogram d e v e l o p e d a f t e r 72 hours showing t h e arrangement of unknowns ( U ^ , U ) and s t a n d a r d s u g a r s o l u t i o n s 2  (1-5).  The s u g a r s g l u c o s e ( g ) , mannose (m) and x y l o s e ( x ) a r e s u f f i c i e n t l y separated f o r photometric a n a l y s i s .  U  2  I  17  d i s s o l v e d i n the w a t e r s a t u r a t e d n - b u t a n o l .  The  presence  o f t h e p h t h a l i c a c i d i n the s o l v e n t was not d e t r i m e n t a l t o the a n a l y s i s of t h e s u g a r s . The chromatograms were i r r i g a t e d w i t h the s o l v e n t by the d e s c e n d i n g f l o w method f o r 72 h o u r s a t room t e m p e r a t u r e .  At the end o f t h i s t i m e t h e s h e e t s  were t r a n s f e r r e d t o a fume hood and a l l o w e d t o d r y f o r a t l e a s t one hour. 15 cms.  The most m o b i l e s u g a r , x y l o e e , r a n about  A l l o f the sugars were s u f f i c i e n t l y s e p a r a t e d  for quantitative  estimation.  F o r c o l o r development,  t h e d r i e d chromatograms  were drawn t h r o u g h a s o l u t i o n o f a n i l i n e i n e t h e r (0.96 a n l l l n e / 1 0 0 mis. e t h e r ) and then h e a t e d a t 105° minutes.  gms.  for 5  Glucose and mannose appeared as brown s p o t s ;  x y l o s e as a p i n k s p o t . IV.  Photometric a n a l y s i s The a r e a I n c l u d i n g t h e mannose and x y l o s e s p o t s  from each chromatographed  s o l u t i o n was  cut out as a  r e c t a n g u l a r s t r i p measuring 3 cms. by 7 cms.  Each  thus c o n t a i n e d one x y l o s e and one mannose s p o t .  strip  These  s t r i p s were p l a c e d i n a m e t a l s u p p o r t (see f i g u r e l ) w h i c h was  s p e c i a l l y d e s i g n e d f o r use w i t h a Beckman Model  B Spectrophotometer.  A l l of the o p t i c a l d e n s i t y r e a d i n g s ,  w h i c h were dependent on the t r a n s m i t t e d l i g h t , were made at 550 mu .  A b l a n k s t r i p from t h e same chromatogram  was  FIG. I  A DIAGRAM OF THE CARRIAGE AND REMOVABLE PAPER STRIP HOLDER ADAPTED FOR USE WITH A B E C K M A N MODEL B SPECTROPHOTOMETER.  18 used f o r o b t a i n i n g a z e r o o p t i c a l d e n s i t y s e t t i n g . the s t r i p was manually  As  moved a c r o s s t h e l i g h t s l i t , t h e  maximum d e n s i t y f o r e a c h spot was r e c o r d e d . From each chromatogram, s t a n d a r d c u r v e s f o r b o t h mannose and x y l o s e were drawn by p l o t t i n g t h e o p t i c a l d e n s i t y r e a d i n g s v e r s u s t h e c o n c e n t r a t i o n o f t h e sugar standards  ( s e e f i g u r e 3).  Once t h e s t a n d a r d c u r v e s were  e s t a b l i s h e d , unknown sugar c o n c e n t r a t i o n s were r e a d i l y determined  from t h e graph. The p e r cent o f mannose and x y l o s e I n t h e wood-  c e l l u l o s e h y d r o l y s a t e s was c o n v e r t e d t o mannan and x y l a n v a l u e s by use o f t h e f o l l o w i n g e q u a t i o n : % polyose  % sugar  mol. wt. o f s u g a r — H 0  x  2  mol. wt. o f sugar % mannan  % mannose  x  0.90  % xylan  % xylose  x  0.88  The p o l y o s e v a l u e was a l s o c o r r e c t e d f o r m o i s t u r e  B.  Enzymatic I.  content.  Studies  Substrate A l l o f t h e c u l t u r e s mentioned i n t h i s  t i o n were grown i n a s y n t h e t i c l i q u i d medium.  investigaThis basal  medium was a s o l u t i o n o f t h e f o l l o w i n g i n o r g a n i c s a l t s i n t a p w a t e r (gm/1) :  NH^Cl 4.3; NH^NOo 1.0; K C l 4.5;  Pig.3  Graph  showing the l i n e a r r e l a t i o n o f  mannose to  (M) and x y l o s e  the o p t i c a l  grammed  spots.  (X) c o n c e n t r a t i o n s  d e n s i t y of t h e i r  chromato-  19  Na HP0^.7H 0 2.7; MgS0^.7H 0 2.5; ZnSO^ .01; FeSO^ .01; 2  2  2  MnSG^ .08; carbon s o u r c e 5 gm./l.  T h i s s o l u t i o n was  a d j u s t e d t o pH 8, (by t h e a d d i t i o n o f sodium h y d r o x i d e ) f i l t e r e d and then s t e r i l i z e d a t 15 p . s . i .  f o r 20 m i n u t e s .  X y l a n , t h e carbon s o u r c e used i n t h e s e e x p e r i ments, was p r e p a r e d by t h e method o f Voss and B u t t e r (35). X y l a n powder was brought i n t o s o l u t i o n i n a m o r t a r w i t h 5 or 6 m i s . o f 2N NaOH p e r gram o f x y l a n , d i l u t e d w i t h water t o 4 t o 5 p e r cent x y l a n and then h e a t e d t o 40-50° f o r h a l f an hour on a water b a t h .  After  centrifugation  from i n s o l u b l e p a r t i c l e s t h e m i l k y s o l u t i o n was d i a l y s e d i n c o l l o d i a n sacks t o a pH o f about 6.5 (2 t o 3 d a y s ) . Ten n i l s , o f t h i s s o l u t i o n were e v a p o r a t e d t o d r y n e s s t o determine t h e x y l a n c o n t e n t .  D i s t i l l e d water was added  to g i v e a f i n a l x y l a n c o n c e n t r a t i o n o f 1 gm./lOO mis. o f solution.  This c o l l o i d a l  s o l u t i o n was s t o r e d i n a b o t t l e  c o n t a i n i n g excess t o l u e n e . To a s c e r t a i n t h e homogeneity  of the xylan a  sample was b o i l e d under r e f l u x f o r 6 hours w i t h IN H S0^. 2  A f t e r n e u t r a l i z a t i o n w i t h barium c a r b o n a t e t h e s o l u t i o n was f i l t e r e d and s p o t t e d on a paper chromatogram.  The  o n l y sugar shown t o be p r e s e n t was x y l o s e . II.  Micro-organisms The f o l l o w i n g organisms were i n c u b a t e d a t 37°  i n l i q u i d media w h i c h c o n t a i n e d c o l l o i d a l  x y l a n as t h e  20 s o l e c a r b o n source: growth Actinomyces  51-3.  good  Actinomyces  £1-36  poor  Actinomyces  52-11  poor  Actinomyces  5?-?  fair  Actinomyces  50=2  poor good  Actinomyces Actinomyces  55-10  poor  Actinomyces  55-13  fair  trifolii  poor  Rhlzoblum  Rhizobium m e l i o t i  poor  Azotobacter v i n e l a n d i i  poor  A z o t o b a c t e r chroococcum  poor  Ghaetomlum globosum Kunze  v e r y good  Chaetomlum elobosum  v e r y good  (NRC V-159)  poor  Escherichia c o l l  At the end o f two weeks t h e e x t e n t o f growth was o b s e r v e d and r o u g h l y e v a l u a t e d as shown above.  The  more a c t i v e organisms were a b l e t o cause a r a p i d c l e a r i n g of t h e opaque media. I n d i c a t i n g s a c c h a r i f i c a t i o n o f t h e c o l l o i d a l xylan. The m i c r o - o r g a n i s m s w h i c h were c a p a b l e of growth i n t h e s y n t h e t i c medium were washed, homogenized to f r e s h s o l u t i o n s of x y l a n .  and added  Sodium c y a n i d e (.003M) was  21  added t o b l o c k the metabolism o f t h e d e g r a d a t i o n p r o d u c t s of x y l a n s u f f i c i e n t l y t o p e r m i t t h e i r d e t e c t i o n by chromatography.  A f t e r i n c u b a t i o n (37° f o r 7 d a y s ) t h e  c u l t u r e s were c o n c e n t r a t e d and s p o t t e d on paper grams.  paper  chromato-  S t a n d a r d sugar s o l u t i o n s were a l s o s p o t t e d on t h e  same chromatograms f o r i d e n t i f i c a t i o n . From t h e s e p r e l i m i n a r y e x p e r i m e n t s t h r e e s o i l m i c r o - o r g a n i s m s were found t o grow r e a d i l y i n t h e s y n t h e t i c medium.  F u r t h e r m o r e , x y l o s e was  resulting organisms:  product  from the h y d r o l y s i s o f t h e x y l a n by t h e s e t h r e e Actinomyces  55-7» Ghaetomlum globosum Kunze  and Chaetomium globosum (NRC Ill.  the p r i n c i p a l  V-159).  Enzyme p r e p a r a t i o n C u l t u r e s o f C. globosum Kunze were I n c u b a t e d a t  25° i n Roux f l a s k s ,  each c o n t a i n i n g 100 mis. o f medium.  As soon as the s u r f a c e s o f t h e media were c o v e r e d (7 t o 10 d a y s ) t h e mycelium was h a r v e s t e d , t w i c e washed w i t h d i s t i l l e d water and c e n t r i f u g e d a t The r e s u l t i n g  mycelium was  0°. then p r e p a r e d f o r  treatment i n a m a g n e t o - o s t r i c t i o n o s c i l l a t o r Raytheon Mfg. Co.).  (50 w a t t , 9 k c  A p r e l i m i n a r y s e p a r a t i o n and b r e a k -  down of t h e c e l l s wae made by m e c h a n i c a l l y g r i n d i n g t h e f u n g i i n a g r o u n d - g l a s s homogenizer.  The  partially  homogenized m y c e l i a were then t r a n s f e r r e d t o t h e h i g h frequency o s c i l l a t o r .  A t e n minute t r e a t m e n t i n the  22  o s c i l l a t o r was walls.  s u f f i c i e n t t o r u p t u r e most o f the  A c l e a r enzyme s o l u t i o n was  d e b r i s by c e n t r i f u g i n g a t 0°.  The  cell  s e p a r a t e d from the  cell  enzyme p r e p a r a t i o n s  were t h e n f r o z e n f o r s t o r a g e . IV.  The d e t e r m i n a t i o n o f enzyme a c t i v i t y Two  mis. o f enzyme p r e p a r a t i o n , 2 mis.  of  c o l l o i d a l x y l a n s o l u t i o n and 1 ml. o f t o l u e n e were shaken v i g o r o u s l y I n a 25 m l . c o r k - s t o p p e r e d Erlenmeyer f l a s k and p l a c e d i n the i n c u b a t o r at 25° f o r f i v e days.  Then,  the m i x t u r e was  was  c e n t r l f u g e d and t h e c l e a r s o l u t i o n  s p o t t e d on a paper chromatogram.  Pure sugar s o l u t i o n s  were s p o t t e d on t h e same sheet f o r i d e n t i f i c a t i o n . was  the predominant sugar p r e s e n t .  Xylose  S e v e r a l p i n k spots o f  r e g u l a r l y d e c r e a s i n g Rf v a l u e s were a t t r i b u t e d t o polymers of x y l o s e such as x y l o b i o s e , x y l o t r l o s e , x y l o t e t r a o s e etc.  (32).  A f t e r d e t e r m i n i n g the a b i l i t y o f t h e enzyme p r e p a r a t i o n t o break down x y l a n , the enzyme e x t r a c t was t e s t e d f o r a c t i v i t y on s e v e r a l w o o d - c e l l u l o s e p r e p a r a t i o n s . The w o o d - c e l l u l o s e sample was  a i r - d r i e d at room tempera-  t u r e and t h e n f l u f f e d I n the manner p r e v i o u s l y d e s c r i b e d . E x a c t l y one gram o f t h i e f l u f f e d sample was 250 ml. Erlenmeyer f l a s k .  added t o a  Twenty mis. o f d i s t i l l e d  were added t o the f l a s k and the m i x t u r e was  water  autoclaved at  I 23  15 p . s . i . f o r 20 minutes. A 10 gram sample o f t h e a i r - d r i e d p u l p was t a k e n f o r m o i s t u r e d e t e r m i n a t i o n a t t h e same time as t h e enzyme sample.  The m o i s t u r e sample was d r i e d a t 1 1 0 ° f o r 8 h o u r s  and weighed.  The p e r cent o f d r y wood c e l l u l o s e i n t h e  sample was c a l c u l a t e d from t h e weight d i f f e r e n c e o f t h e sample b e f o r e and a f t e r d r y i n g . When t h e s t e r i l i z e d  f l a s k was c o o l , 30 m i s . o f  enzyme p r e p a r a t i o n were added a s e p t i c a l l y and mixed t h o r o u g h l y w i t h t h e wood c e l l u l o s e .  As a f u r t h e r p r e -  c a u t i o n a g a i n s t b a c t e r i a l c o n t a m i n a t i o n , t o l u e n e was added to form a t h i n l a y e r over t h e s u r f a c e o f t h e m i x t u r e . I n c u b a t i o n o f t h e p r e p a r a t i o n was c a r r i e d out a t 2 5 ° f o r fourteen  days. At t h e end o f t h e i n c u b a t i o n p e r i o d , t h e  r e m a i n i n g wood c e l l u l o s e was t r a n s f e r r e d t o a f r i t t e d g l a s s f i l t e r and w e l l washed w i t h d i s t i l l e d water. was f i l t e r e d  The f i l t r a t e  t h r o u g h t h e pad o f wood c e l l u l o s e a second  time t o remove any f i b r e s from t h e washings.  The pad was  f i n a l l y washed w i t h acetone and d r i e d a t 1 0 5 ° . The d r i e d sample was s u b s e q u e n t l y  analysed f o r  x y l a n and mannan a c c o r d i n g t o t h e p r e v i o u s l y d e s c r i b e d procedure.  Any s i g n i f i c a n t d i f f e r e n c e i n t h e c a r b o h y d r a t e  content o f t h e enzyme t r e a t e d and u n t r e a t e d  wood-cellulose  p r e p a r a t i o n s I n d i c a t e d t h e e x t e n t o f enzyme a c t i o n on t h e polysaccharides.  24 RESULTS  Table 1.  A n a l y s i s o f S e l e c t e d Commercial Wood P u l p s  Wood source  Process  Use o f pulp  Vise./P.P.  Hemlock  Sulfite  Paper  170  Hemlock  Sulfite  Cellophane  Hemlock  Sulfite  Photo-  #  Alpha cell.fi  Mannan %  Xylan ft  87«7  7.4  2.4  36  90.2  2.5  1.1  447  95-7  1.9  1.4  graphy Fir  Kraft  Paper  38  89.0  6.3  3-4  Fir  Kraft  22  93.6  3.4  2.0  Pine  Prehydrol. sulfate  High tenacity rayon Cellophane  89.6  4.6  2.1  Spruce  Sulfite  603  Rayon  1215  94.4  2.2  2.7  Southern S u l f i t e pine  Tire cord  1380  95.8  2.1  2.8  Western hemlock  Tire cord  1340  96.2  2.9  2.1  Southern S u l f i t e pine  Diesolving  1300  96.2  2.3  1.2  Western hemlock  Diesolving  1300  96.7  2.8  1.3  Sulfite  Sulfite  * i n copper e t h y l e n e d i a m i n e , 1% c e l l u l o s e , 2 5 ° **" c a l c u l a t e d from e q u a t i o n s shown on p. 18  Table 2 .  25  Comparison o f Pentosan and X y l a n A n a l y s e s  Wood source  Use o f pulp  V i s e . i n CED \% c e l l . 2 5 °  Alpha cell.#  Pentosan X y l a n %  Mixed* hardwoods  Paper  40.0  88.5  20.0  Mixed hardwoods  Dissolving  25.0  89.0  3.5  2.9  Mixed hardwoods  Dissolving  60.0  93.0  3.0  3.3  Spruce, f i r , hem.  F i n e paper  175  94.0  3.0  3.1  Spruce, f i r , hem.  Paper  60.0  87.5  8.0  7.3  Poplar aspen  Paper  23.7  85.6  9.9  9.7  Poplar aspen  Paper  18.2  78.2  21.0  19.6  Birch  Paper  24.0  81.3  14.8  14.5  Birch  Paper  18.7  73.5  26.3  20.0  +  18.?  * w h i t e b i r c h , y e l l o w b i r c h , beech, h a r d maple, s o f t maple Table 3 .  An A n a l y s i s  o f Mannan and X y l a n from Wood C e l l u l o s e  B e f o r e and A f t e r Enzyme A c t i o n Wood p u l p sample Spruce  V i s e . i n CED 1% c e l l . 2 5 °  Alpha c e l l . %  60  87.5  Spruce (enzyme added)  +  Mannan %  +  Xylan %  7.4  7.3  7.2  5.0  The o n l y sugar d e t e c t e d i n t h e washings from the enzyme-treated sample was x y l o s e .  26 DISCUSSION  The d e s c r i b e d method f o r q u a n t i t a t i v e l y e s t i m a t i n g x y l a n and mannan i n wood c e l l u l o s e can d e t e r m i n e sugar c o n c e n t r a t i o n s as l o w as 1%.  R e s u l t s are r e a d i l y  d u p l i c a t e d w i t h i n an e r r o r o f - 10% i n such l o w r a n g e s . T h i s s e n s i t i v i t y i s p o s s i b l e because many o f t h e o b j e c t i o n a b l e f e a t u r e s i n e s t i m a t i n g paper chromatograms by p h o t o m e t r i c methods have been e l i m i n a t e d o r c o n s i d e r a b l y reduced.  F o r example,  a drawback t o many d e v e l o p i n g agents  i s t h a t they produce c o l o r e d s p o t s w h i c h a r e t o o u n s t a b l e f o r photometric a n a l y s i s .  The d i s c o l o r a t i o n o r o t h e r  changes i n t h e background o f t h e d e v e l o p e d paper s h e e t s has a l s o been a d i s a d v a n t a g e t o s e v e r a l methods, e s p e c i a l l y those u s i n g o x i d i z i n g agents such as p o t a s s i u m permanganate and m e t a p e r i o d a t e s o l u t i o n s f o r d e v e l o p e r s .  The a n i l i n e  hydrogen p h t h a l a t e d e v e l o p e r used i n t h i s method produces spots which are s t a b l e f o r s e v e r a l hours.  The background  a l s o remains v e r y l i g h t and i n good c o n t r a s t t o t h e c o l o r e d spots.  The s t r i p s o f paper may be k e p t f o r s e v e r a l days  i f they a r e i n d i v i d u a l l y p l a c e d between some s h e e t s o f paper.  However, a n o t i c e a b l e d a r k e n i n g o f t h e background  does o c c u r d u r i n g t h i s time but d e n s i t y r e a d i n g s a r e s t i l l permissible.  A n o t h e r d i s a d v a n t a g e o f some r e p o r t e d  methods i s t h e i r l o w s e n s i t i v i t y , a l t h o u g h good d e t e r m i n -  27  a t l o n s can be made on the d e t e c t a b l e s p o t s . procedure, concentrations detected  In this  of x y l o s e o r mannose can  be  i n amounts as low as 1 mg./ml. when a p p l i e d i n  amounts o f 2 A .  This quantity represents  sugar as low as 0 . 0 0 2 mgms.  amounts o f  The g e o m e t r i c a l  shape of  the  s p o t , w h i c h i s so Important i n s e v e r a l methods u s i n g densitometers,  i s e l i m i n a t e d by t h i s t e c h n i q u e  because  o n l y a s m a l l a r e a o f the whole spot i s scanned by beam o f l i g h t .  The  the  o p t i c a l d e n s i t y , t h e r e f o r e , depends  o n l y on t h e l i g h t t r a n s m i t t e d by a s m a l l p o r t i o n o f the spot r a t h e r than on an average of the whole spot or whole spot p l u s the s u r r o u n d i n g  the  background.  From f i g u r e 3 l t can be seen t h a t the l i g h t t r a n s mittance  v a l u e s f o l l o w Beer's law c l o s e l y w i t h i n the  range o f sugar c o n c e n t r a t i o n s .  Winslow (36)  c o m p a r a t i v e s t u d i e s on r e f l e c t a n c e and techniques  has done  transmittance  f o r e s t i m a t i n g spots on a r e c o r d i n g  photometer.  given  spectro-  The r e s u l t s i n d i c a t e d t h a t measurement by  t r a n s m i t t a n c e was o f the s p o t s and  preferable for quantitative evaluation showed t h a t such t e s t s g i v e  transmittance  d a t a c l o s e l y I n accordance w i t h B e e r ' s law. The  r e s u l t s i n t a b l e 2 show good agreement  between the x y l a n v a l u e s and the c o n v e n t i o n a l values. due  The  pentosan  g e n e r a l l y h i g h e r pentosan r e s u l t s are p o s s i b l y  t o t h e presence o f u r o n i c a c i d s w h i c h a l s o c o n t r i b u t e  t o the pentosan v a l u e .  Corrections for this interference  28 i n p r e s e n t methods are not e n t i r e l y  satisfactory.  The r e s u l t s o f enzymatic a c t i o n on t h e woodc e l l u l o s e p r e p a r a t i o n s i n v e s t i g a t e d show t h a t about 25 per cent o f the x y l a n was removed i n a s p e c i f i c manner. The r e m a i n i n g x y l a n i s p o s s i b l y i n the i n a c c e s s i b l e o r c r y s t a l l i n e p o r t i o n of t h e c e l l u l o s e .  However, t h e  s e l e c t i v e removal o f some o f the c e l l u l o s a n x y l a n i e p o s s i b l e w i t h o u t apparent d e g r a d a t i o n o f t h e o e l l u l o s e . I t i s not assumed i n t h e s e experiments t h a t t h e x y l a n a s e i s n e c e s s a r i l y a s i n g l e enzyme.  Much evidence  has been g i v e n i n support of a s e r i e s o f enzymes, each produced decessor.  o r s t i m u l a t e d by the end p r o d u c t o f i t s p r e These enzymes are s e p a r a t e from t h e  t u i t i v e " enzymes o f t h e c e l l w h i c h a r e  "consti-  themselves  e l a b o r a t e d I r r e s p e c t i v e o f t h e presence o r absence o f homologous s u b s t r a t e s . hand, a r e produced  The a d a p t i v e enzymes,on the o t h e r  as a r e s u l t o f t h e organism coming i n  c o n t a c t w i t h a s p e c i f i c s u b s t r a t e w h i c h i t does not normally u t i l i z e .  F o r example, s u b s t r a t e A i n d u c e s the  f o r m a t i o n o f enzyme A which causes the p r o d u c t i o n o f B. In t u r n , B i n d u c e s t h e f o r m a t i o n o f enzyme B.  This process  i s r e f e r r e d t o as s e q u e n t i a l I n d u c t i o n by K a r s t r o m  (39).  Such a mechanism would f i t t h e breakdown o f x y l a n shown t o o c c u r i n t h e study o f x y l a n a s e by Sorensen  i n whioh e v i d e n c e  i n d i c a t e s t h a t at l e a s t two enzymes are i n v o l v e d (33).  The  29  a b i l i t y o f the x y l a n a s e p r e p a r a t i o n t o produce the s a c c h a r i d e s from x y l a n was  oligo-  not d e s t r o y e d on h e a t i n g whereas  the power t o h y d r o l y s e these o l i g o s a c c h a r i d e s t o x y l o s e was  destroyed. In  k i n e t i c s t u d i e s o f the h y d r o l y s i s o f p o l y -  s a c c h a r i d e s (35»37»38) a l l r e a c t i o n s , whether c h e m i c a l enzymatic,  showed s i m i l a r r a t e c u r v e s .  There was  or  an  i n i t i a l l y r a p i d r a t e f o l l o w e d by a slower c o n s t a n t r e a c t i o n rate.  The  two-phase s t r u c t u r e o f c e l l u l o s e , i . e . the  e x i s t e n c e o f the c e l l u l o s e c h a i n m o l e c u l e s  in crystalline  and amorphous s t a t e s , has been g i v e n i n e x p l a n a t i o n o f these r e s u l t s .  The r e a d i l y a c c e s s i b l e or amorphous r e g i o n s  c o n t a i n most o f the h e m l c e l l u l o s e s w h i c h are hydrolysed.  easily  T h i s r e a c t i o n o c c u r s i n the i n i t i a l phase.  Random c l e a v a g e o f the c e l l u l o s e s t r u c t u r e a l s o o c c u r s . The  slower r e a c t i o n i s b e l i e v e d t o t a k e p l a c e on the s u r -  f a c e o f the c e l l u l o s e c r y s t a l l i t e s . by W a l s e t h ( 3 8 ) ,  I n enzymatic  studies  the enzyme showed some d e c r e a s e i n r e -  a c t i v i t y but not s i g n i f i c a n t l y enough t o account f o r the slow h y d r o l y s i s . M o i s t u r e r e g a i n v a l u e s d e c r e a s e d  i n the  u n d i s s o l v e d r e s i d u e s I n d i c a t i n g t h a t the amorphous r e g i o n s were most r a p i d l y a t t a c k e d . A s t r i k i n g d i f f e r e n c e between a c i d and  enzymlc  h y d r o l y s i s o f wood c e l l u l o s e a l s o has been shown ( 3 8 ) .  In  enzyme d e g r a d a t i o n a r e l a t i v e l y h i g h degree o f p o l y m e r i z a t i o n (D.P.) was  r e t a i n e d i n the r e s i d u a l c e l l u l o s e ,  30  whereas by a c i d h y d r o l y s i s a low D.P. o f u n d i s s o l v e d c e l l u l o s e was n o t i c e d .  The r e l a t i v e s i z e and p e n e t r a t i n g  a b i l i t y o f t h e two h y d r o l y t i c agents was g i v e n i n explanation.  The enzyme may cause r a p i d and complete r e -  d u c t i o n o f t h e c e l l u l o s e i n t h e amorphous r e g i o n t o soluble products.  I t was a l s o suggested t h a t t h e r a p i d  p r o d u c t i o n o f g l u c o s e w i t h o u t a l o w e r i n g o f t h e D.P. may i n d i c a t e t h a t t h e enzyme a t t a c k s t h e ends o f t h e c e l l u l o s e c h a i n s r a t h e r than randomly h y d r o l y s i n g t h e m o l e c u l e s .  On  the o t h e r hand, t h e a c i d i s b e l i e v e d t o p e n e t r a t e t h e c e l l u l o s e s t r u c t u r e t o a g r e a t e r e x t e n t t h a n t h e enzyme and produce s h o r t fragments w h i c h c o u l d not e a s i l y d i f f u s e out o f t h e c e l l u l o s e s t r u c t u r e . The f a c t t h a t t h e r e i s c o n s i d e r a b l e d i f f e r e n c e between a c i d i c and enzymatic h y d r o l y s i s o f wood c e l l u l o s e i n d i c a t e s t h a t i t i s an advantage t o use enzymes f o r s t u d y i n g wood p o l y s a c c h a r i d e s . doubtedly  Enzymatic s t u d i e s w i l l u n -  p r o v i d e a d d i t i o n a l i n f o r m a t i o n on wood  w h i c h i s n o t d e t e c t a b l e by p u r e l y c h e m i c a l means. t h i s Information are used.  polyoses However,  s h o u l d be more c o n c i s e i f s p e c i f i c enzymes  The p r e s e n t method o f o b t a i n i n g such enzymes i s  by s p e c i f i c s u b s t r a t e I n d u c t i o n . The e x p l o r a t o r y work d i s c u s s e d has shown t h a t i t i s p o s s i b l e t o obtain a s p e c i f i c xylanase which i s a c t i v e on a w o o d - c e l l u l o s e  preparation.  T h i s enzyme ( o r enzymes)  c o u l d be o f importance i n t h e study o f wood o e l l u l o s e s t r u c t u r e and c o m p o s i t i o n .  30a  SUMMARY  1.  Mannan and x y l a n from wood c e l l u l o s e can "be  q u a n t i t a t i v e l y determined,  a f t e r a c i d h y d r o l y s i s , by means  of paper p a r t i t i o n chromatography.  The  developed  chromatograms are a n a l y s e d d i r e c t l y u s i n g an adapted m i t t a n c e a c c e s s o r y f o r a Beckman Model B  trans-  spectrophotometer.  The a c c u r a c y of the method i s 5 t o 10 per c e n t . 2.  G e n e r a l l y c l o s e agreement i s shown i n a com-  p a r i s o n o f c o n v e n t i o n a l pentosan  analyses w i t h xylan  d e t e r m i n a t i o n s made by the d e s c r i b e d method.  The  slightly  h i g h e r pentosan v a l u e s are p o s s i b l y due t o t h e i n c l u s i o n of u r o n l c a c i d i n the e s t i m a t i o n s . 3.  An induced x y l a n a s e from Chaetomlum globosum  gunze was  shown t o be capable o f h y d r o l y s i n g x y l a n from  wheat s t r a w .  The main end product of h y d r o l y s i s d e t e c t e d  by means o f paper chromatography was x y l o s e . 4.  T h i s enzyme p r e p a r a t i o n was  a l s o capable of  s p e c i f i c a l l y removing a f r a c t i o n o f the x y l a n from a commercial w o o d - c e l l u l o s e sample w i t h o u t changing the mannose o r g l u c o s e c o n t e n t .  significantly  31  BIBLIOGRAPHY  1.  E. F. H l n n e r , i n " C e l l u l o s e and C e l l u l o s e D e r i v a t i v e s , " E, O t t ed., I n t e r s c i e n c e P u b l i s h e r s , I n c . , New Y o r k , N. Y., 1 9 4 3 , p. 5 1 9 .  2.  F. K l a g e s , Ann., 5 0 9 , 159 ( 1 9 3 * 0 ; .512, 185 ( 1 9 3 * 0 .  3.  W. N. Haworth, R. L. Heath and S. P e a t , J . Chem. S o c , 833  (1941).  4.  G. 0 . A s p i n a l l , E. L. H i r s t , E. G. V. P e r c i v a l and 1. R. W i l l i a m s o n , J . Chem. S o c , 3184 ( 1 9 5 3 ) .  5.  F. G e r h a r d t , P l a n t P h y s i o l . , 4 , 373 ( 1 9 2 9 ) .  6.  S. K. Chandra, E. L. H i r s t and E. G. V. P e r c i v a l , J . Chem. S o c , 1240 ( 1 9 5 D .  2,  7.  A. R o u d l e r and L. E b e r h a r d , T a p p i ,  8.  M. H. O'Dwyer, Biochem. J . ,  9.  L. F. Hawley and A. G. Norman, I n d . Eng. Chem.., 24, 1190  21*  713  38  (1955).  (1939).  (1932).  10.  E. Schmidt, K. M e i n e l , W. Jandebeur and W. Simson, Cellulosechemie, 129 ( 1 9 3 2 ) .  11.  W. J . P o l g l a s e , Advances i n Carbohydrate 10,  Chemistry,  283.  12.  C. Doree, "The Methods o f C e l l u l o s e C h e m i s t r y , " D. Van N o s t r a n d Co., I n c . , New Y o r k , N. Y., 1933t P. 3 5 4 .  13.  C. A. Browne and F. W. Zerban, " P h y s i c a l and C h e m i c a l Methods o f Sugar A n a l y s i s , " John W i l e y and Sons, I n c . , New Y o r k , N. Y., 1 9 4 1 , p. 9 0 4 .  14.  A. W. S c h o r g e r , I n d . Eng. Chem., 2, 748 ( 1 9 1 7 ) .  15.  L. E. Wise, E. K. R a t l i f f and B. L. Browning, A n a l . Chem., 20,  825 (1948).  16.  H. W. Steinmann and B. B. W h i t e , T a p p i , J32. 225 ( 1 9 5 4 ) .  17.  G. N. Kowkabany, Advances i n Carbohydrate 2,  303 ( 1 9 5 4 ) .  Chemistry,  32  18.  L. E. Wise, J . W. Green and R. C. R i t t e n h o u s e , T a p p i , 12, 335 (1949)  19.  J . Sundman, J . S a a r n i o and C. G u s t a f s s o n , P a p e r l J a Puu, B 3 3 . 115  (1951).  20.  B. L. Browning, i n "Wood C h e m i s t r y , " L. E. Wise and E. C. Jahn, eds., R e i n h o l d P u b l i s h i n g Corp., New Y o r k , N. Y., 2nd e d i t i o n , 1952, p. 1 1 6 2 .  21.  C. G u s t a f f s o n , J . Sundman and T. L l n d h , P a p e r i J a Puu,  22.  E. Hagglund, "Chemistry o f Wood," Academic P r e s s I n c . , New York, N. Y., 1951.  23.  R. E. Glegg and D. E i d i n g e r , A n a l . Chem., 2 6 , 1365  24.  J . F. Saeman, W. E. Moore, R. L. M i t c h e l l and M. A. M i l l e t , T a p p i , J Z , 336 ( 1 9 5 4 ) .  25.  F. Cramer, "Paper Chromatography," M a c M i l l a n and Co., L t d . , London, 2nd e d i t i o n , 1954.  26.  R. J . B l o c k , E. L. Durrum and G. Zweig, "Paper Chromatography and Paper E l e c t r o p h o r e s i s , " Academic P r e s s I n c . , New Y o r k , N. Y., 1 9 5 5 ,  27.  E. L e d e r e r and M. L e d e r e r , "Chromatography," P u b l i s h i n g Co., New Y o r k , N. Y., 1 9 5 3 .  28.  J . S a a r n i o , E. N l s k a s a a r i and C. G u s t a f s s o n , Suomen  B 33. 1 (195D.  Kemistelehtl,  (1954).  Elsevier  25 B. 25 ( 1 9 5 2 ) .  29.  R. H. M u l l e r and D. L. C l e g g , A n a l . Chem., 2 J , 397 ( 1 9 5 1 ) .  30.  F. F. McFarren, K. Brand and H. R. R v t k o w s k i , A n a l . Chem., 2 3 , 1146 ( 1 9 5 1 ) .  31.  W. Grassman, R. S t a d l e r and R. Bender, Ann. d. Chemie, i&2.,  20  (1933).  32.  H. Sorensen,  33.  H. Sorensen, N a t u r e , 172, 305 ( 1 9 5 3 ) .  34.  A. P. Yundt, T a p p i , J 4 , 92 ( 1 9 5 1 ) .  35.  W. Voss and G. B u t t e r , Ann. d. Chemie, 5 3 4 . 161  36.  F. H. Winslow and H. A. L l e b h a f s k y , A n a l . Chem., 21,  1338  P h y s i o l o g l a P l a n t arum, J5, 183  (1949).  (1952).  (1938).  33  37.  C. S. W a l s e t h , T a p p i , 21*  2 2 8  38.  C. S. W a l s e t h , T a p p i , 21*  2  39.  H. K a r s t r o m , Ergeb. Enzymforsch., 7_, 350 ( 1 9 3 7 ) .  40.  R. Y. S t a n l e r , J . B a c t . , j£4, 339 ( 1 9 4 7 ) .  33  (195 ). 2  (1952).  

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