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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 of 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 the Department of Biochemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA September, 1956 In presenting t h i s thesis 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 at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available f o r reference and study. I further agree that permission f o r extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representative. I t i s under-stood that copying or publication 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 written permission. Department of Biochemistry The University of B r i t i s h Columbia, Vancouver Canada. Date September 28, 1956 ABSTRACT (11) A method i e described f o r the p r e p a r a t i o n of an induced xylanase from Chaetomlum globosum. This s o i l micro-organism 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 xylan as the sole carbon source. A f t e r i n -cubation at room temperature f o r a s u i t a b l e time, the mycella were harvested, c e n t r i f u g e d and exposed to hig h frequency v i b r a t i o n s which ruptured the 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 to be capable of h y d r o l y s i n g x y l a n from wheat straw. The main end product of h y d r o l y s i s detected by means of paper chromato-graphy was xylose. The a c t i o n of 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 st u d i e d . A considerable f r a c t i o n of the x y l a n was removed without s i g n i f i c a n t l y changing the mannose or glucose content. In connection w i t h the 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 poly-saccharides i n wood-cellulose p r e p a r a t i o n s . D i r e c t photo-metric analyses of the wood hydrolysate spots on paper chromatograms were made w i t h an adapted Beckman spectro-photometer. An a n a l y s i s of s e l e c t e d commercial pulps i s given together w i t h a comparison of the method w i t h conventional pentosan analyses. (v) ACKNOWLEDGEMENT My sincere thanks are extended to Dr. W.J. Po 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 study. A p p r e c i a t i o n i s a l s o extended to the N a t i o n a l Research 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 of t h i s work. The author i s indebted to the f o l l o w i n g companies f o r supplying commercial wood pulp samples: E. I . Du Pont De Nemours & Company Inc . , Benger Laboratory, Waynesboro, V i r g i n i a . Weyerhaeuser Timber Company, Pulp D i v i s i o n , Research Dept., E v e r e t t , Washington. West V i r g i n i a Pulp and Paper Company, Luke, Maryland. 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 Research & Development D i v i s i o n , Sault Ste. Marie, Ontario, Canada. Wheat straw x y l a n was k i n d l y eupplied by Dr. R. L. Wh 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 , Indiana. The c u l t u r e s of (3. globosum Kunze were generously provided by Dr. H. Sj^rensen, Dept. of B a c t e r i o l o g y , State Laboratory of Plant C u l t u r e , Lyngby, Denmark. Dr. D. G. L a i r d , Dept. of S o i l Science, U n i v e r s i t y of B.C. also aided t h i s work by supplying a d d i t i o n a l micro-organisms as w e l l as g i v i n g h e l p f u l suggestions. ( i i i ) TABLE OF CONTENTS Page INTRODUCTION 1 HISTORICAL 3 A. The Q u a n t i t a t i v e Determination of Wood Polysaccharides 3 I . The s t r u c t u r e of the more predominant polysaccharides i n wood 3 I I . The q u a n t i t a t i v e determination of mannan . . 6 I I I . The q u a n t i t a t i v e determination of x y l a n . . 8 B. Hemlcellulases 10 EXPERIMENTAL 13 A. The Q u a n t i t a t i v e Determination of Mannan and Xylan 13 I . H y d r o l y s i s of wood c e l l u l o s e 13 I I . Sugar standards 1^ I I I . Chromatography 16 P l a t e I Photograph of a 72 hour paper chromatogram IV. Photometric a n a l y s i s 17 P l a t e I I A diagram of the c a r r i a g e and removable paper s t r i p holder adapted f o r use w i t h a Beckman Model <T3 spectrophotometer B. Enzyme Studies 18 I . Substrate 18 P l a t e I I I Graph showing the l i n e a r r e l a t i o n of mannose and xylose concentrations to the o p t i c a l d e n s i t y of t h e i r chromatogrammed spots ( i v ) TABLE OF CONTENTS(cont'd.) Page I I . Micro-organisms 19 I I I . Enzyme pr e p a r a t i o n 21 IV. The determination of enzyme a c t i v i t y . . . 22 RESULTS 24 DISCUSSION 26 SUMMARY 3 0 a BIBLIOGRAPHY 31 INTRODUCTION 1 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 of enzymes on the polysaccharides a s s o c i a t e d w i t h wood c e l l u l o s e . In p a r t i c u l a r , t h i s i n v e s t i g a t i o n was concerned w i t h x y l a n and mannan which, next to c e l l u l o s e , are the most abundant carbohydrates of wood. Commercial wood pulps 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 set up t o determine the e f f e c t of induced xylanases on the xylan content of v a r i o u s wood pulps. The methods used were concerned w i t h two aspects of t h i s study: the q u a n t i t a t i v e carbohydrate a n a l y s i s of various wood pulps and the p r e p a r a t i o n of s u i t a b l e enzymes. F i r s t , i n order to estimate changes i n the wood carbohydrate content before and a f t e r enzyme a c t i o n , i t was necessary to develop a s u i t a b l e q u a n t i t a t i v e method of 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 photometric e s t i m a t i o n of the hydrolysed samples spotted on paper chromatograms. Considerable time d u r i n g the experimental work was given to the development of t h i e method and i t s a p p l i c a t i o n to v a r i o u s preparations of wood c e l l u l o s e . Second, i n order to o b t a i n the d e s i r e d enzyme a c t i v i t y the 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 olysaccharide as 2 the sole carbon source. I n t h i s way, enzymes were I n -duced which were capable of me t a b o l i s i n g the p a r t i c u l a r p o lysaccharide. This i n v e s t i g a t i o n i s of value to both funda-mental understanding and i n d u s t r i a l a p p l i c a t i o n i n the c e l l u l o s e f i e l d . At present, no chemical methods are a v a i l a b l e f o r preparing undegraded wood c e l l u l o s e , f r e e from c l o s e l y a s s o c i a t e d polysaccharides. I f s p e c i f i c enzymes could be used to achieve t h i s o b j e c t i v e , i t would provide 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 studies on c e l l u l o s e . In c o n s i d e r i n g p o s s i b l e I n d u s t r i a l a p p l i c a -t i o n s ( e s p e c i a l l y i n the pulp and paper i n d u s t r y ) , pulps from which s p e c i f i c polysaccharides could 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 should prove to be commercially superior i n many r e s p e c t s . Furthermore, the s p e c i f i c removal of wood polysaccharides would enable a r e l i a b l e e v a l u a t i o n of t h e i r c o n t r i b u t i o n to the p r o p e r t i e s of the o r i g i n a l wood-cellulose p r e p a r a t i o n . HISTORICAL 3 A. The Q u a n t i t a t i v e Determination of Wood Polysaccharides I . The s t r u c t u r e of the more predominant poly- saccharides i n wood A b r i e f summary of the s t r u c t u r e of the more important wood polysaccharides or polyoses I s important to an understanding of the a n a l y t i c a l methods used f o r determining them. C e l l u l o s e , by f a r the most abundant wood pol y s a c c h a r i d e , w i l l be discussed f i r s t . The present concept of the molecular s t r u c t u r e of c e l l u l o s e (as obtained from cotton l i n t e r e ) i s a l i n e a r , unbranched polymer of D-glucose anhydride u n i t s , l i n k e d by 4- > 1, B-D-glucosidic bonds ( l ) . In the n a t i v e s t a t e , c e l l u l o s e i s composed of a network of these l i n e a r chains which vary considerably i n the number of component monomeric u n i t e . The i n d i v i d u a l l i n e a r molecules thus form a polymer-homologous s e r i e s w i t h i n any s i n g l e c e l l u l o s e p r e p a r a t i o n . Superimposed on these molecular d i f f e r e n c e s are v a r i a t i o n s i n the arrangement of the chains which 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 orders of l a t t i c e p e r f e c t i o n and degrees of o r i e n t a t i o n . There are formed, t h e r e f o r e , gradations between random molecular arrangement In amorphous regions and nearly 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 ordered p o r t i o n s . k In wood c e l l u l o s e preparations there i s not the chemical homogeneity found i n cotton c e l l u l o s e . This f a c t i s p a r t l y due to the Incomplete removal of such substances as l i g n i n , ash, wood e x t r a c t i v e s , and e s p e c i a l l y the c l o s e l y a s s ociated polyoses, x y l a n and mannan. Furthermore, wood-cellulose preparations have a higher carboxyl-group content than l i n t e r s c e l l u l o s e . These groups are b e l i e v e d to e x i s t as polyuronides or as uroni c anhydride u n i t s i n the c e l l u l o s e molecule or as s o c i a t e d w i t h i t . Among the more important minor polysaccharides 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 apparently v a r i e s considerably w i t h the source (2,3,4). The main p o r t i o n of the polysaccharide c o n s i s t s of D-mannopyranose anhydride 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. In a d d i t i o n , most of the mannans studied contain v a r y i n g amounts of glucose, galactose or arabinose r e s i d u e s . Owing to the l i m i t e d understanding of the exact s t r u c t u r e of mannans i n wood, a r e l i a b l e a n a l y s i s of t h i s polyose i s very d i f f i c u l t and i s g e n e r a l l y confined to the determination of the amount of anhydromannose u n i t s present. The remaining polysaccharide of 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 the pentosan, x y l a n . This polyose i s composed mainly of anhydroxylose u n i t s . In esparto 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 to 80 u n i t s of anhydro-D-xylopyranose Joined by k 1, B-D l i n k a g e . Branching from t h i s chain i s a s i n g l e D-xylose residue Joined by a 3 1 <K-union. Other sources of xylan show the presence of L-arabinose, D-gl u c u r o n l c or methylhexuronic u n i t s i n the polymer (5, 6,7). Uronic a c i d r e s i d u e s are a l s o d e t e c t a b l e . O'Dwyer (8) has shown that many xylans of wood are heterogeneous polysaccharides. Analyses f o r x y l a n i n wood-cellulose preparations are consequently r e s t r i c t e d to the determination of anhydroxylose u n i t s . The polysaccharides mentioned do not e x i s t i n d i s c r e t e l a y e r s or segments i n p l a n t t i s s u e s . On the contrary, much of the d i f f i c u l t y i n a n a l y z i n g and studying 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 to an i n t i m a t e a s s o c i a t i o n of a l l three - an a s s o c i a t i o n which has so f a r d e f i e d a l l chemical means of complete separation and has given r i s e to t h e o r i e s of chemical bonding between the polysaccharides (9.10)<. Several terms based on a s s o c i a t i o n r a t h e r than chemical s t r u c t u r e are used to r e f e r to the polysaccharides i n p l a n t t i s s u e . A l l of the carbohydrates are c o l l e c t -i v e l y o a l l e d h o l o c e l l u l o s e . The n o n - c e l l u l o s i c p o l y -saccharides i n the h o l o c e l l u l o s e are c l a s s e d as hemi-c e l l u l o s e s . These unoriented components form a continuous i n t e r p e n e t r a t i n g system w i t h the c e l l u l o s e s t r u c t u r e . Most of the he m i c e l l u l o s e s are removed by e x t r a c t i o n w i t h d i l u t e a l k a l i e s . 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 subdivided i n t o the amorphous polyuronide h e m i c e l l u l o s e s which 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 , arablnose, glucose and galactose 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 of r e l a t i v e l y simple hexosans or pentosans more or l e s s o r i e n t e d i n the 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 are r e l a t i v e l y s h o r t - c h a i n polysacchar-ides whose pyranose s t r u c t u r e s occupy the same space as the anhydroglucose u n i t s and t h e r e f o r e permit 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 . The same l a t e r a l f o r c e s which are present i n the c e l l u l o s e would a l s o r e t a i n the c e l l u l o s a n s . In commercial wood-c e l l u l o s e p r e p a r a t i o n s , xylan and mannan are the most important c e l l u l o s a n s . I I . The q u a n t i t a t i v e determination of mannan From the preceding c o n s i d e r a t i o n s i t i s easy to a p p r e c i a t e the d i f f i c u l t i e s of a n a l y s i n g 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 given to the a n a l y t i c a l methods used f o r determining only two of these polysaccharides - mannan and x y l a n . 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 not a c t u a l l y determine the mannan or xylan as polymers but 7 r a t h e r determine them I n d i r e c t l y by the amount of an-hydromannoee or anhydroxylose u n i t e found present. Reviews of the l i t e r a t u r e ( 1 1 , 1 2 , 1 3 ) give three general methods f o r es t i m a t i n g mannan i n wood or wood-cellulose preparations. The method of Schorger (Ik), which was modified by Wise, R a t l i f f and Browning ( 1 5 ) . c o n s i s t s of an a c i d h y d r o l y s i s of the mannan to 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 of the mannose as the phenylhydrazone. D i l u t e a c i d s are reported to give incomplete h y d r o l y s i s but stronger a c i d concentrations cause d e s t r u c t i o n of the mannose. Further-more, when mannan i s present i n small amounts (1% or 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 the a n a l y s i s . Large amounts of xylose a l s o i n t e r f e r e by lowering the mannan values. A recent method (16) estimates mannan by the r a t e d i f f e r e n c e of le 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 groupings i n mannan are more r a p i d l y o x i d i z e d than the t r a n s - g l y c o l groupings i n c e l l u l o s e . Con-s t r u c t i o n of a curve showing time versus lead t e t r a -acetate consumption permits e x t r a p o l a t i o n of the f i n a l s t r a i g h t - l i n e p o r t i o n of the curve to zero time f o r an estimate of the mannan present. The use of 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 the most s a t i s f a c t o r y method f o r deter-mining not only mannan but 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 pre p a r a t i o n s . L a t e r , Sundman, Saarnio and G-ustafssbn (19) adapted the method f o r q u a n t i t a t i v e wood sugar determinations. I I I . The q u a n t i t a t i v e determination of x y l a n Before the i n t r o d u c t i o n of chromatographic methods the determination of 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 , xylan estimations were based on "co r r e c t e d " pentosan determinations which were, themselves, p r e v i o u s l y c o r r e c t e d . Pentosans were converted by a c i d h y d r o l y s i s to pentoses and then to 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 determined by c o l o r i m e t r i c , v o l u m e t r i c or g r a v i m e t r i c methods (20). Uronic a c i d s , polyuronides, 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 which are determined as f u r f u r a l . The great value of paper chromatography l i e s i n i t s a b i l i t y to separate and i d e n t i f y microgram q u a n t i t i e s of xylose and mannose i n complex mixtures of carbohydrates q u i c k l y , simply and a c c u r a t e l y . Before chromatographing, the polysaccharides are hydrolysed 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 purpose ( 2 3 ) . To reduce the sugar decomposition to a minimum, Saeman and co-workers (2*0 recommend a two hour i n i t i a l d i s s o l v i n g time i n concentrated a c i d at 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 . A f t e r h y d r o l y s i s and n e u t r a l i z a t i o n ( u s u a l l y w i t h barium carbonate or anion exchange r e s i n s ) the sugar s o l u t i o n s are concen-t r a t e d and then chromatographed. Several e x c e l l e n t reviews of methods and techniques i n paper chromatography have been published ( 2 5 , 2 6 , 2 7 ) . A f t e r the sugar s o l u t i o n s have been chromatographed they can be developed and estimated d i r e c t l y on the paper or, they can be e l u t e d from the sheet and determined by any one of s e v e r a l conventional methods. E l u t i o n techniques can give r i s e to sugar l o s s e s through manipulation or de-composition during e x t r a c t i o n . Pre-treatment of the paper i s o f t e n necessary to avoid e l u t i n g I n t e r f e r i n g substances. The more r a p i d method i s to develop the sugars as colored spots and to determine t h e i r concentra-t i o n by comparison w i t h standards which are run simul-taneously on the same sheet. V i s u a l comparisons are of t e n s a t i s f a c t o r y but photometric methods are more pre-c i s e (28,29 ,30). The photometric determinations are based on the r e l a t i o n s h i p of the spot area, l e n g t h or c o l o r d e n s i t y to sugar c o n c e n t r a t i o n . Unknown sugar 10 concentrations are evaluated from a standard curve which i s p l o t t e d from d e n s i t y readings of the sugar standards run on the same chromatogram as the 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 the s p e c i f i c i t y of the enzymes known as hemcellulases or cytases. The names mannanase, xylanase, arabanase, e t c . used i n the l i t e r a t u r e were a p p l i e d to enzymes whioh hydrolysed 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 the s t u d i e s were c a r r i e d out w i t h crude enzyme prepar-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 to I n t e r p r e t . Furthermore, the l a c k of knowledge of the exact s t r u c t u r e of the v a r i o u s polysaccharide substrates and the d i f f i c u l t y of q u a n t i t a t i v e l y determining them hindered progress i n these enzyme i n v e s t i g a t i o n s . In 1933, G-rassman, S t a d l e r and Bender (31) were able to remove xylanase a c t i v i t y from e x t r a c t s of A s p e r g i l l u s oryzae (Luizym) by adsorption on cha 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 -cated that c e l l u l a s e and xylanase 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 of the mode of a c t i o n of the xylanase was undertaken. Sorensen ( 3 2 ) , i n s t u d i e s on the s p e c i f i c i t y and products of a c t i o n of xylanase on wheat straw x y l a n , 11 has attempted to gi 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 xylanase 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 the r a t e of enzymatic h y d r o l y s i s between xylans from wheat straw and wood. I t was f u r t h e r demonstrated that t h i s xylanase i s adaptive and d i f f e r e n t from mannanase, c e l l u l a s e or d i a s t a s e . Mannanase from the same micro-organism was als o shown to be adaptive. The main products of h y d r o l y s i s by t h i s xylanase 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 of the a b i l i t y to produce xylose from x y l a n , the power to produce intermediate o l i g o -saccharides was not a f f e c t e d . Subsequent studi e s by Sorensen ( 3 3 ) supported the theory that xylanase i s composed of at l e a s t two enzymes. One enzyme behaves as an exoenzyme, capable of randomly s p l i t t i n g o f f low molecular weight o l i g o -saccharides. The other, an endoenzyme, i s capable of s p l i t t i n g x y l o b l o s e . Yundt ( 3 4 ) , i n experiments w i t h c r y s t a l l i n e x y l a n and an enzyme pre 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 that 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 of the xy l a n was fo l l o w e d by a much slower r e a c t i o n . The extent of enzymatic h y d r o l y s i s was determined by the cyanide con-sumption of the aldose end groups. 12 I t was a l s o shown that r e t r o g r a d a t i o n of the polysaccharide 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 of chemical inhomogenelty. EXPERIMENTAL 13 A. The Q u a n t i t a t i v e Determination of Mannan and Xylan I . H y d r o l y s i s of wood c e l l u l o s e Preparatory to h y d r o l y s i s , an a i r - d r i e d sample of 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 10$ moisture) was beaten to a f i n e , f l u f f y pulp i n an o s t e r i z e r . For moisture determination, a ten gram sample of the a l r -d r i e d pulp was weighed out at t h i s time, oven-dried at 1 1 0 ° f o r 8 hours and weighed again. The two-step method of a c i d h y d r o l y s i s of wood c e l l u l o s e developed by L. E. Wise and co-workers ( 15) was subsequently f o l l o w e d . A one gram sample of the f l u f f e d pulp was t r a n s f e r r e d to a 250 ml. round-bottom f l a s k and d i s s o l v e d at 20° i n 10 mis. of 72$ s u l f u r i c a c i d (sp.gr. 1.64). U s u a l l y 2 to 3 hours was s u f f i c i e n t f o r complete s o l u t i o n of the wood polys a c c h a r i d e s . Next, the mixture was d i l u t e d w i t h 140 mis. of water, and the h y d r o l y s i s completed by heating under r e f l u x on a b o i l i n g water bath f o r 4 hours. The hydrolysate was n e u t r a l i z e d w i t h barium carbonate, using congo red paper as an i n d i c a t o r . I t was found that approximately 230 grams of barium carbonate were necessary 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 In a Buchner funnel (Coors 2A) using No. 44 Whatman f i l t e r paper. Pressure a p p l i e d to 14 the top of the f i l t e r cake aided f i l t r a t i o n c o n s i d e r a b l y . R e f i l t e r i n g the s o l u t i o n to o b t a i n a c l e a r f i l t r a t e was found unnecessary i f proper care was given t o the f i r s t f i l t e r i n g operation. A 75 ml. a l i q u o t of the c l e a r f i l t r a t e was concentrated on a 40° water bath under r e -duced pressure to a volume of 1 or 2 mis. The r e s u l t i n g syrupy s o l u t i o n , together w i t h any p r e c i p i t a t e d barium carbonate or s u l f a t e , was t r a n s f e r r e d by means of a mic r o p i p e t t e to a 3 ml. f r i t t e d g l a s s f i l t e r f unnel of medium p o r o s i t y . The f i l t r a t e was c o l l e c t e d i n a 5 ml. volumetric f l a s k . As an a d d i t i o n a l p recaution i n preventing any p r e c i p i t a t e from passing i n t o the f i l t r a t e , a small d i s c of No. 44 Whatman f i l t e r paper, cut out w i t h a s u i t a b l e cork borer, was placed over the f r i t t e d g l a s s surface of the f u n n e l . The f l a s k which contained the concentrated 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 small amounts of d i s t i l l e d water. A l l of these washings were f i l t e r e d i n t o the volumetric f l a s k . Water was added to the f i l t r a t e to b r i n g the volume of the f i l t r a t e to e x a c t l y 5 mis. This r e s u l t i n g s o l u t i o n , which was subsequently analysed by paper chromatography, now rep-resented a 10$ s o l u t i o n of sugars from the hydrolysed wood-cellulose sample. I I . Sugar standarde By experience i t was found that (on paper 15 chromatograms) standard s o l u t i o n s , each c o n t a i n i n g only-one pure sugar, d i d not give 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 mixtures of sugars i n wood-cellulose hy d r o l y s a t e s . Consequently, each standard s o l u t i o n used i n these experiments was a c t u a l l y composed of three pure sugars i n a r a t i o c l o s e l y resembling t h e i r p r o p o r t i o n i n the wood-cellulose hydrolysate s t u d i e d . These three most predominant sugars were glucose, mannose and x y l o s e . However, as p r e v i o u s l y mentioned, only the mannose and xylose contents were of concern i n t h i s study. A l l observations were based on hydrolysed 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 the o r i g i n a l pulp samples. Therefore, the standard sugar s o l u t i o n s were p r o p o r t i o n a l l y prepared so as to be comparable w i t h these d i l u t e d wood-cellulose 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 of glucose i n wood-c e l l u l o s e samples was about 90$. Therefore, the amount of glucose i n a l l of the standards was f i x e d at 9$. In softwood pulps that were studied the xylose content v a r i e d from 1 to 6 per cent whereas the mannose content v a r i e d from 1 to 12 per cent. Sugar standards were prepared covering these approximate ranges. Benzoic a c i d (0.25 gm./lOO mis.) was added as an a n t i s e p t i c to the standard s o l u t i o n s . When necessary, appropriate d i l u -t i o n s were made f o r sugars w i t h concentrations outside these ranges. 16 Standard s o l u t i o n % 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 I I I . Chromatography The hydrolysed wood-cellulose s o l u t i o n s prepared by the preceding method were chromatographed on l a r g e sheets of No. 1 Whatman f i l t e r paper (18£M x 22^"). Along the s t a r t l i n e , a s e r i e s of standard sugar s o l u t i o n s was a l t e r n a t e l y spotted w i t h a prepared hydrolysate (see f i g u r e 2 ) . The spots were spaced at 4 cm. i n t e r v a l s . The amount of chromatographed s o l u t i o n was about 2 N or s u f f i c i e n t to produce a spot 10 mm. i n diameter. A l l a p p l i c a t i o n s of the standard and unknown s o l u t i o n s were made w i t h the same m i c r o p i p e t t e . A f t e r each a p p l i c a t i o n 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 acetone. The solvent system r e p o r t e d by Gustafsson (21) was found to be most s a t i s f a c t o r y f o r t h i s chromato-graphic method. n-Butanol was saturated w i t h water and d i s t i l l e d . P h t h a l l c a c i d (1 .66 gr./lOO mis. s o l v e n t ) , which i s a c t u a l l y a component of the developer, was then ^ 5 u l 4 U l 5 U 2 2 U 2 1 U 2 • • I I • # I f I * I F i g . 2 Photograph of a chromatogram developed a f t e r 72 hours showing the arrangement of unknowns (U^, U 2) and standard sugar s o l u t i o n s (1-5). The sugars glucose ( g ) , mannose (m) and x y l o s e (x) are 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 . 17 d i s s o l v e d i n the water saturated n-butanol. The presence of the p h t h a l i c a c i d i n the solvent was not d e t r i m e n t a l to the a n a l y s i s of the sugars. The chromatograms were i r r i g a t e d w i t h the solvent by the descending flow method f o r 72 hours at room temperature. At the end of t h i s time the sheets were t r a n s f e r r e d to a fume hood and allowed to dry f o r at l e a s t one hour. The most mobile sugar, xyloee, ran about 15 cms. A l l of the sugars were s u f f i c i e n t l y separated f o r q u a n t i t a t i v e e s t i m a t i o n . For c o l o r development, the d r i e d chromatograms were drawn through a s o l u t i o n of a n i l i n e i n ether (0.96 gms. a n l l l n e / 1 0 0 mis. ether) and then heated at 105° f o r 5 minutes. Glucose and mannose appeared as brown spots; xylose as a pink spot. IV. Photometric a n a l y s i s The area I n c l u d i n g the mannose and xylose spots 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 s t r i p thus contained one x y l o s e and one mannose spot. These s t r i p s were placed i n a metal support (see f i g u r e l ) which was s p e c i a l l y designed 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 readings, which 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 blank s t r i p from the same chromatogram was FIG. I A DIAGRAM OF THE CARRIAGE AND REMOVABLE PAPER STRIP HOLDER ADAPTED FOR USE WITH A BECKMAN MODEL B SPECTROPHOTOMETER. 18 used f o r o b t a i n i n g a zero o p t i c a l d e n s i t y s e t t i n g . As the s t r i p was manually moved across the l i g h t s l i t , the maximum d e n s i t y f o r each spot was recorded. From each chromatogram, standard curves f o r both mannose and xylose were drawn by p l o t t i n g the o p t i c a l d e n s i t y readings versus the concentration of the sugar standards (see f i g u r e 3). Once the standard curves were e s t a b l i s h e d , unknown sugar concentrations were r e a d i l y determined from the graph. The per cent of mannose and xylose In the wood-c e l l u l o s e hydrolysates was converted to mannan and xy l a n values by use of the f o l l o w i n g equation: % polyose % sugar x mol. wt. of s u g a r— H 2 0 mol. wt. of sugar % mannan % mannose x 0.90 % xylan % xylose x 0.88 The polyose value was a l s o c o r r e c t e d f o r moisture content. B. Enzymatic Studies I . Substrate A l l of the c u l t u r e s mentioned i n t h i s i n v e s t i g a -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. This b a s a l medium was a s o l u t i o n of the 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 tap water (gm/1) : NH^Cl 4.3; NH^NOo 1.0; K C l 4.5; P i g . 3 Graph showing the l i n e a r r e l a t i o n of mannose (M) and xylose (X) concentrations to the o p t i c a l d e n s i t y of t h e i r chromato-grammed spots. 19 Na 2HP0^.7H 20 2.7; MgS0^.7H20 2.5; ZnSO^ .01; FeSO^ .01; MnSG^ .08; carbon source 5 gm./l. This s o l u t i o n was adjusted to pH 8, (by the a d d i t i o n of sodium hydroxide) f i l t e r e d and then s t e r i l i z e d at 15 p . s . i . f o r 20 minutes. Xyla n , the carbon source used i n these e x p e r i -ments, was prepared by the method of Voss and B u t t e r (35). Xylan powder was brought i n t o s o l u t i o n i n a mortar w i t h 5 or 6 mis. of 2N NaOH per gram of x y l a n , d i l u t e d w i t h water to 4 to 5 per cent xylan and then heated t o 40-50° f o r h a l f an hour on a water bath. A f t e r c e n t r i f u g a t i o n from i n s o l u b l e p a r t i c l e s the milky 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 to a pH of about 6.5 (2 to 3 days). Ten nils, of t h i s s o l u t i o n were evaporated to dryness to determine the xylan content. D i s t i l l e d water was added to give a f i n a l x y l a n concentration of 1 gm./lOO mis. of s o l u t i o n . This c o l l o i d a l s o l u t i o n was stored i n a b o t t l e c o n t a i n i n g excess toluene. To a s c e r t a i n the 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 2S0^. A f t e r n e u t r a l i z a t i o n w i t h barium carbonate the s o l u t i o n was f i l t e r e d and spotted on a paper chromatogram. The only sugar shown to be present was x y l o s e . I I . Micro-organisms The f o l l o w i n g organisms were incubated at 37° i n l i q u i d media which contained c o l l o i d a l x y l a n as the 20 sole carbon source: growth Actinomyces 51-3. good Actinomyces £1-36 poor Actinomyces 52-11 poor Actinomyces 5?-? f a i r Actinomyces 50=2 poor Actinomyces good Actinomyces 55-10 poor Actinomyces 55-13 f a i r Rhlzoblum t r i f o l i i poor Rhizobium m e l i o t i poor Azotobacter v i n e l a n d i i poor Azotobacter chroococcum poor Ghaetomlum globosum Kunze very good Chaetomlum elobosum (NRC V-159) very good E s c h e r i c h i a c o l l poor At the end of two weeks the extent of growth was observed and roughly evaluated as shown above. The more a c t i v e organisms were able to cause a r a p i d c l e a r i n g of the 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 of the c o l l o i d a l x y l a n . The micro-organisms which were capable of growth i n the s y n t h e t i c medium were washed, homogenized and added to f r e s h s o l u t i o n s of x y l a n . Sodium cyanide (.003M) was 21 added to b l o c k the metabolism of the degradation products of x ylan s u f f i c i e n t l y to permit t h e i r d e t e c t i o n by paper chromatography. A f t e r i n c u b a t i o n (37° f o r 7 days) the c u l t u r e s were concentrated and spotted on paper chromato-grams. Standard sugar s o l u t i o n s were a l s o spotted on the same chromatograms f o r i d e n t i f i c a t i o n . From these p r e l i m i n a r y experiments three s o i l micro-organisms were found to grow r e a d i l y i n the s y n t h e t i c medium. Furthermore, xylose was the p r i n c i p a l product r e s u l t i n g from the h y d r o l y s i s of the xylan by these three organisms: Actinomyces 55-7» Ghaetomlum globosum Kunze and Chaetomium globosum (NRC V-159). I l l . Enzyme p r e p a r a t i o n Cultures of C. globosum Kunze were Incubated at 25° i n Roux f l a s k s , each c o n t a i n i n g 100 mis. of medium. As soon as the surfaces of the media were covered (7 to 10 days) the mycelium was harvested, twice 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 at 0°. The r e s u l t i n g mycelium was then prepared f o r treatment i n a magneto-ostriction o s c i l l a t o r (50 watt, 9 kc Raytheon Mfg. Co.). A p r e l i m i n a r y separation and break-down of the c e l l s wae made by mechanically g r i n d i n g the f u n g i i n a ground-glass homogenizer. The p a r t i a l l y homogenized mycelia were then t r a n s f e r r e d t o the high frequency o s c i l l a t o r . A ten minute treatment i n the 22 o s c i l l a t o r was s u f f i c i e n t to rupture most of the c e l l w a l l s . A c l e a r enzyme s o l u t i o n was separated from the c e l l d e b r i s by c e n t r i f u g i n g at 0° . The enzyme preparations were then frozen f o r storage. IV. The determination of enzyme a c t i v i t y Two mis. of 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. of toluene were shaken v i g o r o u s l y In a 25 ml. cork-stoppered Erlenmeyer f l a s k and placed i n the incubator at 25° f o r f i v e days. Then, the mixture was c e n t r l f u g e d and the c l e a r s o l u t i o n was spotted on a paper chromatogram. Pure sugar s o l u t i o n s were spotted on the same sheet f o r i d e n t i f i c a t i o n . Xylose was the predominant sugar present. Several pink spots of r e g u l a r l y decreasing Rf values were a t t r i b u t e d to 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 determining the a b i l i t y of the enzyme pre p a r a t i o n to 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 wood-cellulose p r e p a r a t i o n s . The wood-cellulose sample was a i r - d r i e d at room tempera-tur e and then f l u f f e d In the manner p r e v i o u s l y described. E x a c t l y one gram of t h i e f l u f f e d sample was added to a 250 ml. Erlenmeyer f l a s k . Twenty mis. of d i s t i l l e d water were added to the f l a s k and the mixture was autoclaved at I 23 15 p . s . i . f o r 20 minutes. A 10 gram sample of the a i r - d r i e d pulp was taken f o r moisture determination at the same time as the enzyme sample. The moisture sample was d r i e d at 1 1 0 ° f o r 8 hours and weighed. The per cent of dry wood c e l l u l o s e i n the sample was c a l c u l a t e d from the weight d i f f e r e n c e of the sample before and a f t e r d r y i n g . When the s t e r i l i z e d f l a s k was c o o l , 30 mis. of enzyme pr e p a r a t i o n were added a s e p t i c a l l y and mixed thoroughly w i t h the wood c e l l u l o s e . As a f u r t h e r pre-caution against b a c t e r i a l contamination, toluene was added to form a t h i n l a y e r over the surface of the mixture. Incubation of the p r e p a r a t i o n was c a r r i e d out at 2 5 ° f o r fourteen days. At the end of the i n c u b a t i o n p e r i o d , the remaining wood c e l l u l o s e was t r a n s f e r r e d to 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. The f i l t r a t e was f i l t e r e d through the pad of wood c e l l u l o s e a second time to remove any f i b r e s from the washings. The pad was f i n a l l y washed w i t h acetone and d r i e d at 1 0 5 ° . The d r i e d sample was subsequently analysed f o r xylan and mannan according to the p r e v i o u s l y described 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 the carbohydrate content of the enzyme t r e a t e d and untreated wood-cellulose preparations I n d i c a t e d the extent of enzyme a c t i o n on the polysaccharides. RESULTS 24 Table 1. A n a l y s i s of Selected Commercial Wood Pulps Wood Use of # Alpha Mannan Xylan source Process pulp Vise./P.P. cell.fi % ft Hemlock S u l f i t e Paper 170 87«7 7 . 4 2 . 4 Hemlock S u l f i t e C e l l o - 36 9 0 . 2 2 . 5 1 .1 phane Hemlock S u l f i t e Photo- 447 9 5 - 7 1 .9 1 . 4 graphy F i r K r a f t Paper 38 8 9 . 0 6 . 3 3 - 4 F i r K r a f t High 22 9 3 . 6 3 . 4 2 . 0 t e n a c i t y rayon Pine Pre- C e l l o - 603 8 9 . 6 4 . 6 2 . 1 h y d r o l . phane s u l f a t e Spruce S u l f i t e Rayon 1215 9 4 . 4 2 . 2 2 . 7 Southern S u l f i t e T i r e 1380 9 5 . 8 2 . 1 2 . 8 pine cord Western S u l f i t e T i r e 1340 9 6 . 2 2 . 9 2 . 1 hemlock cord Southern S u l f i t e Die- 1300 9 6 . 2 2 . 3 1 . 2 pine s o l v i n g Western S u l f i t e D i e- 1300 9 6 . 7 2 . 8 1 . 3 hemlock s o l v i n g * i n copper ethylenediamine, 1% c e l l u l o s e , 2 5 ° **" c a l c u l a t e d from equations shown on p. 18 Table 2 . Comparison of Pentosan and Xylan Analyses 25 Wood Use of V i s e . i n CED Alpha Pentosan + X y l a n source pulp \% c e l l . 2 5 ° c e l l . # % Mixed* Paper 40 . 0 8 8 . 5 2 0 . 0 18.? hardwoods Mixed D i s s o l v i n g 2 5 . 0 8 9 . 0 3 . 5 2 . 9 hardwoods Mixed D i s s o l v i n g 6 0 . 0 9 3 . 0 3 . 0 3 . 3 hardwoods Spruce, Fine paper 175 9 4 . 0 3 . 0 3 . 1 f i r , hem. Spruce, Paper 6 0 . 0 8 7 . 5 8 . 0 7 . 3 f i r , hem. Poplar Paper 2 3 . 7 8 5 . 6 9 . 9 9 . 7 aspen Poplar Paper 18 . 2 7 8 . 2 2 1 . 0 1 9 . 6 aspen B i r c h Paper 24 . 0 81.3 14 . 8 14 . 5 B i r c h Paper 18.7 7 3 . 5 2 6 . 3 2 0 . 0 * white b i r c h , y e l l o w b i r c h , beech, hard maple, s o f t maple Table 3 . An A n a l y s i s of Mannan and Xylan from Wood C e l l u l o s e Before and A f t e r Enzyme A c t i o n Wood pulp V i s e . i n CED Alpha c e l l . + Mannan + X y l a n sample 1% c e l l . 2 5 ° % % % Spruce 60 8 7 . 5 7 . 4 7 . 3 Spruce 7 . 2 5 . 0 (enzyme added) The only sugar detected i n the washings from the enzyme-treated sample was x y l o s e . 26 DISCUSSION The described method f o r q u a n t i t a t i v e l y estimat-i n g x y l a n and mannan i n wood c e l l u l o s e can determine sugar concentrations as low 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 of - 10% i n such low ranges. This s e n s i t i v i t y i s p o s s i b l e because many of the o b j e c t i o n -able f e a t u r e s i n e s t i m a t i n g paper chromatograms by photo-metric methods have been e l i m i n a t e d or co n s i d e r a b l y reduced. For example, a drawback to many developing agents i s that they produce c o l o r e d spots which are too unstable 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 or other changes i n the background of the developed paper sheets has a l s o been a disadvantage to 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 potassium permanganate and metaperiodate s o l u t i o n s f o r developers. The a n i l i n e hydrogen phthalate developer 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 very l i g h t and i n good contrast to the c o l o r e d spots. The s t r i p s of paper may be kept f o r s e v e r a l days i f they are i n d i v i d u a l l y placed between some sheets of paper. However, a n o t i c e a b l e darkening of the background does occur during t h i s time but d e n s i t y readings are s t i l l p e r m i s s i b l e . Another disadvantage of some r e p o r t e d methods i s t h e i r low s e n s i t i v i t y , although good determin-27 a t l o n s can be made on the d e t e c t a b l e spots. I n t h i s procedure, concentrations of xylose or mannose can be detected i n amounts as low as 1 mg./ml. when a p p l i e d i n amounts of 2A . This q u a n t i t y represents amounts of sugar as low as 0 . 0 0 2 mgms. The geometrical shape of the spot, which i s so Important i n s e v e r a l methods us i n g densitometers, i s e l i m i n a t e d by t h i s technique because only a small area of the whole spot i s scanned by the beam of l i g h t . 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 only on the l i g h t t r a n s m i t t e d by a small p o r t i o n of the spot r a t h e r than on an average of the whole spot or the whole spot plus the surrounding background. From f i g u r e 3 l t can be seen that the l i g h t t r a n s -mittance values f o l l o w Beer's law c l o s e l y w i t h i n the given range of sugar concentrations. Winslow ( 3 6 ) has done comparative stud i e s on r e f l e c t a n c e and transmittance techniques 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 spectro-photometer. The r e s u l t s i n d i c a t e d that measurement by transmittance was p r e f e r a b l e f o r q u a n t i t a t i v e e v a l u a t i o n of the spots and showed that such t e s t s give transmittance data c l o s e l y In accordance w i t h Beer's law. The r e s u l t s i n t a b l e 2 show good agreement between the xylan values and the conventional pentosan values. The g e n e r a l l y higher pentosan r e s u l t s are p o s s i b l y due to the presence of u r o n i c a c i d s which a l s o c o n t r i b u t e to the pentosan value. C o r r e c t i o n s f o r t h i s i n t e r f e r e n c e 28 i n present methods are not e n t i r e l y s a t i s f a c t o r y . The r e s u l t s of enzymatic a c t i o n on the wood-c e l l u l o s e preparations i n v e s t i g a t e d show that about 25 per cent of the x y l a n was removed i n a s p e c i f i c manner. The remaining xylan 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 or c r y s t a l l i n e p o r t i o n of the c e l l u l o s e . However, the s e l e c t i v e removal of some of 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 without apparent degradation of the o e l l u l o s e . I t i s not assumed i n these experiments that the xylanase 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 given i n support of a s e r i e s of enzymes, each produced or stimulated by the end product of i t s pre-decessor. These enzymes are separate from the " c o n s t i -t u i t i v e " enzymes of the c e l l which are themselves elaborated I r r e s p e c t i v e of the presence or absence of homologous substr a t e s . The adaptive enzymes,on the other hand, are produced as a r e s u l t of the organism coming i n contact w i t h a s p e c i f i c substrate which i t does not normally u t i l i z e . For example, substrate A induces the formation of enzyme A which causes the production of B. In t u r n , B induces the formation of enzyme B. This process i s r e f e r r e d to as s e q u e n t i a l Induction by Karstrom (39). Such a mechanism would f i t the breakdown of x y l a n shown to occur i n the study of xylanase by Sorensen i n whioh evidence i n d i c a t e s that at l e a s t two enzymes are i n v o l v e d (33). The 2 9 a b i l i t y of the xylanase p r e p a r a t i o n to produce the o l i g o -saccharides from x y l a n was not destroyed on h e a t i n g whereas the power to hydrolyse these o l i g o s a c c h a r i d e s t o xylose was destroyed. In k i n e t i c s t u d i e s of the h y d r o l y s i s of p o l y -saccharides (35»37»38) a l l r e a c t i o n s , whether chemical or enzymatic, showed s i m i l a r r a t e curves. There was an i n i t i a l l y r a p i d r a t e followed by a slower constant r e a c t i o n r a t e . The two-phase s t r u c t u r e of c e l l u l o s e , i . e . the existence of the c e l l u l o s e chain molecules i n c r y s t a l l i n e and amorphous s t a t e s , has been given i n e x p l a n a t i o n of 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 region s contain most of the h e m l c e l l u l o s e s which are e a s i l y hydrolysed. This r e a c t i o n occurs i n the i n i t i a l phase. Random cleavage of the c e l l u l o s e s t r u c t u r e a l s o occurs. The slower r e a c t i o n i s b e l i e v e d to take place on the sur-face of the c e l l u l o s e c r y s t a l l i t e s . In enzymatic s t u d i e s by Walseth ( 3 8 ) , the enzyme showed some decrease 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 to account f o r the slow h y d r o l y s i s . Moisture r e g a i n values decreased i n the undissolved residues I n d i c a t i n g that the amorphous regions were most r a p i d l y attacked. 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 of wood c e l l u l o s e a l s o has been shown ( 3 8 ) . In enzyme degradation a r e l a t i v e l y h i g h degree of polymeriz-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. of un 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 of the two h y d r o l y t i c agents was given i n explan a t i o n . The enzyme may cause r a p i d and complete r e -duction of the c e l l u l o s e i n the amorphous r e g i o n to sol u b l e products. I t was a l s o suggested that the r a p i d production of glucose without a lowering of the D.P. may i n d i c a t e t h a t the enzyme attack s the ends of the c e l l u l o s e chains r a t h e r than randomly h y d r o l y s i n g the molecules. On the other hand, the a c i d i s b e l i e v e d to penetrate the c e l l u l o s e s t r u c t u r e to a greater extent than the enzyme and produce short fragments which could not e a s i l y d i f f u s e out of the c e l l u l o s e s t r u c t u r e . The f a c t that there i s considerable 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 of wood c e l l u l o s e i n d i c a t e s that i t i s an advantage to use enzymes f o r studying wood polysaccharides. Enzymatic s t u d i e s w i l l un-doubtedly provide a d d i t i o n a l i n f o r m a t i o n on wood polyoses which i s not det e c t a b l e by purely chemical means. However, t h i s Information should be more concise i f s p e c i f i c enzymes are used. The present method of o b t a i n i n g such enzymes i s by s p e c i f i c substrate Induction. The e x p l o r a t o r y work discussed has shown that i t i s p o s s i b l e to obta i n a s p e c i f i c xylanase which i s a c t i v e on a wood-cellulose p r e p a r a t i o n . This enzyme (or enzymes) could be of importance i n the study of wood o e l l u l o s e s t r u c t u r e and composition. SUMMARY 3 0 a 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 analysed d i r e c t l y using an adapted t r a n s -mittance accessory f o r a Beckman Model B spectrophotometer. The accuracy of the method i s 5 to 10 per cent. 2. Generally c l o s e agreement i s shown i n a com-par i s o n of conventional pentosan analyses w i t h x y l a n determinations made by the described method. The s l i g h t l y higher pentosan values are p o s s i b l y due to the 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 xylanase from Chaetomlum globosum  gunze was shown to be capable of h y d r o l y s i n g x y l a n from wheat straw. The main end product of h y d r o l y s i s detected by means of paper chromatography was x y l o s e . 4. This enzyme pr 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 of the x y l a n from a commercial wood-cellulose sample without s i g n i f i c a n t l y changing the mannose or glucose content. BIBLIOGRAPHY 31 1. E. F. Hlnner, 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, Ott ed., I n t e r s c i e n c e P u b l i s h e r s , Inc., New York, N. Y., 1943, p. 519. 2. F. Klages, Ann., 5 0 9 , 159 ( 1 9 3 *0 ; .512, 185 (193 *0 . 3. W. N. Haworth, R. L. Heath and S. Peat, 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. Williamson, J . Chem. S o c , 3184 (1953). 5 . F. Gerhardt, Plant 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 (195D. 7. A. Roudler and L. Eberhard, Tappi, 2, 38 ( 1 9 5 5 ) . 8. M. H. O'Dwyer, Biochem. J . , 21* 713 ( 1 9 3 9 ) . 9. L. F. Hawley and A. G. Norman, Ind. Eng. Chem.., 24, 1190 ( 1 9 3 2 ) . 10. E. Schmidt, K. Mei 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 Chemistry, 10, 283. 12. C. Doree, "The Methods of C e l l u l o s e Chemistry," D. Van Nostrand Co., Inc., New York, N. Y., 1933t P. 354. 13. C. A. Browne and F. W. Zerban, " P h y s i c a l and Chemical Methods of Sugar A n a l y s i s , " John Wiley and Sons, Inc., New York, N. Y., 1941, p. 904. 14. A. W. Schorger, Ind. Eng. Chem., 2, 748 (1917). 15. L. E. Wise, E. K. R a t l i f f and B. L. Browning, Anal. Chem., 2 0 , 825 (1948). 16. H. W. Steinmann and B. B. White, Tappi, J32. 225 ( 1 9 5 4 ) . 17. G. N. Kowkabany, Advances i n Carbohydrate Chemistry, 2, 303 (1954). 32 18. L. E. Wise, J . W. Green and R. C. Rittenhouse, Tappi, 12, 335 (1949) 19. J . Sundman, J . Saarnio and C. Gustafsson, P a p e r l Ja Puu, B 33. 115 ( 1 9 5 1 ) . 2 0 . B. L. Browning, i n "Wood Chemistry," L. E. Wise and E. C. Jahn, eds., Reinhold P u b l i s h i n g Corp., New York, N. Y., 2nd e d i t i o n , 1952, p. 1162. 21. C. Gustaffson, J . Sundman and T. Llndh, P a p e r i Ja Puu, B 33. 1 (195D. 22. E. Hagglund, "Chemistry of Wood," Academic Press Inc., New York, N. Y., 1951. 23. R. E. Glegg and D. E i d i n g e r , Anal. Chem., 2 6 , 1365 ( 1 9 5 4 ) . 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 , Tappi, JZ , 336 ( 1 9 5 4 ) . 25. F. Cramer, "Paper Chromatography," MacMillan and Co., L t d . , London, 2nd e d i t i o n , 1954. 26. R. J . Block, E. L. Durrum and G. Zweig, "Paper Chromato-graphy and Paper E l e c t r o p h o r e s i s , " Academic Press Inc., New York, N. Y., 1955, 27. E. Lederer and M. Lederer, "Chromatography," E l s e v i e r P u b l i s h i n g Co., New York, N. Y., 1953. 28. J . Saarnio, E. N l s k a s a a r i and C. Gustafsson, Suomen K e m i s t e l e h t l , 25 B. 25 ( 1 9 5 2 ) . 2 9 . R. H. M u l l e r and D. L. Clegg, Anal. Chem., 2 J , 397 ( 1 9 5 1 ) . 30. F. F. McFarren, K. Brand and H. R. Rvtkowski, Anal. Chem., 23, 1146 (1951). 31. W. Grassman, R. S t a d l e r and R. Bender, Ann. d. Chemie, i&2., 20 (1933). 32. H. Sorensen, P h y s i o l o g l a Plant arum, J5, 183 (1952). 33. H. Sorensen, Nature, 172, 305 ( 1 9 5 3 ) . 34. A. P. Yundt, Tappi, J 4 , 92 (1951) . 35. W. Voss and G. B u t t e r , Ann. d. Chemie, 534. 161 ( 1 9 3 8 ) . 36. F. H. Winslow and H. A. Llebhafsky, Anal. Chem., 21, 1338 ( 1 9 4 9 ) . 33 3 7 . C. S. Walseth, Tappi, 21* 2 2 8 ( 1 9 5 2 ) . 3 8 . C. S. Walseth, Tappi, 21* 2 3 3 ( 1 9 5 2 ) . 3 9 . H. Karstrom, Ergeb. Enzymforsch., 7_, 350 ( 1 9 3 7 ) . 40. R. Y. Stanler, J . Bact., j£4, 339 ( 1 9 4 7 ) . 

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