Plant Cell Wall Polymers - American Chemical Society

Chapter 47 Production and Purification of Xylanases

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 29, 2016 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch047

David J . Senior, Paul R. Mayers, and John N. Saddler Biotechnology and Chemistry Department, Forintek Canada Corporation, 800 Montreal Road, Ottawa, Ontario K1G 3Z5, Canada

Various groups have recently indicated that xylanase enzymes can be used to upgrade pulps. To be fully effective, it is essential that the enzymes are free of cellulase activity and have a high specific activity. Strategies followed to produce xylanases without contaminating cellulase activity include selective inactivation of the cellulase by heat and heavy metals and cloning of the xylanase gene into a cellulase-free host. We have shown that xylanase production by Trichoderma harzanium can be enhanced by selection of a suitable hemicellulase-rich growth substrate. A purified xylanase could be obtained by subsequent ultrafiltration and ion exchange treatment of the culturefiltrate.Xylanase treatment of pulps could significantly decrease the xylan content while leaving the cellulose intact. X y l a n a s e p r o d u c t i o n has been r e p o r t e d t o o c c u r i n a w i d e s p e c t r u m o f o r g a n i s m s . A l t h o u g h absent i n vertebrate a n i m a l s , x y l a n a s e s are p r o d u c e d i n m a n y forms o f b a c t e r i a , f u n g i a n d yeasts, crustaceans, algae a n d p l a n t seeds. C u r r e n t interest i n xylanases has been focused p r i m a r i l y o n t h e enzymes p r o d u c e d b y f u n g i a n d b a c t e r i a a n d , t o a lesser e x t e n t , yeasts. T h e h i g h yields a n d r e l a t i v e ease o f p r o d u c t i o n have m a d e these systems the most p r o m i s i n g for future c o m m e r c i a l i z a t i o n . O n e o f the m a j o r expenses i n c u r r e d i n t h e a p p l i c a t i o n o f enzymes for bioconversion processes is the cost o f e n z y m e p r o d u c t i o n (1). T h e t o t a l cost of p r o d u c t i o n includes the cost o f fermentative p r o d u c t i o n as w e l l as d o w n s t r e a m processing requirements. B o t h o f these factors m u s t be o p t i m i z e d a n d integrated for m a x i m u m cost-effectiveness. Several different approaches have been followed t o p r o d u c e x y l a n a s e s . Highest levels o f e x t r a c e l l u l a r xylanases have been p r o d u c e d i n yeast a n d

0097-6156/89/0399-0641$06.00/0 o 1989 American Chemical Society

Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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f u n g a l systems u s i n g x y l a n i n d u c i n g g r o w t h substrates. I n c o m p a r i s o n , b a c t e r i a l a n d cloned systems have p r o d u c e d moderate q u a n t i t i e s of cellulasefree xylanases. T w o b r o a d areas of a p p l i c a t i o n for x y l a n o l y t i c enzymes have been i d e n tified (1). T h e first involves the use of xylanases w i t h other h y d r o l y t i c enzymes i n the bioconversion of wastes such as those f r o m the forest a n d a g r i c u l t u r a l i n d u s t r i e s , a n d i n the c l a r i f i c a t i o n a n d l i q u i f i c a t i o n of j u i c e s , vegetables a n d f r u i t s . F o r these purposes, the enzyme p r e p a r a t i o n s need o n l y to be filtered a n d concentrated as essentially no f u r t h e r p u r i f i c a t i o n is r e q u i r e d . Several specific examples of a p p l i c a t i o n s i n v o l v i n g crude x y l a n a s e p r e p a r a t i o n s i n c l u d e : bioconversion of cellulosic m a t e r i a l s for subsequent f e r m e n t a t i o n (2); h y d r o l y s i s of p u l p waste liquors; a n d w o o d e x t r a c t i v e s to m o n o m e r i c sugars for subsequent p r o d u c t i o n of single cell p r o t e i n (3-5). X y l o s e p r o d u c e d b y the a c t i o n of xylanases c a n be used for subsequent p r o d u c t i o n of higher value c o m p o u n d s such as e t h a n o l (6), x y l u l o s e (7) a n d x y l o n i c a c i d (8-9). T h e second area of a p p l i c a t i o n involves the use of cellulase-free x y lanases for removal of hemicellulose f r o m p u l p s (10-20) a n d p l a n t fibres (21). It is essential t h a t these xylanase p r e p a r a t i o n s are free of c o n t a m i n a t i n g cellulase a c t i v i t y or d a m a g e to the cellulose fibres a n d consequently the p r o d u c t q u a l i t y w i l l result. Here we e x a m i n e the recent progress w h i c h has been m a d e i n the p r o d u c t i o n a n d p u r i f i c a t i o n of xylanases. Xylanase P r o d u c t i o n by Yeasts B i e l y et ai (22) have s h o w n t h a t x y l a n a s e p r o d u c t i o n can be i n d u c e d b y use of a p p r o p r i a t e substrates i n the yeast Cryptococcus albidus. W o o d x y l a n s a n d x y l a n oligomers were f o u n d to increase e x t r a c e l l u l a r x y l a n a s e p r o d u c t i o n b y two orders of m a g n i t u d e w h e n the yeast was g r o w n o n these m a t e r i a l s rather t h a n o n a s i m p l e glucose m e d i u m . X y l o b i o s e was identified as the o n l y x y l o o l i g o m e r w h i c h was not degraded e x t r a c e l l u l a r l y a n d i t was concluded t h a t xylobiose is either a n a t u r a l inducer of x y l a n a s e or its i m m e d i a t e precursor. O t h e r x y l o p y r a n o s i d e s have also been investigated for their x y l a n a s e i n d u c i n g p o t e n t i a l i n t h i s yeast. M e t h y l - / ? - D - x y l o p y r a n o s i d e i n d u c e d x y l a n a s e p r o d u c t i o n at levels c o m p a r a b l e to t h a t o b t a i n e d after g r o w t h o n x y l a n (23). M o r o s o l i et al. (24) d e m o n s t r a t e d t h a t the i n d u c t i o n effect was manifested at the t r a n s c r i p t i o n a l level i n t h i s o r g a n i s m . Y a s u i et ai o b t a i n e d a 15-20 f o l d greater increase i n x y l a n a s e p r o d u c t i o n u s i n g m e t h y l - / ? - D - x y l o p y r a n o s i d e t h a n when x y l a n was used as a n i n d u c e r i n Cryptococcus flavus (25). L e a t h e r s et al. (26) showed t h a t xylose, xylobiose a n d arabinose were inducers of xylanases i n Aureobasidium pullulans. F o r x y l a n a s e a p p l i c a t i o n s w h i c h require xylanases of h i g h s e l e c t i v i t y (e.g., b i o p u l p i n g ) , any c o n t a m i n a t i n g cellulases can be d e t r i m e n t a l to the t r e a t m e n t s . T h e c o n s t i t u t i v e levels of cellulases i n yeasts is generally very low i n r e l a t i o n to x y l a n a s e levels t h u s i n d i c a t i n g yeast x y l a n a s e p r e p a r a t i o n s are p r o m i s i n g for the selective h y d r o l y s i s of x y l a n s . However, e x t r a c e l l u l a r yeast xylanases are t y p i c a l l y p r o d u c e d i n the order of 1 u n i t per m i l l i l i t r e


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of c u l t u r e filtrate, w h i c h is very low for cost-effective p r o d u c t i o n . R e c e n t l y , L e a t h e r s has r e p o r t e d the o v e r p r o d u c t i o n of a cellulase-free x y l a n a s e o f very h i g h specific a c t i v i t y f r o m a colour v a r i a n t s t r a i n of A. pullulans (27). T h e level of e x t r a c e l l u l a r x y l a n a s e i n the c u l t u r e filtrate was 373 I U / m L w h i c h is c o m p a r a b l e to the most p r o l i f i c x y l a n a s e - p r o d u c i n g f u n g a l s t r a i n s so far identified.


Xylanase P r o d u c t i o n by C l o n e d Systems A n o t h e r a l t e r n a t i v e for p r o d u c t i o n of cellulase-free x y l a n a s e p r e p a r a t i o n s has been to clone the x y l a n a s e genes f r o m x y l a n a s e p r o d u c i n g b a c t e r i a . O n e x y l a n a s e p r o d u c i n g s t r a i n , Bacillus subtilis P A P 1 1 5 , was chosen as a x y l a n a s e gene donor because of its r e l a t i v e l y h i g h level o f e x t r a c e l l u l a r x y l a n a s e p r o d u c t i o n (0.9 I U / m L c u l t u r e filtrate) a n d the t h o r o u g h c h a r a c t e r i z a t i o n of the e n z y m e (28). T h e gene was successfully inserted i n t o Escherichia coli ( W A 8 0 2 ) a n d the x y l a n a s e p r o d u c e d was i s o l a t e d a n d p a r t i a l l y c h a r a c t e r i z e d . O n l y 2 5 % of the o r i g i n a l e x t r a c e l l u l a r x y l a n a s e a c t i v i t y was recovered a n d t h i s was l o c a l i z e d w i t h i n the cells (29). T h e same gene was i n s e r t e d i n t o E. coli J M I 0 5 a n d the new clones p r o d u c e d m o r e x y lanase t h a n the o r i g i n a l Bacillus (10). U n l i k e the first clone, a b o u t h a l f o f the x y l a n a s e a c t i v i t y was f o u n d e x t r a c e l l u l a r l y a n d y i e l d s were increased significantly. S i m i l a r approaches to c l o n i n g of b a c t e r i a l xylanases have been used for genes f r o m Clostridium acetobutylicum (30), Bacillus polymyxa (31), Bacteroides succinogenes (32), Clostridium thermocellum (33) a n d Pseudomonas fluorescens subsp. cellulosa (34). I n each case the x y l a n a s e s were p r e d o m i n a n t l y l o c a t e d i n t r a c e l l u l a r l y a n d the levels of x y l a n a s e s p r o d u c e d f r o m cloned s y s t e m s were, i n general, very low i n c o m p a r i s o n to yeast a n d f u n g a l s y s t e m s . A c o m p a r i s o n of the p r o d u c t i o n y i e l d s a n d extent o f ext r a c e l l u l a r p r o d u c t i o n for v a r i o u s cloned x y l a n a s e genes is f o u n d i n T a b l e I. U n l i k e most other cloned systems, the x y l a n a s e p r e p a r a t i o n f r o m B. succinogenes was c o n t a m i n a t e d w i t h cellulase a c t i v i t y . H o n d a et al. have r e p o r t e d a x y l a n a s e gene f r o m a n a l k a l o p h i l i c Bacillus sp. C I 2 5 i n E. coli (35-36). A b o u t 8 0 % of the x y l a n a s e a c t i v i t y was secreted i n t o the e x t r a c e l l u l a r m e d i u m a n d the a c t i v i t y was s l i g h t l y h i g h e r t h a n t h a t p r o d u c e d b y the o r i g i n a l Bacillus ( a p p r o x i m a t e l y 0.6 I U / m L ) . A x y l a n a s e was p r o d u c e d f r o m a x y l a n a s e gene cloned f r o m a l k a l o p h i l i c Aeromonas sp. 212 at a level a b o u t 80 f o l d higher t h a n i n the o r i g i n a l b a c t e r i u m . U n f o r t u n a t e l y , none of the e n z y m e was secreted e x t r a c e l l u l a r l y (37). H a m m a m o t o a n d H o r i k o s h i have recently succeeded i n the c o n s t r u c t i o n of a secretion vector c o n t a i n i n g the x y l a n a s e gene f r o m a l k a l o p h i l i c Bacillus sp. C 1 2 5 , w h i c h was t h e n cloned i n t o E. coli (38). T r a n s f o r m a n t s w h i c h c a r r i e d these genes secreted a significant a m o u n t o f e x t r a c e l l u l a r x y lanase i n c o m p a r i s o n to clones w h i c h d i d not c o n t a i n the secretion vector. It has been suggested t h a t the secretion o f the x y l a n a s e is d i r e c t e d b y the characteristics of the p r o t e i n itself. F u t u r e w o r k m a y i d e n t i f y i m p r o v e m e n t s to secretory sequences w h i c h w i l l increase the e x t r a c e l l u l a r q u a n t i t i e s of x y lanases f r o m cloned systems.


644


T a b l e I. X y l a n a s e s f r o m C l o n e d Systems


X y l a n a s e G e n e Source Aeromonas sp. 212 Bacillus polymyxa Bacillus subtilis Bacillus subiilus Bacillus sp. C 1 2 5 Bacillus sp. C 1 2 5 (secretion vector) Clostridium acetobutylicum Clostridium thermocellum Streptomyces lividans

Enzyme Yield (IU/ml) 1.63 0.037 0.5 2.2 0.75 0.7

%

Extracellular 0 70 50

Reference (37) (31) (29) (10) (35-36) (38)

0

(30)

11.2

—

(33)

1200

100

(40)

64

A l t h o u g h most cloned xylanases are t o t a l l y free f r o m cellulase c o n t a m i n a t i o n , the recovery y i e l d s r e m a i n i n general r e l a t i v e l y low w h e n c o m p a r e d t o f u n g a l systems. M o r e effort i n the areas of i m p r o v e d x y l a n a s e p r o d u c t i o n y i e l d s a n d secretion m a y make the b a c t e r i a l systems appear m o r e p r o m i s i n g for b i o t e c h n o l o g i c a l a p p l i c a t i o n s . R e c e n t l y a n e x t r a c e l l u l a r , cellulase-free x y l a n a s e e n z y m e was prepared b y homologous c l o n i n g of a x y l a n a s e gene f r o m Streptomyces lividans i n t o a xylanase-cellulase-negative m u t a n t of S. lividans (39). A considerable o v e r p r o d u c t i o n of t h i s e n z y m e (up to 1200 I U / m L i n the c u l t u r e filtrate) has also been d e m o n s t r a t e d w h e n g r o w n o n x y l a n or h e m i c e l l u l o s e - r i c h substrates (40). Fungal Xylanase

Production

T h e use of f i l a m e n t o u s f u n g i for p r o d u c t i o n of xylanases was i n i t i a l l y a t t r a c t i v e because the enzymes are released e x t r a c e l l u l a r l y t h u s e l i m i n a t i n g the need for cell lysis procedures. I n a d d i t i o n , x y l a n a s e levels i n f u n g a l c u l ture filtrates are t y p i c a l l y i n m u c h higher concentrations t h a n f r o m yeasts a n d b a c t e r i a . M a n y examples of xylanases p r o d u c e d f r o m f u n g i are l i s t e d i n the review b y D e k k e r (41). It has been proposed t h a t the p r o d u c t i o n of xylanases a n d cellulases is under separate r e g u l a t o r y c o n t r o l i n some filamentous f u n g i (1). H r m o v a et ai (42) reached a s i m i l a r conclusion after m o n i t o r i n g the d a i l y p r o d u c t i o n of these enzymes i n Trichoderma reesei Q M 9414. X y l a n a s e a n d cellulase a c t i v i t i e s followed independent p r o d u c t i o n profiles d u r i n g f u n g a l g r o w t h . T h e same effect has been observed i n b a t c h cultures o f T. harzianum. We have observed peak x y l a n a s e a c t i v i t y o n the t h i r d d a y of g r o w t h whereas the cellulase a c t i v i t y peaked after d a y five or s i x ( u n p u b l i s h e d ) . T h e r e are m a n y recent examples o f x y l a n a s e i n d u c t i o n i n f u n g i g r o w n o n m e d i a c o n t a i n i n g x y l a n , h e m i c e l l u l o s e - r i c h m a t e r i a l a n d low m o l e c u l a r weight c a r b o h y d r a t e s (42-66). It has generally been observed t h a t higher


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levels of x y l a n a s e are i n d u c e d i n systems c o n t a i n i n g higher x y l a n concent r a t i o n s i n the g r o w t h m e d i u m . A c o m p a r i s o n of x y l a n a s e a n d cellulase p r o d u c t i o n f r o m v a r i o u s f u n g i is s h o w n i n T a b l e I I . M o s t c u l t u r e s g r o w n o n x y l a n y i e l d e d higher levels of x y l a n a s e t h a n w h e n g r o w n o n s i m i l a r concent r a t i o n s of cellulose. W i t h the e x c e p t i o n of Schizophyllum commune (45), the highest p r o d u c t i o n of xylanases by various species has been the result of g r o w t h o n x y l a n . A l t h o u g h a t t e m p t s have been m a d e to s t a n d a r d i z e x y l a n a s e assays (67), some of the v a r i a t i o n i n e n z y m e a c t i v i t i e s r e p o r t e d must be a t t r i b u t e d to v a r i a t i o n s i n assay procedures a m o n g workers. U s i n g a x y l a n - f r e e cellulose f r o m Acetobacter xylinum as a g r o w t h s u b s t r a t e , H r m o v a et al. d e m o n s t r a t e d t h a t s m a l l levels of c o n s t i t u t i v e l y p r o duced x y l a n a s e c o u l d be a t t r i b u t e d to cellulase a c t i v i t y i n T. reesei Q M 9 4 1 4 (42). A s the c o n c e n t r a t i o n of x y l a n i n the m e d i u m was i n c r e a s e d , higher x y l a n a s e p r o d u c t i o n r e s u l t e d . U s i n g a series of s t e a m t r e a t e d aspen w o o d samples, we have observed t h a t the r a t i o of x y l a n a s e to cellulase a c t i v i ties p r o d u c e d o n these m a t e r i a l s increased d i r e c t l y w i t h a n increase i n the r a t i o of x y l a n to cellulose i n the m e d i u m (measured as x y l a n to g l u c a n r a tio) ( F i g u r e 1). D e t e r m i n a t i o n of these ratios for A v i c e l , S o l k a F l o e a n d oat spelts x y l a n l a r g e l y s u p p o r t e d t h i s r e l a t i o n s h i p . S o m e d e v i a t i o n e n countered between these g r o w t h substrates was likely due to v a r i a t i o n s i n c o m p o s i t i o n a n d accessibility of the s u b s t r a t e . It is therefore a p p a r e n t t h a t w i t h Trichoderma fungal systems, m a x i m a l levels of xylanases can be achieved b y g r o w i n g the cells i n x y l a n c o n t a i n i n g m e d i a . T h e i n d u c t i o n of xylanases i n f u n g i g r o w n o n x y l a n m e d i u m , however, cannot be realized i n a l l f u n g i w i t h equivalent success. U n l i k e the level of xylanases i n d u c e d i n most f u n g i , the x y l a n a s e a c t i v i t y p r o d u c e d f r o m S. commune g r o w n on cellulose was u n u s u a l l y h i g h (See T a b l e I I ) . B i e l y et al. (68) a n d M a c k e n z i e a n d B i l o u s (69) have observed m a r k e d l y higher p r o d u c t i o n of x y l a n a s e i n S. commune w h e n g r o w n o n cellulose r a t h e r t h a n o n x y l a n . T h e h i g h cost of u s i n g p u r i f i e d x y l a n s as substrates for x y l a n a s e i n d u c t i o n are p r o h i b i t i v e for large-scale x y l a n a s e p r o d u c t i o n . A s a n a l t e r n a t i v e , m a n y groups have investigated the effectiveness of other h e m i c e l l u l o s e - r i c h m a t e r i a l s for t h i s purpose. E x a m p l e s of these i n c l u d e : a g r i c u l t u r a l wastes such as straws (2,45,47,50, 52,56,57,70,71), b r a n (47,56,57,60), sugar cane bagasse (57,61), c o r n cob a n d oak dust (70); p u l p (2,45,52); p u l p m i l l waste (3) a n d s t e a m treated w o o d (53,59). A l t e r n a t i v e m e t h o d s for more cost-effective x y l a n a s e p r o d u c t i o n i n f u n g i i n c l u d e the development of c a t a b o l i t e derepressed m u t a n t s , c e l l u lase negative m u t a n t s , genetically engineered s t r a i n s a n d h i g h - x y l a n a s e p r o d u c i n g c o n s t i t u t i v e m u t a n t s . T h e l a t t e r case c o u l d afford h i g h x y l a n a s e y i e l d s u s i n g soluble sugars as g r o w t h substrates w h i c h are b o t h i n e x p e n s i v e and easy to h a n d l e i n c o n v e n t i o n a l f e r m e n t a t i o n e q u i p m e n t . T h e vast m a j o r i t y of xylanases t h a t have been p r o d u c e d are q u i t e l a b i l e above 45-50°C. It w o u l d be of great advantage to e m p l o y xylanases w h i c h r e t a i n a h i g h specific a c t i v i t y above t h i s t e m p e r a t u r e range. A n u m b e r of t h e r m o s t a b l e xylanases have been p r e p a r e d to v a r y i n g


646


Oat Spelts Xylan ·


200

ϋ

Plant Cell Wall Polymers - American Chemical Society

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