ANALYTICALBIOCHEMISTRY206, 50-52 (1992)
Activity Staining of Endoglucanases in Polyacrylamide Gels R o y M a t h e w a n d K. K o t e s w a r a R a o
Department of Microbiology and Biotechnology Centre, Faculty of Science, M. S. University of Baroda, Baroda 390 002, India Received February 24, 1992
T h e e n d o g l u c a n a s e s o f Penicillium funiculosum w e r e analyzed for the presence of multiple forms using a modified version of the Congo red method. Postelectrophoretic slab gels were directly incubated in a solution of c a r b o x y m e t h y l c e l l u l o s e f o r a p e r i o d as s h o r t a s 1 5 m i n and then the activities were visualized by staining with C o n g o r e d . T e n d i s t i n c t b a n d s o f c l e a r a n c e s w e r e obt a i n e d i n d i c a t i n g t h e p r e s e n c e o f at l e a s t a s m a n y m u l t i p l e f o r m s . ©1992 Academic Press, Inc.
Endo-l,4-/~-D-glucanases (EC 3.2.1.4) are im port ant in the hydrolysis of cellulose. T hough these enzymes are known to exist in multiple forms in fungal culture filtrates, the reason(s) for this multiplicity is still not very clear (1). Analysis of these enzymes could be done by detecting the cellulolytic activities on postelectrophoretic polyacrylamide gels. Various methods have been described for this purpose. These include mainly (a) cutting of the gels and assaying for activity (2), (b) use of agar replicas (3,4), and (c) incorporation of cellulose into the polyacrylamide gels (5). T he last two methods use Congo red-based procedures for visualizing the endoglucanase activities. T h e first two methods are time consuming, while the last method has the disadvantage t h a t a p ar t of the same gel cannot be used for glycoprotein staining because of the presence of the polysaccharide. In this paper, we report a modified version of the Congo red staining procedure for analyzing the multiplicity of endoglucanase forms in a preparation from Penicillium funiculosum. This method utilizes a so far unreported property of carboxymethylcellulose to adhere to polyacrylamide, thus permitting a rapid visualization of the enzymes acting on carboxymethylcellulose (CMC) 1 by subsequent staining with Congo red. xAbbreviations used: CMC, carboxymethylcellulose;PAGE, polyacrylamide gel electrophoresis;SDS, sodium dodecylsulfate. 50
Our results suggest the presence o f t e n forms of endoglucanases in this preparation.
MATERIALS AND METHODS
Chemicals. CMC P. [uniculosum cellulase (Endo1,4-fl-D-glucanase), bovine serum albumin, and acrylamide were obtained from Sigma Chemical Co. (St. Louis, MO). Congo red was obtained from B D H (Poole, England). Preparation of sample [or electrophoresis. Sample for nondenaturing polyacrylamide gel electrophoresis (PAGE) was prepared by directly dissolving the enzyme powder in distilled water. Samples for S D S - P A G E were prepared by heating t he enzyme in sample buffer (6) at 50°C for 2 h or 65°C for 45 min. Boiling of the enzyme in sample buffer for 1 h led to complete loss of enzymatic activity. PAGE. Nondenaturing PAGE was carried out at a const ant current of 20 mA in 7.5% gels (1.5 mm thick) by the method of Davis (7). During the run the gels were maintained at 15°C using a water circulator. However, T r i s - H C l buffer in the resolving gel was used at half the strength. Gels were prerun for at least 4 h at 10 mA. Neither the strength of the buffer nor the prerun were found to affect the binding of CMC to the gel or the actual reaction. However, we found t hat these conditions gave better separation of the bands. Electrophoresis was continued till the dye-front just emerged from the gel. S D S - P A G E was carried out by the method of Laemmli (6). Staining for endoglucanase activity. Gels after nondenaturing PAGE were directly transferred to 1% CMC in 0.1 M citrate buffer (pH 4.8) at 50°C and incubated for different time intervals as indicated in Fig. 1. T h e gels were then t ransferred to 0.1 g% solution of Congo red and incubated at 25°C, with constant shaking for 10 min. T h e gels were destained by washing with 1 M sodium chloride. Activity bands were clearly visible as yel0003-2697/92 $5.00 Copyright © 1992 b y Academic Press, Inc. All rights of r e p r o d u c t i o n in a n y form reserved.
ACTIVITY STAINING OF ENDOGLUCANASES
?
1
2
3
4
5
6
FIG. 1. Comparison of activity and protein, and glycoprotein staining of a preparation from Penicillium funiculosum. Lanes 1 and 2, activity staining after 5 and 15 min incubation in CMC solution; lanes 3 and 4, protein staining; lanes 5 and 6, glycoprotein staining. Lanes 4 and 6 contained 200 t~g each of the preparation while all others contained 100 #g each (100 tLgof the preparation corresponded to 23 ug of protein with a specific activity of 17 U/mg protein). Arrowheads indicate bands obtained on activity staining.
lowish c l e a r a n c e s a g a i n s t a deep red b a c k g r o u n d b y the e n d of 10 m i n of destaining. I n c r e a s e in c o n t r a s t bet w e e n the b a n d s were o b t a i n e d b y t r a n s f e r r i n g the gels to mildly acidified s o d i u m chloride solution a f t e r t h e d e s t a i n i n g procedure. Gels which were e l e c t r o p h o r e s e d u n d e r d e n a t u r i n g conditions were first rinsed with distilled w a t e r a n d t h e n left o v e r n i g h t in 0.01 M citrate buffer ( p H 4.8) before t r a n s f e r r i n g to C M C solution. Protein staining. Gels were stained for p r o t e i n by the C o o m a s s i e brilliant blue R-250 m e t h o d . Glycoprotein staining. G l y c o p r o t e i n s t a i n i n g was done by t h e m e t h o d of Z a c h a r i u s et al. (8). Protein estimation. P r o t e i n c o n t e n t of the cellulase p r e p a r a t i o n was e s t i m a t e d b y the m e t h o d of B r a d ford (9).
RESULTS AND DISCUSSION T h e activity staining p a t t e r n o b t a i n e d with P. funiculosum cellulase ( E n d o - l , 4 - ~ - D - g l u c a n a s e ) u n d e r n o n d e n a t u r i n g conditions is s h o w n in Fig. 1. T e n distinct activity b a n d s were o b t a i n e d by the e n d of 15 m i n of incubation in the C M C solution. T h e fact t h a t earlier c h a r a c t e r i z a t i o n of the e n d o g l u c a n a s e s of P. funiculosum (10) h a d revealed only four f o r m s suggests t h a t this m e t h o d could p r o v e to be m o r e sensitive in d e t e c t i o n of t h e s e enzymes. N o p r o t e i n b a n d s are seen c o r r e s p o n d ing to the last two activity b a n d s ( L a n e 2, Fig. 1) f u r t h e r suggesting t h a t this staining m e t h o d is sensitive e n o u g h
51
to d e t e c t activities which h a v e low p r o t e i n c o n t e n t . T h e fact t h a t only t h r e e o f the activity b a n d s c o r r e s p o n d s to g l y c o p r o t e i n b a n d s suggests t h a t differential glycosylation could be a c a u s e of t h e m u l t i p l i c i t y of e n d o g l u c a n ase forms. T h e possibility t h a t differences in glycosylation could c a u s e at least c h r o m a t o g r a p h i c a l l y distinct f o r m s h a s b e e n r e p o r t e d in case of o t h e r s y s t e m s also (11). H o w e v e r , e l e c t r o p h o r e s i s on S D S - P A G s h o w e d a single activity b a n d . S i m i l a r r e s u l t s h a v e b e e n o b t a i n e d b y o t h e r w o r k e r s in case of Aspergillus terreus w h e r e i n m u l t i p l e activity b a n d s of e n d o g l u c a n a s e were o b t a i n e d on isoelectric focussing w h e r e a s H P L C r e v e a l e d only one p e a k (12). In o r d e r to verify t h e a u t h e n t i c i t y of this s t a i n i n g m e t h o d , v a r i o u s c o n t r o l e x p e r i m e n t s were also c a r r i e d out as described below. Since t h e effectiveness of this m e t h o d would d e p e n d on t h e b i n d i n g of C M C to t h e p o l y a c r y l a m i d e gel, a c o n t r o l w a s p e r f o r m e d b y i n c u b a t ing a p o l y a c r y l a m i d e gel in buffer w i t h o u t C M C at 50°C. It was seen t h a t in this case, t h o u g h t h e r e w a s initial b i n d i n g of C o n g o red to t h e gel, d e s t a i n i n g with 1 M s o d i u m chloride led to a l m o s t c o m p l e t e r e m o v a l of t h e red color as c o m p a r e d to gels which h a d b e e n i n c u b a t e d in CMC. T h e p r e s e n c e of C M C on t h e gel s u r f a c e w a s f u r t h e r c o n f i r m e d by placing d r o p s o f e n d o g l u c a n a s e solution on the surface of p o l y a c r y l a m i d e gels w h i c h h a d b e e n i m m e r s e d in C M C solution b e f o r e h a n d . In this case i n c u b a t i o n for 15 min, followed b y t h e activity s t a i n i n g p r o c e d u r e gave c l e a r a n c e s which c o r r e s p o n d e d to t h e d r o p s p l a c e d on the gel. As a c o n t r o l for n o n s p e cific clearances, a gel with different c o n c e n t r a t i o n s o f b o v i n e s e r u m a l b u m i n was r u n (up to 500 #g p e r well). N o c l e a r a n c e was obtained. H e a t - i n a c t i v a t e d e n z y m e also s h o w e d no clearance, t h o u g h t h e p r o t e i n m i g r a t i o n p a t t e r n r e m a i n e d t h e s a m e as in t h e active s a m p l e . T h e modified m e t h o d of s t a i n i n g for e n d o g l u c a n a s e s offers a distinct a d v a n t a g e o v e r existing m e t h o d s . T h e a g a r replica m e t h o d is k n o w n to be h a m p e r e d b y low sensitivity a n d loss of r e s o l u t i o n (13). T h e a g a r replica m e t h o d p e r f o r m e d b y us could reveal only 2 of t h e 10 b a n d s o b t a i n e d b y t h e modified p r o c e d u r e . T h e subs t r a t e inclusion m e t h o d was equally as sensitive as o u r m e t h o d b u t t h e addition of c a r b o h y d r a t e s u b s t r a t e into the gel would i n t e r f e r e in g l y c o p r o t e i n staining. Also inclusion of p o l y m e r i c s u b s t r a t e s d e c r e a s e s t h e m o b i l i t y of all p r o t e i n s due to i n c r e a s e d viscosity of t h e gel (5). Our m e t h o d also avoids t h e use of t h e m o r e e x p e n s i v e f l u o r e s c e n t s u b s t r a t e s like m e t h y l u m b e l l i f e r y l derivatives (14).
ACKNOWLEDGMENTS The authors thank the Council of Scientific and Industrial Research (CSIR), India, for the award of a Senior Research Fellowship to R.M.
52
MATHEW AND RAO
REFERENCES 1. Coughlan, M. P. (1985) in Biotechnology and Genetic Engineering Reviews, (Russell, G. E., Ed.), Vol. 3, pp. 39-109, Intercept, Newcastle -Upon-Tyne. 2. Saddler, J. N., and Khan, A. W. (1981) Can. d. Microbiol. 27, 288-294. 3. McHale, A., and Coughlan, M. P. (1981) Biochem. d. 199, 267268. 4. B6guin, P. (1983) Anal. Biochem. 131,333-336. 5. Schwarz, W. H., Bronnenmeir, K., Gr~ibnitz, F., and Staudenbauer, W. L. (1987) Anal. Biochem. 164, 72-77. 6. Laemmli, U. K. (1970) Nature 227,680-685. 7. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427.
8. Zacharius, R. M., Zell, T. E., Morrison, J. H., and Woodlock, J. J. (1969) Anal. Biochem. 30, 148-152. 9. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 10. Wood, T. M., McCrae, S. I., and MacFarlane, C. C. (1980) Biochem. J. 189, 51-65. 11. Moloney, A. P., McCrae, S. I., Wood, T. M., and Coughlan, M. P. (1985) Biochem. J. 225, 365-374. 12. Hrmovh, M., Biely, P., and Vr§anskfi, M. (1989) Enzyme Microb. Technol. 11,610-616. 13. Lacks, S. A., and Springhorn, S. S. (1980) J. Biol. Chem. 255, 7467-7473. 14. Claeyssens, M. (1988) in Biochemistry and Genetics of Cellulose Degradation (Aubert, J. P., Beguin, P., and Millet, J., Eds.), pp. 393-397. Academic Press, New York.
Activity Staining of Endoglucanases in Polyacrylamide Gels R o y M a t h e w a n d K. K o t e s w a r a R a o
Department of Microbiology and Biotechnology Centre, Faculty of Science, M. S. University of Baroda, Baroda 390 002, India Received February 24, 1992
T h e e n d o g l u c a n a s e s o f Penicillium funiculosum w e r e analyzed for the presence of multiple forms using a modified version of the Congo red method. Postelectrophoretic slab gels were directly incubated in a solution of c a r b o x y m e t h y l c e l l u l o s e f o r a p e r i o d as s h o r t a s 1 5 m i n and then the activities were visualized by staining with C o n g o r e d . T e n d i s t i n c t b a n d s o f c l e a r a n c e s w e r e obt a i n e d i n d i c a t i n g t h e p r e s e n c e o f at l e a s t a s m a n y m u l t i p l e f o r m s . ©1992 Academic Press, Inc.
Endo-l,4-/~-D-glucanases (EC 3.2.1.4) are im port ant in the hydrolysis of cellulose. T hough these enzymes are known to exist in multiple forms in fungal culture filtrates, the reason(s) for this multiplicity is still not very clear (1). Analysis of these enzymes could be done by detecting the cellulolytic activities on postelectrophoretic polyacrylamide gels. Various methods have been described for this purpose. These include mainly (a) cutting of the gels and assaying for activity (2), (b) use of agar replicas (3,4), and (c) incorporation of cellulose into the polyacrylamide gels (5). T he last two methods use Congo red-based procedures for visualizing the endoglucanase activities. T h e first two methods are time consuming, while the last method has the disadvantage t h a t a p ar t of the same gel cannot be used for glycoprotein staining because of the presence of the polysaccharide. In this paper, we report a modified version of the Congo red staining procedure for analyzing the multiplicity of endoglucanase forms in a preparation from Penicillium funiculosum. This method utilizes a so far unreported property of carboxymethylcellulose to adhere to polyacrylamide, thus permitting a rapid visualization of the enzymes acting on carboxymethylcellulose (CMC) 1 by subsequent staining with Congo red. xAbbreviations used: CMC, carboxymethylcellulose;PAGE, polyacrylamide gel electrophoresis;SDS, sodium dodecylsulfate. 50
Our results suggest the presence o f t e n forms of endoglucanases in this preparation.
MATERIALS AND METHODS
Chemicals. CMC P. [uniculosum cellulase (Endo1,4-fl-D-glucanase), bovine serum albumin, and acrylamide were obtained from Sigma Chemical Co. (St. Louis, MO). Congo red was obtained from B D H (Poole, England). Preparation of sample [or electrophoresis. Sample for nondenaturing polyacrylamide gel electrophoresis (PAGE) was prepared by directly dissolving the enzyme powder in distilled water. Samples for S D S - P A G E were prepared by heating t he enzyme in sample buffer (6) at 50°C for 2 h or 65°C for 45 min. Boiling of the enzyme in sample buffer for 1 h led to complete loss of enzymatic activity. PAGE. Nondenaturing PAGE was carried out at a const ant current of 20 mA in 7.5% gels (1.5 mm thick) by the method of Davis (7). During the run the gels were maintained at 15°C using a water circulator. However, T r i s - H C l buffer in the resolving gel was used at half the strength. Gels were prerun for at least 4 h at 10 mA. Neither the strength of the buffer nor the prerun were found to affect the binding of CMC to the gel or the actual reaction. However, we found t hat these conditions gave better separation of the bands. Electrophoresis was continued till the dye-front just emerged from the gel. S D S - P A G E was carried out by the method of Laemmli (6). Staining for endoglucanase activity. Gels after nondenaturing PAGE were directly transferred to 1% CMC in 0.1 M citrate buffer (pH 4.8) at 50°C and incubated for different time intervals as indicated in Fig. 1. T h e gels were then t ransferred to 0.1 g% solution of Congo red and incubated at 25°C, with constant shaking for 10 min. T h e gels were destained by washing with 1 M sodium chloride. Activity bands were clearly visible as yel0003-2697/92 $5.00 Copyright © 1992 b y Academic Press, Inc. All rights of r e p r o d u c t i o n in a n y form reserved.
ACTIVITY STAINING OF ENDOGLUCANASES
?
1
2
3
4
5
6
FIG. 1. Comparison of activity and protein, and glycoprotein staining of a preparation from Penicillium funiculosum. Lanes 1 and 2, activity staining after 5 and 15 min incubation in CMC solution; lanes 3 and 4, protein staining; lanes 5 and 6, glycoprotein staining. Lanes 4 and 6 contained 200 t~g each of the preparation while all others contained 100 #g each (100 tLgof the preparation corresponded to 23 ug of protein with a specific activity of 17 U/mg protein). Arrowheads indicate bands obtained on activity staining.
lowish c l e a r a n c e s a g a i n s t a deep red b a c k g r o u n d b y the e n d of 10 m i n of destaining. I n c r e a s e in c o n t r a s t bet w e e n the b a n d s were o b t a i n e d b y t r a n s f e r r i n g the gels to mildly acidified s o d i u m chloride solution a f t e r t h e d e s t a i n i n g procedure. Gels which were e l e c t r o p h o r e s e d u n d e r d e n a t u r i n g conditions were first rinsed with distilled w a t e r a n d t h e n left o v e r n i g h t in 0.01 M citrate buffer ( p H 4.8) before t r a n s f e r r i n g to C M C solution. Protein staining. Gels were stained for p r o t e i n by the C o o m a s s i e brilliant blue R-250 m e t h o d . Glycoprotein staining. G l y c o p r o t e i n s t a i n i n g was done by t h e m e t h o d of Z a c h a r i u s et al. (8). Protein estimation. P r o t e i n c o n t e n t of the cellulase p r e p a r a t i o n was e s t i m a t e d b y the m e t h o d of B r a d ford (9).
RESULTS AND DISCUSSION T h e activity staining p a t t e r n o b t a i n e d with P. funiculosum cellulase ( E n d o - l , 4 - ~ - D - g l u c a n a s e ) u n d e r n o n d e n a t u r i n g conditions is s h o w n in Fig. 1. T e n distinct activity b a n d s were o b t a i n e d by the e n d of 15 m i n of incubation in the C M C solution. T h e fact t h a t earlier c h a r a c t e r i z a t i o n of the e n d o g l u c a n a s e s of P. funiculosum (10) h a d revealed only four f o r m s suggests t h a t this m e t h o d could p r o v e to be m o r e sensitive in d e t e c t i o n of t h e s e enzymes. N o p r o t e i n b a n d s are seen c o r r e s p o n d ing to the last two activity b a n d s ( L a n e 2, Fig. 1) f u r t h e r suggesting t h a t this staining m e t h o d is sensitive e n o u g h
51
to d e t e c t activities which h a v e low p r o t e i n c o n t e n t . T h e fact t h a t only t h r e e o f the activity b a n d s c o r r e s p o n d s to g l y c o p r o t e i n b a n d s suggests t h a t differential glycosylation could be a c a u s e of t h e m u l t i p l i c i t y of e n d o g l u c a n ase forms. T h e possibility t h a t differences in glycosylation could c a u s e at least c h r o m a t o g r a p h i c a l l y distinct f o r m s h a s b e e n r e p o r t e d in case of o t h e r s y s t e m s also (11). H o w e v e r , e l e c t r o p h o r e s i s on S D S - P A G s h o w e d a single activity b a n d . S i m i l a r r e s u l t s h a v e b e e n o b t a i n e d b y o t h e r w o r k e r s in case of Aspergillus terreus w h e r e i n m u l t i p l e activity b a n d s of e n d o g l u c a n a s e were o b t a i n e d on isoelectric focussing w h e r e a s H P L C r e v e a l e d only one p e a k (12). In o r d e r to verify t h e a u t h e n t i c i t y of this s t a i n i n g m e t h o d , v a r i o u s c o n t r o l e x p e r i m e n t s were also c a r r i e d out as described below. Since t h e effectiveness of this m e t h o d would d e p e n d on t h e b i n d i n g of C M C to t h e p o l y a c r y l a m i d e gel, a c o n t r o l w a s p e r f o r m e d b y i n c u b a t ing a p o l y a c r y l a m i d e gel in buffer w i t h o u t C M C at 50°C. It was seen t h a t in this case, t h o u g h t h e r e w a s initial b i n d i n g of C o n g o red to t h e gel, d e s t a i n i n g with 1 M s o d i u m chloride led to a l m o s t c o m p l e t e r e m o v a l of t h e red color as c o m p a r e d to gels which h a d b e e n i n c u b a t e d in CMC. T h e p r e s e n c e of C M C on t h e gel s u r f a c e w a s f u r t h e r c o n f i r m e d by placing d r o p s o f e n d o g l u c a n a s e solution on the surface of p o l y a c r y l a m i d e gels w h i c h h a d b e e n i m m e r s e d in C M C solution b e f o r e h a n d . In this case i n c u b a t i o n for 15 min, followed b y t h e activity s t a i n i n g p r o c e d u r e gave c l e a r a n c e s which c o r r e s p o n d e d to t h e d r o p s p l a c e d on the gel. As a c o n t r o l for n o n s p e cific clearances, a gel with different c o n c e n t r a t i o n s o f b o v i n e s e r u m a l b u m i n was r u n (up to 500 #g p e r well). N o c l e a r a n c e was obtained. H e a t - i n a c t i v a t e d e n z y m e also s h o w e d no clearance, t h o u g h t h e p r o t e i n m i g r a t i o n p a t t e r n r e m a i n e d t h e s a m e as in t h e active s a m p l e . T h e modified m e t h o d of s t a i n i n g for e n d o g l u c a n a s e s offers a distinct a d v a n t a g e o v e r existing m e t h o d s . T h e a g a r replica m e t h o d is k n o w n to be h a m p e r e d b y low sensitivity a n d loss of r e s o l u t i o n (13). T h e a g a r replica m e t h o d p e r f o r m e d b y us could reveal only 2 of t h e 10 b a n d s o b t a i n e d b y t h e modified p r o c e d u r e . T h e subs t r a t e inclusion m e t h o d was equally as sensitive as o u r m e t h o d b u t t h e addition of c a r b o h y d r a t e s u b s t r a t e into the gel would i n t e r f e r e in g l y c o p r o t e i n staining. Also inclusion of p o l y m e r i c s u b s t r a t e s d e c r e a s e s t h e m o b i l i t y of all p r o t e i n s due to i n c r e a s e d viscosity of t h e gel (5). Our m e t h o d also avoids t h e use of t h e m o r e e x p e n s i v e f l u o r e s c e n t s u b s t r a t e s like m e t h y l u m b e l l i f e r y l derivatives (14).
ACKNOWLEDGMENTS The authors thank the Council of Scientific and Industrial Research (CSIR), India, for the award of a Senior Research Fellowship to R.M.
52
MATHEW AND RAO
REFERENCES 1. Coughlan, M. P. (1985) in Biotechnology and Genetic Engineering Reviews, (Russell, G. E., Ed.), Vol. 3, pp. 39-109, Intercept, Newcastle -Upon-Tyne. 2. Saddler, J. N., and Khan, A. W. (1981) Can. d. Microbiol. 27, 288-294. 3. McHale, A., and Coughlan, M. P. (1981) Biochem. d. 199, 267268. 4. B6guin, P. (1983) Anal. Biochem. 131,333-336. 5. Schwarz, W. H., Bronnenmeir, K., Gr~ibnitz, F., and Staudenbauer, W. L. (1987) Anal. Biochem. 164, 72-77. 6. Laemmli, U. K. (1970) Nature 227,680-685. 7. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427.
8. Zacharius, R. M., Zell, T. E., Morrison, J. H., and Woodlock, J. J. (1969) Anal. Biochem. 30, 148-152. 9. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 10. Wood, T. M., McCrae, S. I., and MacFarlane, C. C. (1980) Biochem. J. 189, 51-65. 11. Moloney, A. P., McCrae, S. I., Wood, T. M., and Coughlan, M. P. (1985) Biochem. J. 225, 365-374. 12. Hrmovh, M., Biely, P., and Vr§anskfi, M. (1989) Enzyme Microb. Technol. 11,610-616. 13. Lacks, S. A., and Springhorn, S. S. (1980) J. Biol. Chem. 255, 7467-7473. 14. Claeyssens, M. (1988) in Biochemistry and Genetics of Cellulose Degradation (Aubert, J. P., Beguin, P., and Millet, J., Eds.), pp. 393-397. Academic Press, New York.
