- Email: [email protected]
β-d -Glucosidases from Sclerotium rolfsii
424
CELLULOSE
[49]
exoenzymes. TM Sequential removal of cellobiose by fl-glucosidase enhances the efficiency of the cellulose degradation process. ~9 Adequate levels of fl-glucosidase activity are essential when glucose is wanted as the major product of cellulose hydrolysis, z° 18 T. M. W o o d a n d S. I. M c C r a e , Proc. Symp. Bioconversion Cell. Subst. Energy, Chem. Microb. Protein, 1st p. 111 (1978). ~9 B. S. M o n t e n e c o u r t a n d D. E. Eveleigh, Appl. Environ. Microbiol. 34, 777 (1977). 2o C. Mishra, M. Rao, R. Seeta, M. C. Srinivasan, and V. D e s h p a n d e , Biotechnol. Bioeng. 26, 370 (1984).
[49] f l - D - G l u c o s i d a s e s f r o m Sclerotium rolfsii
By JAI C. SADANA, RAJKUMARV. PATIL, and JAIPRAKASH G. S H E W A L E Sclerotium rolfsii produces at least four different extracellular fl-glucosidases when grown on cellulose.~,2 Assay Method
Principle. fl-D-Glucosidase (fl-I)-glucoside glucohydrolase, EC 3.2.1.21) activity is measured as the amount of glucose released from cellobiose or p-nitrophenol from p-nitrophenyl-fl-D-glucopyranoside (PNPG). The glucose released is determined by the glucose o×idaseperoxidase test 3 using Glox glucose reagent. The amount ofp-nitrophenol released is calculated from the molar absorbance of 18,500 for nitrophenol at 410 nm. 4 With Cellobiose as Substrate Reagents Citrate buffer, 50 mM, pH 4.5 D(+)-Cellobiose, 50 mM in 50 mM citrate buffer, pH 4.5 J J. G. Shewale a n d J. C. Sadana, Arch. Biochem. Biophys. 207, 185 (1981). 2 j. C. Sadana, J. G. Shewale, and R. V. Patil, Carbohydr. Res. 118, 205 (1983). 3 H. U. B e r g m e y e r , K. G a w e h n , and M. Grassl, Methods Enzymatic Anal. 1, 457 (1974). 4 D. Herr, F. B a u m e r , and H. Dellweg, Eur. J. Appl. Microbiol. Biotechnol. 5, 29 (1978).
METHODS IN ENZYMOLOGY, VOL. 160
Copyright © 1988by AcademicPress. Inc. All rights of reproduction in any form reserved.
[49]
S. rolfsii fl-D-GLUCOSIDASES
425
Glox glucose reagent: one packet of Glox is dissolved in 500 ml of 50 mM phosphate buffer, pH 7.0. The reagent is freshly prepared to avoid high blank values Sulfuric acid, 41% Procedure. The enzyme is diluted just before assay in 50 mM citrate buffer, pH 4.5, to obtain a concentration of 0.05-0.3 unit of enzyme per milliliter (see definition below). The reaction is initiated by adding enzyme. The enzyme (0.1 ml) is added to 0.9 ml of cellobiose solution in a Corning test tube and the reaction mixture is incubated at 65 ° for 30 min. The reaction is stopped by heating the tubes in a boiling water bath for 5 min. After cooling 5 ml of Glox (glucose oxidase-peroxidase) reagent is added and the reaction mixture incubated for 1 hr at 30°. At the end of incubation, 4 ml of 41% H 2 5 0 4 is added and the color developed is measured at 530 nm. The color is developed immediately and is stable for 2 hr. A control is run with boiled enzyme. Under the conditions of assay, the enzyme shows linearity up to 0.4 mg of glucose produced.
With P N P G as Substrate Reagents Citrate buffer, 50 mM, pH 4.2 p-Nitrophenyl-/3-D-glucopyranoside (PNPG), 1 mg/ml in 50 mM citrate buffer, pH 4.2 Sodium carbonate, 2% in distilled water Procedure. The enzyme is diluted just before assay in 50 mM citrate buffer, pH 4.2, to obtain a concentration of 0.01-0.05 unit of enzyme per milliliter (see definition below). The reaction is initiated by adding 0.1 ml of enzyme to 0.9 ml of PNPG in a Coming test tube. The reaction mixture is incubated at 68 ° for 30 min. The reaction is stopped by adding 1 ml of 2% NazCO3 and the color developed is measured at 410 nm. The color is developed immediately after addition of sodium carbonate and is stable for at least 24 hr. A control is run with boiled enzyme. The enzyme shows linearity up to 12/xg of p-nitrophenol produced. Definitions o f Unit and Specific Activity. One unit of/3-D-glucosidase activity is defined as the amount of enzyme that releases 1 t~mol of glucose from cellobiose at 65 °, pH 4.5 or I/xmol ofp-nitrophenol from PNPG at 68 °, pH 4.2, per minute. In the assay with cellobiose, 2 mol of glucose is released from 1 tool of cellobiose but the glucose values have been given as such without dividing by a factor of 2. This is according to the recom5 Glox, glucose reagent (glucose oxidase-peroxidase reagent), was purchased from AB Kabi Diagnostica, Stockholm, Sweden.
426
CELLULOSE
[49]
mendations of the Commission on Biotechnology, International Union of Pure and Applied Chemistry. 6 The specific activity is defined as the number of units per milligram of protein. Protein is determined by the procedure of Lowry et al. 7
Purification Procedure Three of the purification steps, ammonium sulfate precipitation (step I), fractionation by gel chromatography on Sephadex G-75 (step 2), and ultrafiltration of Fraction A (step 3) are identical to the steps for cellobiose dehydrogenase as described in this volume [53]. During gel filtration on Sephadex G-75 (step 2)/3-glucosidase is eluted along with some highmolecular-weight cellulases, after the void volume (Fraction A) and ahead of low-molecular-weight cellulases (Fraction B). Fraction A (110-165 ml) contains about 95-98% of fl-glucosidase activity and about 70% endo-flglucanase activity, whereas Fraction B (170-265 ml) contains about 30% cellulase and 2%/3-glucosidase activity. Step 4. DEAE-Sephadex A-50 Ion-Exchange Chromatography. Fraction A after concentration on Amicon (step 3) is dialyzed in a collodion bag against 0.05 M phosphate buffer, pH 7.3, and chromatographed on DEAE-Sephadex column (1.8 x 100 cm) equilibrated with 0.05 M phosphate buffer, pH 7.3. The column is washed with the equilibration buffer. Fractions of 2 ml are collected at a flow rate of 12-15 ml/hr./3-Glucosidase and endo-/3-glucanase activities are not adsorbed on the column. fl-Glucosidase comes just after the void volume and forms the first peak; it is almost free of endo-fl-glucanase (Fig. 1). The dark brown pigment present in the culture filtrate is removed in this step. Fractions 6-7 containing fl-glucosidase of 44-50 specific activity are pooled and the pH is adjusted to 4.5 with 0.1 M citric acid. This is freeze-dried and then redissolved in 5 ml of 0.05 M citrate buffer, pH 4.5, and dialyzed against the same buffer. The fl-glucosidase fraction at this stage gives one band in disc gel electrophoresis at pH 8.9. However, electrophoresis of the enzyme at pH 4.3 resolves it into four bands of proteins. Step 5. Preparative Isoelectric Focusing. The fl-glucosidase (Fractions 6-7) from step 4 is dialyzed overnight against 1 mM citrate buffer, pH 4.5, to reduce the salt concentration and is purified further by preparative isoelectric focusing. A gradient of sucrose from 50 to 5% is prepared
6 I U P A C , Comm. Biotechn. 59, 257 (1987).
70. H. Lowry, N. J. Rosebrough, A. L. Fan', and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
S. rolfsii fl-D-OLUCOSIDASES
[49]
427
100 I
700 600
II
~ C
//O~.CM
80
MCcise
E
500. E
"" D
~60
"" 400 • L O t~
40 E
c13
o 30o u
200
3o
, 8 4o
2o ~_
& ZO
100
0
0
~
,
4
8
12
0
16
20
FRACTION NUMBER FIG. I. Ion-exchangechromatography on DEAE-Sepbadex A-50 of Fraction A from step 3. Peak I comprises fractions 6-7 and Peak II Fractions 14-18. (O)/3-D-Olucosidase (BO),
(0) CMCase (endo-/3-glucanase), (A) protein. From Shewale and Sadana. ~
in a 110 ml LKB electrofocusing column. 8 Ampholyte solution (final concentration of I%), pH 4-6, and/3-glucosidase enzyme solution are mixed with light and dense solutions before making the gradient. The anode solution contains 0.16 M phosphoric acid in 60% sucrose, whereas the cathode solution employs 0.25 N sodium hydroxide. The cathode is at the top during the run. The voltage at the end of run (72 hr) is 500 V and the current 2 mA. Fractions of 1 ml are collected and are immediately processed for pH (5-7°), activity, and protein determination. Enzyme is freed from sucrose by extensive dialysis against 0.05 M citrate buffer, pH 4.5. fl-Glucosidase activity is resolved into four separate peaks at pH 4.10, 4.55, 5.10, and 5.55 (Fig. 2) and they are designated BG-1, BG-2, BG-3, and BG-4, respectively. The observation is reproducible. The results of a typical purification of fl-glucosidase enzymes are given in Table I. Purity. The purified enzymes individually show one protein band on polyacrylamide gel electrophoresis at pH 4.3 and 8.9, in the presence or absence of SDS, and on isoelectric focusing in 7.5% polyacrylamide gel s
Instruction manual, LKB Preparative Isoelectric Focusing, Uppsala, Sweden.
428
CELLULOSE
[49] 6.0
2SO
i
E
5.0
20O
4.0
-~
)so
o.2s
uJ
Protei~ 0.20 uJ
~00, o.~o m,
50
0 20
03 n~ 0
<
i
30
,,
.
~
i
40
50
0.00 60
FRACTION NUMBER
FIG. 2. Isoelectric focusing of Peak I from DEAE-Sephadex chromatography. ((3) fl-Glucosidase, (A) absorbance at 280 nm, (0) pH. From Shewale and Sadana.
over the pH range 3.5-10. The purified fl-glucosidases are free of contaminating endo-fl-glucanase activity as determined viscometrically. 9 Stability. fl-Glucosidases are stable for several months when stored in 50 mM citrate buffer, pH 4.5, at -15 °, and on repeated freezing and thawing. Properties
Physical Properties. The relative molecular masses of the BG-1, BG-2, BG-3, and BG-4 /3-glucosidases, estimated by gel filtration on BioGel P-150 according to the procedure of Andrews, 1° are 90,000, 90,000, 107,000, and 92,000, respectively, and by migration in SDSpolyacrylamide gel N are 95,500, 95,500, 106,000, and 95,000, respectively. The molecular weight of BG-3 fl-glucosidase, determined by electrophoresis using the slope method, 12 is 100,000. Subunit Structure. The four/3-glucosidases from S. rolfsii are comprised of single polypeptide chains since the reduced carboxymethylated 9 p. L. Hurst, J. Nielsen, P. A. Sullivan, and M. G. Shepherd, Biochem. J. 165, 33 (1977). 10 p. Andrews, Biochem. J. 91, 222 (1964). 11 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). 12 j. L. Hedrick and A. J. Smith, Arch. Biochem. Biophys. 126, 155 (1968).
S. rolfsii /3-O-GLOCOSIDASES
[49]
429
TABLE I PURIFICATIONOF fl-GLucOSIDASESFROMS. rolfsii~ fl-Glucosidase
Fraction Culture filtrate Ammonium sulfate, 0-3.4 M saturation Sephadex G-75 Fraction A Fraction B Ultrafiltration of fraction A (Amicon XM-50) DEAE-Sephadex A-50 Peak I Peak II Preparative isoelectric focusing of Peak I BG-1 BG-2 BG-3 BG-4
Total protein (mg)
Total units
Specific activity (U/mg Recovery protein) (%)
16,150 10,750
17,720 15,930
1.1 1.4
7,580 918 3,800
11,400 74 9,210
1.5 0.004 2.4
23 550
1,125 127
0.37 0.20 1.80 0.59
10 10 120 14
CMCase b
100 90
64 0.4 52
Specific activity (U/mg Recovery protein) (%)
Total units 355,488 284,740
22 26
100 80
109,290 49,680 57,260
14 54 15
30 14 16
48 0.23
6.3 0.71
110 28,359
27 50 57 25
0.05 0.05 0.57 0.07
0 0 0 0
4.7 51
0 0 0 0
0.03 7.9
0 0 0 0
a From Shewale and Sadana. j b CMCase, Endo-fl-glucanase. One unit of endo-/3-glucanase activity is defined as the amount of enzyme that releases 180/.~gof reducing sugars (expressed as glucose equivalent) per minute from carboxymethylcellulose. form of each enzyme shows one protein band on SDS-gel electrophoresis w i t h r e l a t i v e m o l e c u l a r m a s s e s c o r r e s p o n d i n g to n a t i v e p r o t e i n s . Chemical Properties. A l l f o u r S. rolfsii f l - g l u c o s i d a s e s a r e g l y c o p r o t e i n s . M o n i t o r i n g t h e v a l u e s o f t h e M i c h a e l i s c o n s t a n t (Kin) a n d m a x i m u m v e l o c i t y (Vma×) a s a f u n c t i o n o f p H f o r BG-3 s u g g e s t s i n v o l v e m e n t o f a c a r b o x y l a t e g r o u p in t h e f o r m a t i o n a n d d i s s o c i a t i o n o f t h e e n z y m e - s u b strate complex. Enzymatic Properties. T h e optimum p H a n d temperature for activity f o r all f o u r f l - g l u c o s i d a s e s a r e p H 4.2 a n d 68 ° w i t h P N P G as s u b s t r a t e , a n d p H 4.5 a n d 65 ° w i t h c e l l o b i o s e a s s u b s t r a t e , r e s p e c t i v e l y . T h e a c t i v a t i o n e n e r g i e s , c a l c u l a t e d f r o m A r r h e n i u s p l o t s , a r e 12.2, 14.9, 11.5, a n d 18.3 k c a l / m o l w i t h P N P G a n d 6.5, 7.6, 6.9, a n d 6.4 k c a l / m o l w i t h c e l l o b i o s e as substrate for BG-1, BG-2, BG-3, and BG-4 enzymes, respectively.
430
CELLULOSE
[49]
Kinetics. The Km values for PNPG, p-nitrophenyl fl-D-cellobioside, cellobiose, and cellodexterins are given in Table II. The Vmaxvalues for cellobiose as substrate, calculated from Lineweaver-Burk plots, for BG-I, BG-2, BG-3, and BG-4/3-glucosidases are 55, 78, 175, and 51/~mol glucose/mg protein/min at 65°, pH 4.5. The gm values of all four /3glucosidases decrease with the chain length of the cellodextrins upto cellopentaose. Inhibitors. Glucose, glucono-l,5-8-1actone, and nojirimycin inhibit all four S. rolfsii fl-glucosidases. The Ki values of BG-3/3-glucosidase, with cellobiose as substrate, for glucose, glucono-l,5-8-1actone, and nojirimycin are 0.55 mM, 0.01 mM, and 1.0/.~M, respectively. The ratio of Km to Ki for glucono-l,5-8-1actone, 530, and for nojirimycin, 5840, suggests that these are strong inhibitors of cellobiase activity. The inhibitory effect of glucose is more marked with cellobioase as substrate when compared with higher molecular weight cellodextrins and decreases with the increase in chain length of cellodextrins. Specificity. The specificity of the enzyme is not restricted to the /3-D-(1 ~ 4) linkage, and all four/3-glucosidases hydrolyze disaccharides having/3-D-(1 --~ 6), /3-D-(1 ~ 3) and/3-D-(1 ~ 2) linkages. Laminaribiose [/3-(1 ~ 3)] is hydrolyzed at a faster rate than gentibiose [/3-(1 ~ 6)] and sophorose [/3-(1 ~ 2)], The enzymes require a strictly/3-D-glucopyranosyl configuration for activity. Neither the glucosides with a-configuration nor the galactosides or xylosides are hydrolyzed. The enzymes can tolerate a T A B L E II M1CHAELIS CONSTANTS FOR S. rolfsii fl-D-GLucOSIDASES I - I V ~ B-D-Glucosidases (mM) Substrate
I
II
III
IV
III b
p -Nitrophenyl-fl-D-glucopyranoside p -Nitrophenyl-fl-D-cellobioside Cellobiose Cellotriose Cellotetraose Cellopentaose Cellohexaose
1.07 c 3.65 1.00 0.50 0.49 0.62
1.38 c 3.07 1.23 0.85 0.40 0.37
0.89 0.38 5.84 1.98 0.83 0.76 0.37
0.79 c 4.15 0.70 0.50 0.55 0.67
0.51 0.35 3.65 1.13 0.75 0.60 0.50
Kinetic studies are done with the standard a s s a y s y s t e m s , at 65 ° and p H 4.5, and varying the substrate concentration. Km values are calculated from L i n e w e a v e r - B u r k plots, w h i c h are linear in all cases. F r o m S a d a n a et al. 2 b A s s a y is carried o u t at 65 ° a n d p H 4.5, in the presence o f bovine s e r u m albumin (0.5 nag/ ml). c N o t determined. a
[49]
S. rolfsii
fl-D-GLUCOSIDASES
431
wide variety of aglycon though the rate of hydrolysis depends on the nature of the aglycon moiety. Phenyl-fl-D-glucopyranoside and o- or pnitrophenyl-/3-D-glucopyranosides are hydrolyzed 10-20 times faster than methyl-/3-D-glucopyranoside. Hydrolysis of Cellodextrins and fl-o-Glucans. S. rolfsff fl-glucosidases, as indicated earlier, hydrolyze cellodextrins in addition to cellobiose. The initial higher rates of hydrolysis of cellodextrins up to cellopentaose and lower Km values for higher molecular weight cellodextrins (Table II) indicate cellopentaose as the preferred substrate for all four/3glucosidase enzymes. The reduced cellodextrins, cellotetraitol, and cellopentaitol are hydrolyzed to almost the same extent as those for the corresponding unreduced cellodextrins. Cellobiotol, however, is completely resistant. None of the four/3-glucosidases acts on highly ordered substrates such as Avicel, but the enzymes slowly hydrolyze disordered substrates such as phosphoric acid-treated Avicel and carboxymethylcellulose but no measurable decrease in viscosity of a carboxymethylcellulose solution is observed in presence of these enzymes. Products of Hydrolysis. Analysis of the products formed as a result of enzyme action on cellodextrins, reduced cellodextrins, and phosphoric acid-treated Avicel shows that the fl-glucosidases from S. rolfsii act by cleaving D-glucosyl groups from the nonreducing end of the chain of the substrates. Characterization of/3-Glucosidases. An enzyme having the capacity to attack long-chain polymers, such as phosphoric acid-swollen cellulose, carboxymethylcellulose, and cellodextrins, is classified, according to the definition of Reese et al.,13 as an exoglucanase. The /3-glucosidase enzymes of S. rolfsii, thus, behave rather as exo-/3-D-glucan glucohydrolases. Role of fl-Glucosidase in Cellulose Hydrolysis. The higher reaction rate of the four S. rolfsii/3-glucosidases with higher molecular weight cellodextrins as compared to cellobiose, the decrease in their Km values, and the decrease in the inhibitory effect of D-glucose on the rate of hydrolysis with the increase in chain length of cellodextrins indicate that higher molecular weight cellodextrins, and not cellobiose, are the major route of D-glucose formation from cellulose.
~3 E. T. Reese, A. H. Maguire, and F. W. Parrish, Can. J. Biochem. 46, 25 (1968).
CELLULOSE
[49]
exoenzymes. TM Sequential removal of cellobiose by fl-glucosidase enhances the efficiency of the cellulose degradation process. ~9 Adequate levels of fl-glucosidase activity are essential when glucose is wanted as the major product of cellulose hydrolysis, z° 18 T. M. W o o d a n d S. I. M c C r a e , Proc. Symp. Bioconversion Cell. Subst. Energy, Chem. Microb. Protein, 1st p. 111 (1978). ~9 B. S. M o n t e n e c o u r t a n d D. E. Eveleigh, Appl. Environ. Microbiol. 34, 777 (1977). 2o C. Mishra, M. Rao, R. Seeta, M. C. Srinivasan, and V. D e s h p a n d e , Biotechnol. Bioeng. 26, 370 (1984).
[49] f l - D - G l u c o s i d a s e s f r o m Sclerotium rolfsii
By JAI C. SADANA, RAJKUMARV. PATIL, and JAIPRAKASH G. S H E W A L E Sclerotium rolfsii produces at least four different extracellular fl-glucosidases when grown on cellulose.~,2 Assay Method
Principle. fl-D-Glucosidase (fl-I)-glucoside glucohydrolase, EC 3.2.1.21) activity is measured as the amount of glucose released from cellobiose or p-nitrophenol from p-nitrophenyl-fl-D-glucopyranoside (PNPG). The glucose released is determined by the glucose o×idaseperoxidase test 3 using Glox glucose reagent. The amount ofp-nitrophenol released is calculated from the molar absorbance of 18,500 for nitrophenol at 410 nm. 4 With Cellobiose as Substrate Reagents Citrate buffer, 50 mM, pH 4.5 D(+)-Cellobiose, 50 mM in 50 mM citrate buffer, pH 4.5 J J. G. Shewale a n d J. C. Sadana, Arch. Biochem. Biophys. 207, 185 (1981). 2 j. C. Sadana, J. G. Shewale, and R. V. Patil, Carbohydr. Res. 118, 205 (1983). 3 H. U. B e r g m e y e r , K. G a w e h n , and M. Grassl, Methods Enzymatic Anal. 1, 457 (1974). 4 D. Herr, F. B a u m e r , and H. Dellweg, Eur. J. Appl. Microbiol. Biotechnol. 5, 29 (1978).
METHODS IN ENZYMOLOGY, VOL. 160
Copyright © 1988by AcademicPress. Inc. All rights of reproduction in any form reserved.
[49]
S. rolfsii fl-D-GLUCOSIDASES
425
Glox glucose reagent: one packet of Glox is dissolved in 500 ml of 50 mM phosphate buffer, pH 7.0. The reagent is freshly prepared to avoid high blank values Sulfuric acid, 41% Procedure. The enzyme is diluted just before assay in 50 mM citrate buffer, pH 4.5, to obtain a concentration of 0.05-0.3 unit of enzyme per milliliter (see definition below). The reaction is initiated by adding enzyme. The enzyme (0.1 ml) is added to 0.9 ml of cellobiose solution in a Corning test tube and the reaction mixture is incubated at 65 ° for 30 min. The reaction is stopped by heating the tubes in a boiling water bath for 5 min. After cooling 5 ml of Glox (glucose oxidase-peroxidase) reagent is added and the reaction mixture incubated for 1 hr at 30°. At the end of incubation, 4 ml of 41% H 2 5 0 4 is added and the color developed is measured at 530 nm. The color is developed immediately and is stable for 2 hr. A control is run with boiled enzyme. Under the conditions of assay, the enzyme shows linearity up to 0.4 mg of glucose produced.
With P N P G as Substrate Reagents Citrate buffer, 50 mM, pH 4.2 p-Nitrophenyl-/3-D-glucopyranoside (PNPG), 1 mg/ml in 50 mM citrate buffer, pH 4.2 Sodium carbonate, 2% in distilled water Procedure. The enzyme is diluted just before assay in 50 mM citrate buffer, pH 4.2, to obtain a concentration of 0.01-0.05 unit of enzyme per milliliter (see definition below). The reaction is initiated by adding 0.1 ml of enzyme to 0.9 ml of PNPG in a Coming test tube. The reaction mixture is incubated at 68 ° for 30 min. The reaction is stopped by adding 1 ml of 2% NazCO3 and the color developed is measured at 410 nm. The color is developed immediately after addition of sodium carbonate and is stable for at least 24 hr. A control is run with boiled enzyme. The enzyme shows linearity up to 12/xg of p-nitrophenol produced. Definitions o f Unit and Specific Activity. One unit of/3-D-glucosidase activity is defined as the amount of enzyme that releases 1 t~mol of glucose from cellobiose at 65 °, pH 4.5 or I/xmol ofp-nitrophenol from PNPG at 68 °, pH 4.2, per minute. In the assay with cellobiose, 2 mol of glucose is released from 1 tool of cellobiose but the glucose values have been given as such without dividing by a factor of 2. This is according to the recom5 Glox, glucose reagent (glucose oxidase-peroxidase reagent), was purchased from AB Kabi Diagnostica, Stockholm, Sweden.
426
CELLULOSE
[49]
mendations of the Commission on Biotechnology, International Union of Pure and Applied Chemistry. 6 The specific activity is defined as the number of units per milligram of protein. Protein is determined by the procedure of Lowry et al. 7
Purification Procedure Three of the purification steps, ammonium sulfate precipitation (step I), fractionation by gel chromatography on Sephadex G-75 (step 2), and ultrafiltration of Fraction A (step 3) are identical to the steps for cellobiose dehydrogenase as described in this volume [53]. During gel filtration on Sephadex G-75 (step 2)/3-glucosidase is eluted along with some highmolecular-weight cellulases, after the void volume (Fraction A) and ahead of low-molecular-weight cellulases (Fraction B). Fraction A (110-165 ml) contains about 95-98% of fl-glucosidase activity and about 70% endo-flglucanase activity, whereas Fraction B (170-265 ml) contains about 30% cellulase and 2%/3-glucosidase activity. Step 4. DEAE-Sephadex A-50 Ion-Exchange Chromatography. Fraction A after concentration on Amicon (step 3) is dialyzed in a collodion bag against 0.05 M phosphate buffer, pH 7.3, and chromatographed on DEAE-Sephadex column (1.8 x 100 cm) equilibrated with 0.05 M phosphate buffer, pH 7.3. The column is washed with the equilibration buffer. Fractions of 2 ml are collected at a flow rate of 12-15 ml/hr./3-Glucosidase and endo-/3-glucanase activities are not adsorbed on the column. fl-Glucosidase comes just after the void volume and forms the first peak; it is almost free of endo-fl-glucanase (Fig. 1). The dark brown pigment present in the culture filtrate is removed in this step. Fractions 6-7 containing fl-glucosidase of 44-50 specific activity are pooled and the pH is adjusted to 4.5 with 0.1 M citric acid. This is freeze-dried and then redissolved in 5 ml of 0.05 M citrate buffer, pH 4.5, and dialyzed against the same buffer. The fl-glucosidase fraction at this stage gives one band in disc gel electrophoresis at pH 8.9. However, electrophoresis of the enzyme at pH 4.3 resolves it into four bands of proteins. Step 5. Preparative Isoelectric Focusing. The fl-glucosidase (Fractions 6-7) from step 4 is dialyzed overnight against 1 mM citrate buffer, pH 4.5, to reduce the salt concentration and is purified further by preparative isoelectric focusing. A gradient of sucrose from 50 to 5% is prepared
6 I U P A C , Comm. Biotechn. 59, 257 (1987).
70. H. Lowry, N. J. Rosebrough, A. L. Fan', and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
S. rolfsii fl-D-OLUCOSIDASES
[49]
427
100 I
700 600
II
~ C
//O~.CM
80
MCcise
E
500. E
"" D
~60
"" 400 • L O t~
40 E
c13
o 30o u
200
3o
, 8 4o
2o ~_
& ZO
100
0
0
~
,
4
8
12
0
16
20
FRACTION NUMBER FIG. I. Ion-exchangechromatography on DEAE-Sepbadex A-50 of Fraction A from step 3. Peak I comprises fractions 6-7 and Peak II Fractions 14-18. (O)/3-D-Olucosidase (BO),
(0) CMCase (endo-/3-glucanase), (A) protein. From Shewale and Sadana. ~
in a 110 ml LKB electrofocusing column. 8 Ampholyte solution (final concentration of I%), pH 4-6, and/3-glucosidase enzyme solution are mixed with light and dense solutions before making the gradient. The anode solution contains 0.16 M phosphoric acid in 60% sucrose, whereas the cathode solution employs 0.25 N sodium hydroxide. The cathode is at the top during the run. The voltage at the end of run (72 hr) is 500 V and the current 2 mA. Fractions of 1 ml are collected and are immediately processed for pH (5-7°), activity, and protein determination. Enzyme is freed from sucrose by extensive dialysis against 0.05 M citrate buffer, pH 4.5. fl-Glucosidase activity is resolved into four separate peaks at pH 4.10, 4.55, 5.10, and 5.55 (Fig. 2) and they are designated BG-1, BG-2, BG-3, and BG-4, respectively. The observation is reproducible. The results of a typical purification of fl-glucosidase enzymes are given in Table I. Purity. The purified enzymes individually show one protein band on polyacrylamide gel electrophoresis at pH 4.3 and 8.9, in the presence or absence of SDS, and on isoelectric focusing in 7.5% polyacrylamide gel s
Instruction manual, LKB Preparative Isoelectric Focusing, Uppsala, Sweden.
428
CELLULOSE
[49] 6.0
2SO
i
E
5.0
20O
4.0
-~
)so
o.2s
uJ
Protei~ 0.20 uJ
~00, o.~o m,
50
0 20
03 n~ 0
<
i
30
,,
.
~
i
40
50
0.00 60
FRACTION NUMBER
FIG. 2. Isoelectric focusing of Peak I from DEAE-Sephadex chromatography. ((3) fl-Glucosidase, (A) absorbance at 280 nm, (0) pH. From Shewale and Sadana.
over the pH range 3.5-10. The purified fl-glucosidases are free of contaminating endo-fl-glucanase activity as determined viscometrically. 9 Stability. fl-Glucosidases are stable for several months when stored in 50 mM citrate buffer, pH 4.5, at -15 °, and on repeated freezing and thawing. Properties
Physical Properties. The relative molecular masses of the BG-1, BG-2, BG-3, and BG-4 /3-glucosidases, estimated by gel filtration on BioGel P-150 according to the procedure of Andrews, 1° are 90,000, 90,000, 107,000, and 92,000, respectively, and by migration in SDSpolyacrylamide gel N are 95,500, 95,500, 106,000, and 95,000, respectively. The molecular weight of BG-3 fl-glucosidase, determined by electrophoresis using the slope method, 12 is 100,000. Subunit Structure. The four/3-glucosidases from S. rolfsii are comprised of single polypeptide chains since the reduced carboxymethylated 9 p. L. Hurst, J. Nielsen, P. A. Sullivan, and M. G. Shepherd, Biochem. J. 165, 33 (1977). 10 p. Andrews, Biochem. J. 91, 222 (1964). 11 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). 12 j. L. Hedrick and A. J. Smith, Arch. Biochem. Biophys. 126, 155 (1968).
S. rolfsii /3-O-GLOCOSIDASES
[49]
429
TABLE I PURIFICATIONOF fl-GLucOSIDASESFROMS. rolfsii~ fl-Glucosidase
Fraction Culture filtrate Ammonium sulfate, 0-3.4 M saturation Sephadex G-75 Fraction A Fraction B Ultrafiltration of fraction A (Amicon XM-50) DEAE-Sephadex A-50 Peak I Peak II Preparative isoelectric focusing of Peak I BG-1 BG-2 BG-3 BG-4
Total protein (mg)
Total units
Specific activity (U/mg Recovery protein) (%)
16,150 10,750
17,720 15,930
1.1 1.4
7,580 918 3,800
11,400 74 9,210
1.5 0.004 2.4
23 550
1,125 127
0.37 0.20 1.80 0.59
10 10 120 14
CMCase b
100 90
64 0.4 52
Specific activity (U/mg Recovery protein) (%)
Total units 355,488 284,740
22 26
100 80
109,290 49,680 57,260
14 54 15
30 14 16
48 0.23
6.3 0.71
110 28,359
27 50 57 25
0.05 0.05 0.57 0.07
0 0 0 0
4.7 51
0 0 0 0
0.03 7.9
0 0 0 0
a From Shewale and Sadana. j b CMCase, Endo-fl-glucanase. One unit of endo-/3-glucanase activity is defined as the amount of enzyme that releases 180/.~gof reducing sugars (expressed as glucose equivalent) per minute from carboxymethylcellulose. form of each enzyme shows one protein band on SDS-gel electrophoresis w i t h r e l a t i v e m o l e c u l a r m a s s e s c o r r e s p o n d i n g to n a t i v e p r o t e i n s . Chemical Properties. A l l f o u r S. rolfsii f l - g l u c o s i d a s e s a r e g l y c o p r o t e i n s . M o n i t o r i n g t h e v a l u e s o f t h e M i c h a e l i s c o n s t a n t (Kin) a n d m a x i m u m v e l o c i t y (Vma×) a s a f u n c t i o n o f p H f o r BG-3 s u g g e s t s i n v o l v e m e n t o f a c a r b o x y l a t e g r o u p in t h e f o r m a t i o n a n d d i s s o c i a t i o n o f t h e e n z y m e - s u b strate complex. Enzymatic Properties. T h e optimum p H a n d temperature for activity f o r all f o u r f l - g l u c o s i d a s e s a r e p H 4.2 a n d 68 ° w i t h P N P G as s u b s t r a t e , a n d p H 4.5 a n d 65 ° w i t h c e l l o b i o s e a s s u b s t r a t e , r e s p e c t i v e l y . T h e a c t i v a t i o n e n e r g i e s , c a l c u l a t e d f r o m A r r h e n i u s p l o t s , a r e 12.2, 14.9, 11.5, a n d 18.3 k c a l / m o l w i t h P N P G a n d 6.5, 7.6, 6.9, a n d 6.4 k c a l / m o l w i t h c e l l o b i o s e as substrate for BG-1, BG-2, BG-3, and BG-4 enzymes, respectively.
430
CELLULOSE
[49]
Kinetics. The Km values for PNPG, p-nitrophenyl fl-D-cellobioside, cellobiose, and cellodexterins are given in Table II. The Vmaxvalues for cellobiose as substrate, calculated from Lineweaver-Burk plots, for BG-I, BG-2, BG-3, and BG-4/3-glucosidases are 55, 78, 175, and 51/~mol glucose/mg protein/min at 65°, pH 4.5. The gm values of all four /3glucosidases decrease with the chain length of the cellodextrins upto cellopentaose. Inhibitors. Glucose, glucono-l,5-8-1actone, and nojirimycin inhibit all four S. rolfsii fl-glucosidases. The Ki values of BG-3/3-glucosidase, with cellobiose as substrate, for glucose, glucono-l,5-8-1actone, and nojirimycin are 0.55 mM, 0.01 mM, and 1.0/.~M, respectively. The ratio of Km to Ki for glucono-l,5-8-1actone, 530, and for nojirimycin, 5840, suggests that these are strong inhibitors of cellobiase activity. The inhibitory effect of glucose is more marked with cellobioase as substrate when compared with higher molecular weight cellodextrins and decreases with the increase in chain length of cellodextrins. Specificity. The specificity of the enzyme is not restricted to the /3-D-(1 ~ 4) linkage, and all four/3-glucosidases hydrolyze disaccharides having/3-D-(1 --~ 6), /3-D-(1 ~ 3) and/3-D-(1 ~ 2) linkages. Laminaribiose [/3-(1 ~ 3)] is hydrolyzed at a faster rate than gentibiose [/3-(1 ~ 6)] and sophorose [/3-(1 ~ 2)], The enzymes require a strictly/3-D-glucopyranosyl configuration for activity. Neither the glucosides with a-configuration nor the galactosides or xylosides are hydrolyzed. The enzymes can tolerate a T A B L E II M1CHAELIS CONSTANTS FOR S. rolfsii fl-D-GLucOSIDASES I - I V ~ B-D-Glucosidases (mM) Substrate
I
II
III
IV
III b
p -Nitrophenyl-fl-D-glucopyranoside p -Nitrophenyl-fl-D-cellobioside Cellobiose Cellotriose Cellotetraose Cellopentaose Cellohexaose
1.07 c 3.65 1.00 0.50 0.49 0.62
1.38 c 3.07 1.23 0.85 0.40 0.37
0.89 0.38 5.84 1.98 0.83 0.76 0.37
0.79 c 4.15 0.70 0.50 0.55 0.67
0.51 0.35 3.65 1.13 0.75 0.60 0.50
Kinetic studies are done with the standard a s s a y s y s t e m s , at 65 ° and p H 4.5, and varying the substrate concentration. Km values are calculated from L i n e w e a v e r - B u r k plots, w h i c h are linear in all cases. F r o m S a d a n a et al. 2 b A s s a y is carried o u t at 65 ° a n d p H 4.5, in the presence o f bovine s e r u m albumin (0.5 nag/ ml). c N o t determined. a
[49]
S. rolfsii
fl-D-GLUCOSIDASES
431
wide variety of aglycon though the rate of hydrolysis depends on the nature of the aglycon moiety. Phenyl-fl-D-glucopyranoside and o- or pnitrophenyl-/3-D-glucopyranosides are hydrolyzed 10-20 times faster than methyl-/3-D-glucopyranoside. Hydrolysis of Cellodextrins and fl-o-Glucans. S. rolfsff fl-glucosidases, as indicated earlier, hydrolyze cellodextrins in addition to cellobiose. The initial higher rates of hydrolysis of cellodextrins up to cellopentaose and lower Km values for higher molecular weight cellodextrins (Table II) indicate cellopentaose as the preferred substrate for all four/3glucosidase enzymes. The reduced cellodextrins, cellotetraitol, and cellopentaitol are hydrolyzed to almost the same extent as those for the corresponding unreduced cellodextrins. Cellobiotol, however, is completely resistant. None of the four/3-glucosidases acts on highly ordered substrates such as Avicel, but the enzymes slowly hydrolyze disordered substrates such as phosphoric acid-treated Avicel and carboxymethylcellulose but no measurable decrease in viscosity of a carboxymethylcellulose solution is observed in presence of these enzymes. Products of Hydrolysis. Analysis of the products formed as a result of enzyme action on cellodextrins, reduced cellodextrins, and phosphoric acid-treated Avicel shows that the fl-glucosidases from S. rolfsii act by cleaving D-glucosyl groups from the nonreducing end of the chain of the substrates. Characterization of/3-Glucosidases. An enzyme having the capacity to attack long-chain polymers, such as phosphoric acid-swollen cellulose, carboxymethylcellulose, and cellodextrins, is classified, according to the definition of Reese et al.,13 as an exoglucanase. The /3-glucosidase enzymes of S. rolfsii, thus, behave rather as exo-/3-D-glucan glucohydrolases. Role of fl-Glucosidase in Cellulose Hydrolysis. The higher reaction rate of the four S. rolfsii/3-glucosidases with higher molecular weight cellodextrins as compared to cellobiose, the decrease in their Km values, and the decrease in the inhibitory effect of D-glucose on the rate of hydrolysis with the increase in chain length of cellodextrins indicate that higher molecular weight cellodextrins, and not cellobiose, are the major route of D-glucose formation from cellulose.
~3 E. T. Reese, A. H. Maguire, and F. W. Parrish, Can. J. Biochem. 46, 25 (1968).