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Endoglucanase from Clostridium thermocellum
[38]
ENDOGLUCANASE FROMC. thermocellum
351
the abscission zone. In bean, it appears to function in the shedding of various organs such as fruits, flowers, and leaves. Acidic Cellulases. Less is known about the acidic forms of cellulase, since they have not been purified sufficiently. Their pH optima are in the 5.0-6.0 range. Their isoelectric points are 4.5 and 4.8. They are induced by auxin 9 and appear in high levels in young, rapidly expanding tissue. 5 These cellulases may function in loosening the cellulose fibrils of the cell wall to allow expansion and growth. Membrane-Bound Cellulase. Cellulase is also found to be associated with the plasma membrane. 3 It is present before abscission, but declines in the abscission zone during the abscission process. The membrane cellulase has a pI of 4.5 and a pH optimum of 5.1. It is believed to be a glycoprotein, because it binds to Sepharose-concanavalin A and can be eluted with 0.2 M methyl a-D-mannopyranoside. The enzyme is partially inhibited by dextran, 14 and is strongly inhibited by D-gluconic acid lactone. The function of this form of cellulase and its relation to the soluble forms of acidic cellulase are not yet known. ~4 E. del Campillo and L. N. Lewis, J. Cell. Biochem., Suppl. 10B, 20 (1986).
[38] E n d o g l u c a n a s e f r o m C l o s t r i d i u m t h e r m o c e l l u m By THOMAS K. NG and J. G. ZEIKUS Cellulose + H20 ~ cellodextrins
Clostridium thermocellum produces several endoglucanases which are part of a cellulase complex termed cellulosome.1 Five of the endoglucanases have been purified and characterized. 2-6 Most interestingly, two of these endoglucanases from C. thermocellum are produced by Escherichia coli strains carrying the genes that encode for the proteins. 5,6 The nucleotide sequence of the genes has also been determined. 7,8 Although i R. Lamed and E. A. Bayer, this volume [57]. 2 T. K. Ng and J. G. Zeikus, Biochem. J. 199, 341 (1981). 3 N. Creuzet and C. Frixon, Biochimie 65, 149 (1983). 4 j. P~tre, R. Longin, and J. Millet, Biochimie 63, 629 (1981). P. B6guin, P. Comet, and J. Millet, Biochimie 65, 495 (1983). 6 D. Petre, J. Millet, R. Longin, P. B6guin, H. Girard, and J.-P. Aubert, Biochirnie 68, 687 (1986). 7 p. B6guin, P. Cornet, and J.-P. Aubert, J. Bacteriol. 162, 102 (1985). O. Grepinet and P. B6guin, Nucleic Acids Res. 14, 1791 (1986).
METHODS IN ENZYMOLOGY, VOL. 160
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
352
CELLULOSE
[38]
an exoglucanase from C. thermocellum has not been identified, all the endoglucanases reported so far contain exoglucanase activity. 2-6 One of these five endoglucanases, termed endoglucanase I, will be described here. Assay Method
Principle. The formation of free, reducing C-1 hydroxyl groups generated by internal cleavage of/3-1,4 bonds is measured with dinitrosalicylic acid (DNS) using glucose as the standard. Reagents Carboxymethylcellulose 7HS (Hercules) DNS (3,5-dinitrosalicylic acid) reagent: 10 g NaOH, 200 g potassium sodium tartrate, 2 g phenol, 0.5 g sodium sulfite, and l0 g DNS dissolved in the given order in I liter of distilled water. The reagent is stored under nitrogen gas in the dark at 4° for up to 3 months 0.05 M sodium acetate, pH 5.0, with 0.03% NaN3 Procedure. Before the assay, a 1% solution of carboxymethylcellulose in acetate buffer is prepared by stirring the suspension at room temperature for 24 hr. Aliquots of 1 ml are dispensed into 13 x 100-mm tubes and kept frozen at - 2 0 °. The reaction is started by the addition of 1 ml enzyme solution to test tubes with the frozen substrate. Tubes with enzyme are vortexed and incubated at 60° for 5, 10, and 15 min. The reaction is stopped by immersion in an ice-water bath and the addition of 3 ml DNS reagent. The tubes are then heated in a boiling water bath for 15 min and cooled immediately to 4 ° in an ice-water bath. The intensity of the color is measured spectrophotometrically at 640 nm. The amount of reducing sugars is determined using glucose as the standard. Definition of Unit and Specific Activity. One unit of endoglucanase is defined as the micromoles of reducing sugars as glucose equivalent released per minute under the conditions described. Specific activity is expressed as units per milligram of protein determined by the Lowry procedure. 9 Purification Procedure All operations except where otherwise specified are performed at 0 4°. All buffers contain 3/xM of dithiothreitol. 9 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
[38]
ENDOGLUCANASE FROMC. thermocellum
353
Step 1. Growth of Bacteria. Clostridium thermocellum strain LQRI (available as strain DSM 2360, German Culture Collection, G6ttingen, FRG) is cultured under strict anaerobic condition as described by Ng et al. ~oFor the purification of endoglucanase I, C. thermocellum is grown in a 14-liter fermenter with 12 liters of medium for 24 hr at 60° under constant stirring at 100 rpm and gassing with oxygen-free nitrogen at 100 ml/min. The culture medium contains per liter of distilled water: K2HPO4, 0.5 g; KHzPO4 • 3HzO, 1.0 g; urea, 2 g; MgCI2 • 6H20, 0.5 g; CaClz • 2H20, 0.05 g; FeSO4-5H20, 0.5 mg; 4-morpholinepropanesulfonic acid, 10 g; cysteine hydrochloride hydrate, 1 g; cellobiose, 5 g; and yeast extract, 2 g. Yeast extract used in the medium is pretreated by diafiltration to remove proteins with molecular weight greater than 10,000. 2 Step 2. Preparation of Crude Enzyme. After 24 hr of growth, the culture broth is centrifuged at 10,000 g for 30 min and the supernatant is concentrated 10-fold by ultrafiltration with a 10,000 molecular weight cutoff membrane. Ammonium sulfate is added to the concentrated supernatant with gentle stirring at 80% of saturation (0.516 g/ml). After 30 min, the precipitate is collected by centrifugation, redissolved in minimal volume of 20 mM ammonium acetate buffer, pH 5.0, and dialyzed exhaustively by ultrafiltration against the same buffer. Step 3. Ion-Exchange Chromatography. Dialyzed crude enzyme (400 ml with 3.34 g of protein) is applied to a 4.5 × 50 cm column of DEAESephadex A-50 preequilibrated in 50 mM ammonium acetate buffer, pH 5.0. The column is eluted in steps with 750 ml of 50 mM, 600 ml of 100 mM, 500 ml of 600 mM, and 750 ml of 800 mM of the same buffer. Fractions eluted at 50 mM (270-830 ml) which contain the highest specific and total endoglucanase activity are pooled, concentrated by ultrafiltration, and reapplied to a 2.6 x 30 cm column of DEAE-Sephadex A-50 in 50 mM Tris-HC1 buffer, pH 7.2. The column is eluted with a stepwise increase of NaCI (600 ml of 0 mM, 800 ml of 50 mM, 600 ml of 100 mM, and 600 ml of 150 mM) in the same buffer. The protein eluted at 50 mM NaCI contains 60% of the total endoglucanase activity. Fractions are pooled and refractionated on a 2.6 x 30 cm SP-Sephadex C-50 column in 20 mM sodium citrate buffer, pH 3.5. The column is eluted with a continuous NaCI gradient (0-300 mM) in 400 ml of citrate buffer and protein with the highest activity elutes at approximately 200 mM NaCI. The fractions are pooled, dialyzed against 50 mM sodium acetate buffer, pH 5.0, and concentrated 20-fold by ultrafiltration. Step 4. Column Electrophoresis. Electrophoretic fractionation is performed with a preparative electrophoresis apparatus described else10 T. K. Ng, P. J. Weimer, and J. G. Zeikus, Arch. Microbiol. 114, 1 (1977).
354
CELLULOSE
[38]
TABLE I PURIFICATION OF ENDOGLUCANASEI FROM C. t h e r m o c e l l u m a
Procedure Cell-free supernatant Ultrafiltrate
(NH4)2SO4 ppt. DEAE-Sephadex A50-1 DEAE-Sephadex A50-II SP-Sephadex C-50 Preparative gel electrophoresis a
Volume (ml)
Total activity (units)
Total protein (mg)
Specific activity (units/mg)
Yield (%)
12,000 1,000 400 400 60 25 10
20,300 17,800 13,800 6,400 2,750 975 514
6,720 4,750 3,370 624 119 21 7.9
3.0 3.7 4.2 10.3 23.0 45.9 65.1
-87.6 68.3 31.6 13.6 4.8 2.5
From Ng and Zeikus. 2 Reprinted by permission of the Biochemical Society, London.
where. ~t.12A 4 cm length of 8% separating gel with a 0.5 cm section of 4% stacking gel is used. The polyacrylamide gels are prepared by mixing solutions of 0.3 M Tris-HCl, pH 7.5, buffer with 0.23% of N,N,N',N'tetramethylenediamine, 40% acrylamide with 0.8% of N,N'-methylenebisacrylamide, water, and 0.14% ammonium persulfate in the ratio of 1 : 2 : I : 4 for 8% gel and 1 : 1 : 2 : 4 for 4% gel. The upper reservoir buffer consists of 0.8 M Tris-sulfate, pH 8.0, and the lower reservoir buffer is twice that strength. Both reservoirs are fed constantly with buffer at 1.0 ml/min, while the elution chamber is swept with 0.36 M Tris-HC1 buffer, pH 7.6, at 1.0 ml/min. A 2-ml sample with 43 mg of protein in 10% glycerol is applied to the column and the potential across the electrodes is set at 400 V ( - 3 4 mA); 2.5-ml fractions are collected. After 16 hr, endoglucanase I is eluted between fractions 384 and 389. The purification procedure is summarized in Table I. A 22-fold increase in specific activity with 2.5% recovery in total activity is achieved. Properties
Physical Properties. The endoglucanase I is a glycoprotein with - 1 1 % carbohydrate. The molecular weights determined by sedimentation equilibrium analysis, amino acid composition analysis, and polyacrylamide gel electrophoresis are 83,000, 88,000, and 94,000, respectively. The molar absorption coefficient at 280 nm is 53,750 M -~ cm -1 and the isoelectric ii T. Jovin, A. Chambach, and M. A. Naughton, A n a l . B i o c h e m . 9, 351 (1964). ~2A commercial apparatus is available as PolyPrep 200 from Buchler, Fort Lee. New Jersey.
ENDOGLUCANASE D OF C. thermocellum
[39]
355
point is 6.72. The pH and temperature optima are 5.2 and 62°. The enzyme contains 2.5% methionine but no cysteine. Specificity. The enzyme catalyzes most actively the hydrolysis of carboxymethylceUulose. Activity toward cellodextrins increases with the degree of polymerization, with cellopentaose having the highest activity. Cellotriose and cellobiose are not hydrolyzed. The enzyme-substrate complex formation prefers cellotriosyl units over cellobiosyl units and that from glucosyl units is rare. Avicel and acid-swollen cellulose are attacked but the activity is very low. Kinetics Constants. The [S]0.sv and V m a x for cellopentaose and cellohexaose are 2.30 × 10 -3 M, 39.3/xmol/min/mg protein (54.5 S -l assuming a molecular weight of 83,000), and 0.56 × 10-3 M, 58.7 /xmol/min/mg protein (81.2 S - 1), respectively.
[39] C r y s t a l l i n e E n d o g l u c a n a s e D o f Clostridiurn thermocellurn O v e r p r o d u c e d in Escherichia coli
By PIERRE BI~GUIN, GWENNA~L JOLIFF, MICHEL JUY, ADOLFO G. AMIT, JACQUELINE MILLET, ROBERTO J. POLIAK, and JEAN-PAUL AUBERT Introduction
Clostridium thermocellum, a thermophilic anaerobe, has been shown to produce a very active cellulase complex I composed of a large number of individual enzymes. 2 Attempts to resolve the complex into its active subunits have not been successful and only two endoglucanases (EG) (EC 3.2.1.4, cellulase) have been purified from culture supernatant. 3,4 Construction of C. thermocellum genomic libraries in Escherichia c o l i 5-7 showed that, as a general rule, C. thermocellum genes were readily expressed in E. coli. Consequently, it appeared that it was easier to purify E. A. Johnson, M. Sakajoh, G. Halliwell, A. Madia, and A. Demain, Appl. Environ. Microbiol. 43, 1125 (1982). 2 R. Lamed, E. Setter, and E. A. Bayer, J. Bacteriol. 156, 828 (1983). 3 j. P6tre, R. Longin, and J. Millet, Biochimie 63, 629 (1981). 4 T. K. Ng and J. G. Zeikus, Biochem. J. 199, 341 (1981). 5 p. Comet, D. Tronik, J. Millet, and J.-P. Aubert, FEMS Microbiol. Lett. 16, 137 (1983). 6 j. Millet, D. P6tr6, P. B6guin, O. Raynaud, and J.-P. Aubert, FEMS Microbiol. Lett. 29, 145 (1985). 7 W. Schwarz, K. Bronnenmeier, and W. L. Staudenbauer, Biotechnol. Lett. 7, 859 (1985).
METHODS IN ENZYMOLOGY, VOL. 160
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENDOGLUCANASE FROMC. thermocellum
351
the abscission zone. In bean, it appears to function in the shedding of various organs such as fruits, flowers, and leaves. Acidic Cellulases. Less is known about the acidic forms of cellulase, since they have not been purified sufficiently. Their pH optima are in the 5.0-6.0 range. Their isoelectric points are 4.5 and 4.8. They are induced by auxin 9 and appear in high levels in young, rapidly expanding tissue. 5 These cellulases may function in loosening the cellulose fibrils of the cell wall to allow expansion and growth. Membrane-Bound Cellulase. Cellulase is also found to be associated with the plasma membrane. 3 It is present before abscission, but declines in the abscission zone during the abscission process. The membrane cellulase has a pI of 4.5 and a pH optimum of 5.1. It is believed to be a glycoprotein, because it binds to Sepharose-concanavalin A and can be eluted with 0.2 M methyl a-D-mannopyranoside. The enzyme is partially inhibited by dextran, 14 and is strongly inhibited by D-gluconic acid lactone. The function of this form of cellulase and its relation to the soluble forms of acidic cellulase are not yet known. ~4 E. del Campillo and L. N. Lewis, J. Cell. Biochem., Suppl. 10B, 20 (1986).
[38] E n d o g l u c a n a s e f r o m C l o s t r i d i u m t h e r m o c e l l u m By THOMAS K. NG and J. G. ZEIKUS Cellulose + H20 ~ cellodextrins
Clostridium thermocellum produces several endoglucanases which are part of a cellulase complex termed cellulosome.1 Five of the endoglucanases have been purified and characterized. 2-6 Most interestingly, two of these endoglucanases from C. thermocellum are produced by Escherichia coli strains carrying the genes that encode for the proteins. 5,6 The nucleotide sequence of the genes has also been determined. 7,8 Although i R. Lamed and E. A. Bayer, this volume [57]. 2 T. K. Ng and J. G. Zeikus, Biochem. J. 199, 341 (1981). 3 N. Creuzet and C. Frixon, Biochimie 65, 149 (1983). 4 j. P~tre, R. Longin, and J. Millet, Biochimie 63, 629 (1981). P. B6guin, P. Comet, and J. Millet, Biochimie 65, 495 (1983). 6 D. Petre, J. Millet, R. Longin, P. B6guin, H. Girard, and J.-P. Aubert, Biochirnie 68, 687 (1986). 7 p. B6guin, P. Cornet, and J.-P. Aubert, J. Bacteriol. 162, 102 (1985). O. Grepinet and P. B6guin, Nucleic Acids Res. 14, 1791 (1986).
METHODS IN ENZYMOLOGY, VOL. 160
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
352
CELLULOSE
[38]
an exoglucanase from C. thermocellum has not been identified, all the endoglucanases reported so far contain exoglucanase activity. 2-6 One of these five endoglucanases, termed endoglucanase I, will be described here. Assay Method
Principle. The formation of free, reducing C-1 hydroxyl groups generated by internal cleavage of/3-1,4 bonds is measured with dinitrosalicylic acid (DNS) using glucose as the standard. Reagents Carboxymethylcellulose 7HS (Hercules) DNS (3,5-dinitrosalicylic acid) reagent: 10 g NaOH, 200 g potassium sodium tartrate, 2 g phenol, 0.5 g sodium sulfite, and l0 g DNS dissolved in the given order in I liter of distilled water. The reagent is stored under nitrogen gas in the dark at 4° for up to 3 months 0.05 M sodium acetate, pH 5.0, with 0.03% NaN3 Procedure. Before the assay, a 1% solution of carboxymethylcellulose in acetate buffer is prepared by stirring the suspension at room temperature for 24 hr. Aliquots of 1 ml are dispensed into 13 x 100-mm tubes and kept frozen at - 2 0 °. The reaction is started by the addition of 1 ml enzyme solution to test tubes with the frozen substrate. Tubes with enzyme are vortexed and incubated at 60° for 5, 10, and 15 min. The reaction is stopped by immersion in an ice-water bath and the addition of 3 ml DNS reagent. The tubes are then heated in a boiling water bath for 15 min and cooled immediately to 4 ° in an ice-water bath. The intensity of the color is measured spectrophotometrically at 640 nm. The amount of reducing sugars is determined using glucose as the standard. Definition of Unit and Specific Activity. One unit of endoglucanase is defined as the micromoles of reducing sugars as glucose equivalent released per minute under the conditions described. Specific activity is expressed as units per milligram of protein determined by the Lowry procedure. 9 Purification Procedure All operations except where otherwise specified are performed at 0 4°. All buffers contain 3/xM of dithiothreitol. 9 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
[38]
ENDOGLUCANASE FROMC. thermocellum
353
Step 1. Growth of Bacteria. Clostridium thermocellum strain LQRI (available as strain DSM 2360, German Culture Collection, G6ttingen, FRG) is cultured under strict anaerobic condition as described by Ng et al. ~oFor the purification of endoglucanase I, C. thermocellum is grown in a 14-liter fermenter with 12 liters of medium for 24 hr at 60° under constant stirring at 100 rpm and gassing with oxygen-free nitrogen at 100 ml/min. The culture medium contains per liter of distilled water: K2HPO4, 0.5 g; KHzPO4 • 3HzO, 1.0 g; urea, 2 g; MgCI2 • 6H20, 0.5 g; CaClz • 2H20, 0.05 g; FeSO4-5H20, 0.5 mg; 4-morpholinepropanesulfonic acid, 10 g; cysteine hydrochloride hydrate, 1 g; cellobiose, 5 g; and yeast extract, 2 g. Yeast extract used in the medium is pretreated by diafiltration to remove proteins with molecular weight greater than 10,000. 2 Step 2. Preparation of Crude Enzyme. After 24 hr of growth, the culture broth is centrifuged at 10,000 g for 30 min and the supernatant is concentrated 10-fold by ultrafiltration with a 10,000 molecular weight cutoff membrane. Ammonium sulfate is added to the concentrated supernatant with gentle stirring at 80% of saturation (0.516 g/ml). After 30 min, the precipitate is collected by centrifugation, redissolved in minimal volume of 20 mM ammonium acetate buffer, pH 5.0, and dialyzed exhaustively by ultrafiltration against the same buffer. Step 3. Ion-Exchange Chromatography. Dialyzed crude enzyme (400 ml with 3.34 g of protein) is applied to a 4.5 × 50 cm column of DEAESephadex A-50 preequilibrated in 50 mM ammonium acetate buffer, pH 5.0. The column is eluted in steps with 750 ml of 50 mM, 600 ml of 100 mM, 500 ml of 600 mM, and 750 ml of 800 mM of the same buffer. Fractions eluted at 50 mM (270-830 ml) which contain the highest specific and total endoglucanase activity are pooled, concentrated by ultrafiltration, and reapplied to a 2.6 x 30 cm column of DEAE-Sephadex A-50 in 50 mM Tris-HC1 buffer, pH 7.2. The column is eluted with a stepwise increase of NaCI (600 ml of 0 mM, 800 ml of 50 mM, 600 ml of 100 mM, and 600 ml of 150 mM) in the same buffer. The protein eluted at 50 mM NaCI contains 60% of the total endoglucanase activity. Fractions are pooled and refractionated on a 2.6 x 30 cm SP-Sephadex C-50 column in 20 mM sodium citrate buffer, pH 3.5. The column is eluted with a continuous NaCI gradient (0-300 mM) in 400 ml of citrate buffer and protein with the highest activity elutes at approximately 200 mM NaCI. The fractions are pooled, dialyzed against 50 mM sodium acetate buffer, pH 5.0, and concentrated 20-fold by ultrafiltration. Step 4. Column Electrophoresis. Electrophoretic fractionation is performed with a preparative electrophoresis apparatus described else10 T. K. Ng, P. J. Weimer, and J. G. Zeikus, Arch. Microbiol. 114, 1 (1977).
354
CELLULOSE
[38]
TABLE I PURIFICATION OF ENDOGLUCANASEI FROM C. t h e r m o c e l l u m a
Procedure Cell-free supernatant Ultrafiltrate
(NH4)2SO4 ppt. DEAE-Sephadex A50-1 DEAE-Sephadex A50-II SP-Sephadex C-50 Preparative gel electrophoresis a
Volume (ml)
Total activity (units)
Total protein (mg)
Specific activity (units/mg)
Yield (%)
12,000 1,000 400 400 60 25 10
20,300 17,800 13,800 6,400 2,750 975 514
6,720 4,750 3,370 624 119 21 7.9
3.0 3.7 4.2 10.3 23.0 45.9 65.1
-87.6 68.3 31.6 13.6 4.8 2.5
From Ng and Zeikus. 2 Reprinted by permission of the Biochemical Society, London.
where. ~t.12A 4 cm length of 8% separating gel with a 0.5 cm section of 4% stacking gel is used. The polyacrylamide gels are prepared by mixing solutions of 0.3 M Tris-HCl, pH 7.5, buffer with 0.23% of N,N,N',N'tetramethylenediamine, 40% acrylamide with 0.8% of N,N'-methylenebisacrylamide, water, and 0.14% ammonium persulfate in the ratio of 1 : 2 : I : 4 for 8% gel and 1 : 1 : 2 : 4 for 4% gel. The upper reservoir buffer consists of 0.8 M Tris-sulfate, pH 8.0, and the lower reservoir buffer is twice that strength. Both reservoirs are fed constantly with buffer at 1.0 ml/min, while the elution chamber is swept with 0.36 M Tris-HC1 buffer, pH 7.6, at 1.0 ml/min. A 2-ml sample with 43 mg of protein in 10% glycerol is applied to the column and the potential across the electrodes is set at 400 V ( - 3 4 mA); 2.5-ml fractions are collected. After 16 hr, endoglucanase I is eluted between fractions 384 and 389. The purification procedure is summarized in Table I. A 22-fold increase in specific activity with 2.5% recovery in total activity is achieved. Properties
Physical Properties. The endoglucanase I is a glycoprotein with - 1 1 % carbohydrate. The molecular weights determined by sedimentation equilibrium analysis, amino acid composition analysis, and polyacrylamide gel electrophoresis are 83,000, 88,000, and 94,000, respectively. The molar absorption coefficient at 280 nm is 53,750 M -~ cm -1 and the isoelectric ii T. Jovin, A. Chambach, and M. A. Naughton, A n a l . B i o c h e m . 9, 351 (1964). ~2A commercial apparatus is available as PolyPrep 200 from Buchler, Fort Lee. New Jersey.
ENDOGLUCANASE D OF C. thermocellum
[39]
355
point is 6.72. The pH and temperature optima are 5.2 and 62°. The enzyme contains 2.5% methionine but no cysteine. Specificity. The enzyme catalyzes most actively the hydrolysis of carboxymethylceUulose. Activity toward cellodextrins increases with the degree of polymerization, with cellopentaose having the highest activity. Cellotriose and cellobiose are not hydrolyzed. The enzyme-substrate complex formation prefers cellotriosyl units over cellobiosyl units and that from glucosyl units is rare. Avicel and acid-swollen cellulose are attacked but the activity is very low. Kinetics Constants. The [S]0.sv and V m a x for cellopentaose and cellohexaose are 2.30 × 10 -3 M, 39.3/xmol/min/mg protein (54.5 S -l assuming a molecular weight of 83,000), and 0.56 × 10-3 M, 58.7 /xmol/min/mg protein (81.2 S - 1), respectively.
[39] C r y s t a l l i n e E n d o g l u c a n a s e D o f Clostridiurn thermocellurn O v e r p r o d u c e d in Escherichia coli
By PIERRE BI~GUIN, GWENNA~L JOLIFF, MICHEL JUY, ADOLFO G. AMIT, JACQUELINE MILLET, ROBERTO J. POLIAK, and JEAN-PAUL AUBERT Introduction
Clostridium thermocellum, a thermophilic anaerobe, has been shown to produce a very active cellulase complex I composed of a large number of individual enzymes. 2 Attempts to resolve the complex into its active subunits have not been successful and only two endoglucanases (EG) (EC 3.2.1.4, cellulase) have been purified from culture supernatant. 3,4 Construction of C. thermocellum genomic libraries in Escherichia c o l i 5-7 showed that, as a general rule, C. thermocellum genes were readily expressed in E. coli. Consequently, it appeared that it was easier to purify E. A. Johnson, M. Sakajoh, G. Halliwell, A. Madia, and A. Demain, Appl. Environ. Microbiol. 43, 1125 (1982). 2 R. Lamed, E. Setter, and E. A. Bayer, J. Bacteriol. 156, 828 (1983). 3 j. P6tre, R. Longin, and J. Millet, Biochimie 63, 629 (1981). 4 T. K. Ng and J. G. Zeikus, Biochem. J. 199, 341 (1981). 5 p. Comet, D. Tronik, J. Millet, and J.-P. Aubert, FEMS Microbiol. Lett. 16, 137 (1983). 6 j. Millet, D. P6tr6, P. B6guin, O. Raynaud, and J.-P. Aubert, FEMS Microbiol. Lett. 29, 145 (1985). 7 W. Schwarz, K. Bronnenmeier, and W. L. Staudenbauer, Biotechnol. Lett. 7, 859 (1985).
METHODS IN ENZYMOLOGY, VOL. 160
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.