APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3505-3510
Vol. 56, No. 11
0099-2240/90/113505-06$02.00/0 Copyright © 1990, American Society for Microbiology
Purification and Characterization of Thermostable P-Mannanase and oL-Galactosidase from Bacillus stearothermophilus G. TALBOT AND J. SYGUSCH*
Departement de Biochimie, Faculte de Medecine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada JJH 5N4 Received 18 April 1990/Accepted 17 August 1990
Bacillus stearothermophilus secretes ,-mannanase and oa-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of aL-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified P-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The P-D-mannanase activity exhibited thermostabiity, retaining nearly full activity after incubation for 24 h at 70°C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for V.. and Km of 455 60 U/mg and 1.5 0.3 mg/ml, respectively, at 70°C and pH 6.5. The a-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The a-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60°C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl a-D-galactose. Kinetics of p-nitrophenyl a-D-galactose hydrolysis 0.02 mM, yielded linear reciprocal plots corresponding to Vmax and Km of 195 + 10 U/mg and 0.25 respectively, at 60°C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps. The extraction of lignin from wood fibers is
an
essential
step in bleaching of dissolving pulps. Pulp pretreatment
under alkaline conditions hydrolyzes hemicelluloses covalently bound to lignin and thus facilitates subsequent removal of lignin. There is a drawback to alkaline treatment of wood pulps, however, in that it creates an environmental pollution problemn (8). The alternate use of hemicellulases equally facilitates lignin removal in pulp bleaching and yields results comparable to alkaline pretreatment (14). Consequently, to substitute within a pulp-bleaching sequence an enzymatic pretreatment for the ultrahot alkaline extraction stage (14) offers the possibility of significant reduction in environmental pollution and thus is of considerable interest to the pulp and paper industry. To be feasible, however, enzymatic bleaching requires that hemicellulase treatment not impair pulp quality by attacking cellulose fibers. Softwoods from which the majority of pulps are derived contain as much as 15 to 20% hemicellulose in the form of galactomannan (11). Hemicellulases having substrate specificities for galactomannan constituents would make excellent candidates for use in enzymatic bleaching of softwood pulps (14). Since pulping is best carried out at elevated temperatures, thermophilic hemicellulases could offer significant advantages over mesophilic hemicellulases in terms of their higher intrinsic stability and catalytic efficiencies at such elevated temperatures. Bacillus strains are capable of utilizing lignocellulose fractions as carbon sources by secreting hydrolytic enzymes which depolymerize the hemicellulose and cellulose compo-
*
nents (4, 9). This article describes the purification and characterization of two thermostable hemicellulases from Bacillus stearothermophilus. In the presence of galactomannan, B. stearothermophilus produces both a P-D-mannanase and an a-D-galactosidase activity. Both enzymes retain activity for more than 24 h at temperatures in excess of 60°C. The high specific activity at 70°C of the P-D-mannanase makes the enzyme extremely attractive for use in enzymatic bleaching.
MATERIALS AND METHODS
Microorganism. B. stearothermophilus was obtained from the American Type Culture Collection (ATCC; catalog ascension no. 266). Materials. Locust bean gum, guar gum,
mannan
from
Saccharomyces cerevisiae, xylan (oat spelts), Sigmacell, carboxymethyl cellulose, melibiose, stachyose, raffinose, p-nitrophenyl ot-D-galactopyranoside, and other p-nitrophenyl derivatives were purchased from Sigma Chemical Co. Avicel (microcrystalline cellulose) was obtained from Fluka. Enzyme production. The bacterial culture was first amplified at 55°C in ATCC 266 liquid medium and then grown on a defined minimal agar medium (12), using 1% (wt/vol) locust bean gum (Sigma) as carbon source. Colonies grown on this agar medium were first amplified by vigorous shaking at 55°C in 3 ml of ATCC 266 liquid medium. The overnight cultures were then further amplified under identical conditions in 6 liters of the same medium until growth had reached stationary phase. After centrifugation at 10,000 x g for 20 min, the bacterial pellet was gently transferred into 3 liters of the defined minimal liquid medium (12) containing 1% (wtlvol) locust
Corresponding author. 3505
3506
TALBOT AND SYGUSCH
bean gum. Maximum hemicellulase activities occurred within 48 h and were monitored as described below. Purification. Mannanase and a-galactosidase activities secreted into the extracellular medium were harvested by centrifugation of the culture broth to remove cells and solid residues. The clarified supernatant was brought to 20% ammonium sulfate saturation and centrifuged at 4°C and 40,000 x g for 15 min. All subsequent fractionation steps involving ammonium sulfate were carried out at 4°C. After centrifugation, the pellet was discarded and the supernatant was brought to 50% (NH4)2SO4 saturation and pelleted again at 40,000 x g for 20 min. The a-galactosidase activity was recovered in the pellet and dissolved in 20 ml of 100 mM KH2PO4-150 mM KCl, pH 6.5. The supernatant was then brought to a 95% (NH4)2SO4 final saturation and centrifuged at 45,000 x g for 30 min. Mannanase activity was recovered in the pellet and dissolved in 20 ml of 100 mM KH2PO4-150 mM KCl, pH 6.5. Mannanase and oa-galactosidase fractions were then each subjected to gel filtration, using Sephacryl S-200 (Pharmacia) previously equilibrated in 100 mM KH2PO4-150 mM NaCl, pH 6.5. Active fractions from the Sephacryl S-200 gel filtration step were pooled and further purified by ion-exchange chromatography: an anion exchanger, Mono Q HR 5/5 (Pharmacia), previously equilibrated in 20 mM MES (morpholineethanesulfonic acid), pH 6.5, was utilized to purify mannanase activity, while a cation exchanger, Mono S HR 5/5 (Pharmacia), equilibrated with 20 mM Bis-Tris, pH 6.0, was used for a-galactosidase purification. For both ion exchangers, enzyme activity was eluted by using a 0 to 1 M NaCl gradient at a flow rate of 1 ml/min. Fractions having ax-galactosidase activity were pooled and dialyzed against 25 mM Bis-Tris, pH 6.3, buffer before being applied to a chromatofocusing Mono P HR 5/20 column (Pharmacia) equilibrated with the same buffer. A pH gradient was developed with 10% Polybuffer 74 (Pharmacia) and eluted a-galactosidase activity at pH 5.0. To ensure solubility of a-galactosidase activity, 4% (wt/vol) taurine (Sigma) was added in buffers used for dialysis and chromatofocusing. Hydrophobic interaction chromatography was then used to purify both mannanase and ot-galactosidase activities further. Mannanase activity was dialyzed against 0.4 M (NH4)2SO4-50 mM KH2PO4, pH 6.5, and absorbed onto a fast protein liquid chromatography phenyl-Superose HR 5/5 column (Pharmacia). Retention of a-galactosidase activity on the hydrophobic interaction column required only 0.25 M (NH4)2SO4 made up in the same phosphate buffer. Enzymatic activities were eluted by using respective gradients of 0.4 to 0 M (NH4)2SO4 and 0.25 M to 0 M (NH4)2SO4. In the case of the mannanase purification, activity could be detected in three distinct fractions. The active fractions for both enzymes were concentrated with polyethylene glycol 20,000 by placing each enzyme in a dialysis bag which was in contact with the polymer. Each active fraction was then applied to a gel filtration column, Superose 12 HR 10/30 (Pharmacia), equilibrated with 100 mM KH2PO4-50 mM KCI, pH 6.5. Resulting enzymatic activities were recovered in a single peak and can be stored in 50% glycerol at -20°C for at least 6 months without significant loss of activity. Protein assay. Proteins were measured by using bicinchonic acid protein assay reagent obtained from Pierce. Enzyme assays. Mannanase activity was estimated by the 3,5-dinitrosalicylic acid method as described previously (1), using locust bean gum as substrate. A 500-,lI assay containing 200 RIu of a 0.5% (wt/vol) substrate suspension, 50 pl of 500 mM phosphate buffer (pH 6.5), and the desired dilution
APPL. ENVIRON. MICROBIOL.
of enzyme was incubated for 5 min at 55°C. The reaction was stopped by addition of 500 RI of 3,5-dinitrosalicylic acid solution. ox-Galactosidase was assayed in a 1-ml total volume by mixing 50 ,I of a 20 mM p-nitrophenyl a-D-galactopyranoside solution with the appropriate enzyme dilution in a 20 mM final concentration of phosphate buffer for 5 min at 55°C. The reaction was stopped by addition of 30 RI of 2 N NaOH and incubated in a iced water bath to minimize hydrolysis of the substrate. A unit of mannanase or oa-galactosidase activity was defined as the amount of enzyme which liberates 1 ,umol of mannose or p-nitrophenol per min under the given assay conditions. Whenever indicated, error bars signify the standard error about the mean value, which was calculated from a minimum of three different activity assays. Error bars were not shown whenever the width of the error bar was less than the symbol sizes in the figures. Polyacrylamide gel electrophoresis (PAGE). Molecular weight estimates of the purified enzymes were determined on the basis of migration on 12.5% denaturing polyacrylamide gels (5). Nondenaturating gel electrophoresis was carried out with a 10 to 15% polyacrylamide gradient separating gel and molecular weight markers from Pharmacia. Kinetic analysis. Kinetic constants and associated standard deviations were evaluated by linear regression of reciprocal velocity plots. Product chromatography. Polyacrylamide gel, Bio-Gel P-4
Vol. 56, No. 11
0099-2240/90/113505-06$02.00/0 Copyright © 1990, American Society for Microbiology
Purification and Characterization of Thermostable P-Mannanase and oL-Galactosidase from Bacillus stearothermophilus G. TALBOT AND J. SYGUSCH*
Departement de Biochimie, Faculte de Medecine, Universite de Sherbrooke, Sherbrooke, Quebec, Canada JJH 5N4 Received 18 April 1990/Accepted 17 August 1990
Bacillus stearothermophilus secretes ,-mannanase and oa-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of aL-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified P-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The P-D-mannanase activity exhibited thermostabiity, retaining nearly full activity after incubation for 24 h at 70°C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for V.. and Km of 455 60 U/mg and 1.5 0.3 mg/ml, respectively, at 70°C and pH 6.5. The a-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The a-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60°C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl a-D-galactose. Kinetics of p-nitrophenyl a-D-galactose hydrolysis 0.02 mM, yielded linear reciprocal plots corresponding to Vmax and Km of 195 + 10 U/mg and 0.25 respectively, at 60°C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps. The extraction of lignin from wood fibers is
an
essential
step in bleaching of dissolving pulps. Pulp pretreatment
under alkaline conditions hydrolyzes hemicelluloses covalently bound to lignin and thus facilitates subsequent removal of lignin. There is a drawback to alkaline treatment of wood pulps, however, in that it creates an environmental pollution problemn (8). The alternate use of hemicellulases equally facilitates lignin removal in pulp bleaching and yields results comparable to alkaline pretreatment (14). Consequently, to substitute within a pulp-bleaching sequence an enzymatic pretreatment for the ultrahot alkaline extraction stage (14) offers the possibility of significant reduction in environmental pollution and thus is of considerable interest to the pulp and paper industry. To be feasible, however, enzymatic bleaching requires that hemicellulase treatment not impair pulp quality by attacking cellulose fibers. Softwoods from which the majority of pulps are derived contain as much as 15 to 20% hemicellulose in the form of galactomannan (11). Hemicellulases having substrate specificities for galactomannan constituents would make excellent candidates for use in enzymatic bleaching of softwood pulps (14). Since pulping is best carried out at elevated temperatures, thermophilic hemicellulases could offer significant advantages over mesophilic hemicellulases in terms of their higher intrinsic stability and catalytic efficiencies at such elevated temperatures. Bacillus strains are capable of utilizing lignocellulose fractions as carbon sources by secreting hydrolytic enzymes which depolymerize the hemicellulose and cellulose compo-
*
nents (4, 9). This article describes the purification and characterization of two thermostable hemicellulases from Bacillus stearothermophilus. In the presence of galactomannan, B. stearothermophilus produces both a P-D-mannanase and an a-D-galactosidase activity. Both enzymes retain activity for more than 24 h at temperatures in excess of 60°C. The high specific activity at 70°C of the P-D-mannanase makes the enzyme extremely attractive for use in enzymatic bleaching.
MATERIALS AND METHODS
Microorganism. B. stearothermophilus was obtained from the American Type Culture Collection (ATCC; catalog ascension no. 266). Materials. Locust bean gum, guar gum,
mannan
from
Saccharomyces cerevisiae, xylan (oat spelts), Sigmacell, carboxymethyl cellulose, melibiose, stachyose, raffinose, p-nitrophenyl ot-D-galactopyranoside, and other p-nitrophenyl derivatives were purchased from Sigma Chemical Co. Avicel (microcrystalline cellulose) was obtained from Fluka. Enzyme production. The bacterial culture was first amplified at 55°C in ATCC 266 liquid medium and then grown on a defined minimal agar medium (12), using 1% (wt/vol) locust bean gum (Sigma) as carbon source. Colonies grown on this agar medium were first amplified by vigorous shaking at 55°C in 3 ml of ATCC 266 liquid medium. The overnight cultures were then further amplified under identical conditions in 6 liters of the same medium until growth had reached stationary phase. After centrifugation at 10,000 x g for 20 min, the bacterial pellet was gently transferred into 3 liters of the defined minimal liquid medium (12) containing 1% (wtlvol) locust
Corresponding author. 3505
3506
TALBOT AND SYGUSCH
bean gum. Maximum hemicellulase activities occurred within 48 h and were monitored as described below. Purification. Mannanase and a-galactosidase activities secreted into the extracellular medium were harvested by centrifugation of the culture broth to remove cells and solid residues. The clarified supernatant was brought to 20% ammonium sulfate saturation and centrifuged at 4°C and 40,000 x g for 15 min. All subsequent fractionation steps involving ammonium sulfate were carried out at 4°C. After centrifugation, the pellet was discarded and the supernatant was brought to 50% (NH4)2SO4 saturation and pelleted again at 40,000 x g for 20 min. The a-galactosidase activity was recovered in the pellet and dissolved in 20 ml of 100 mM KH2PO4-150 mM KCl, pH 6.5. The supernatant was then brought to a 95% (NH4)2SO4 final saturation and centrifuged at 45,000 x g for 30 min. Mannanase activity was recovered in the pellet and dissolved in 20 ml of 100 mM KH2PO4-150 mM KCl, pH 6.5. Mannanase and oa-galactosidase fractions were then each subjected to gel filtration, using Sephacryl S-200 (Pharmacia) previously equilibrated in 100 mM KH2PO4-150 mM NaCl, pH 6.5. Active fractions from the Sephacryl S-200 gel filtration step were pooled and further purified by ion-exchange chromatography: an anion exchanger, Mono Q HR 5/5 (Pharmacia), previously equilibrated in 20 mM MES (morpholineethanesulfonic acid), pH 6.5, was utilized to purify mannanase activity, while a cation exchanger, Mono S HR 5/5 (Pharmacia), equilibrated with 20 mM Bis-Tris, pH 6.0, was used for a-galactosidase purification. For both ion exchangers, enzyme activity was eluted by using a 0 to 1 M NaCl gradient at a flow rate of 1 ml/min. Fractions having ax-galactosidase activity were pooled and dialyzed against 25 mM Bis-Tris, pH 6.3, buffer before being applied to a chromatofocusing Mono P HR 5/20 column (Pharmacia) equilibrated with the same buffer. A pH gradient was developed with 10% Polybuffer 74 (Pharmacia) and eluted a-galactosidase activity at pH 5.0. To ensure solubility of a-galactosidase activity, 4% (wt/vol) taurine (Sigma) was added in buffers used for dialysis and chromatofocusing. Hydrophobic interaction chromatography was then used to purify both mannanase and ot-galactosidase activities further. Mannanase activity was dialyzed against 0.4 M (NH4)2SO4-50 mM KH2PO4, pH 6.5, and absorbed onto a fast protein liquid chromatography phenyl-Superose HR 5/5 column (Pharmacia). Retention of a-galactosidase activity on the hydrophobic interaction column required only 0.25 M (NH4)2SO4 made up in the same phosphate buffer. Enzymatic activities were eluted by using respective gradients of 0.4 to 0 M (NH4)2SO4 and 0.25 M to 0 M (NH4)2SO4. In the case of the mannanase purification, activity could be detected in three distinct fractions. The active fractions for both enzymes were concentrated with polyethylene glycol 20,000 by placing each enzyme in a dialysis bag which was in contact with the polymer. Each active fraction was then applied to a gel filtration column, Superose 12 HR 10/30 (Pharmacia), equilibrated with 100 mM KH2PO4-50 mM KCI, pH 6.5. Resulting enzymatic activities were recovered in a single peak and can be stored in 50% glycerol at -20°C for at least 6 months without significant loss of activity. Protein assay. Proteins were measured by using bicinchonic acid protein assay reagent obtained from Pierce. Enzyme assays. Mannanase activity was estimated by the 3,5-dinitrosalicylic acid method as described previously (1), using locust bean gum as substrate. A 500-,lI assay containing 200 RIu of a 0.5% (wt/vol) substrate suspension, 50 pl of 500 mM phosphate buffer (pH 6.5), and the desired dilution
APPL. ENVIRON. MICROBIOL.
of enzyme was incubated for 5 min at 55°C. The reaction was stopped by addition of 500 RI of 3,5-dinitrosalicylic acid solution. ox-Galactosidase was assayed in a 1-ml total volume by mixing 50 ,I of a 20 mM p-nitrophenyl a-D-galactopyranoside solution with the appropriate enzyme dilution in a 20 mM final concentration of phosphate buffer for 5 min at 55°C. The reaction was stopped by addition of 30 RI of 2 N NaOH and incubated in a iced water bath to minimize hydrolysis of the substrate. A unit of mannanase or oa-galactosidase activity was defined as the amount of enzyme which liberates 1 ,umol of mannose or p-nitrophenol per min under the given assay conditions. Whenever indicated, error bars signify the standard error about the mean value, which was calculated from a minimum of three different activity assays. Error bars were not shown whenever the width of the error bar was less than the symbol sizes in the figures. Polyacrylamide gel electrophoresis (PAGE). Molecular weight estimates of the purified enzymes were determined on the basis of migration on 12.5% denaturing polyacrylamide gels (5). Nondenaturating gel electrophoresis was carried out with a 10 to 15% polyacrylamide gradient separating gel and molecular weight markers from Pharmacia. Kinetic analysis. Kinetic constants and associated standard deviations were evaluated by linear regression of reciprocal velocity plots. Product chromatography. Polyacrylamide gel, Bio-Gel P-4
