APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1994, p. 3774-3780 Vol. 60, No. 10 0099-2240/94/$04.00+0 Copyright ) 1994, American Society for Microbiology Production, Purification, and Properties of a Thermostable 13-Glucosidase from a Color Variant Strain of Aureobasidium pullulans BADAL C. SAHA,t* SHELBY N. FREER, AND RODNEY J. BOTHAST Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department ofAgriculture, Peoria, Illinois 61604 Received 24 January 1994/Accepted 22 July 1994 A color variant strain of Aureobasidium pullulans (NRRL Y-12974) produced 0-glucosidase activity when grown in liquid culture on a variety of carbon sources, such as , xylose, arabinose, lactose, sucrose, maltose, , xylitol, xylan, , starch, and pullulan. An extracellular 0-glucosidase was purified 129-fold to homogeneity from the cell-free culture broth of the organism grown on corn bran. The purification protocol included ammonium sulfate treatment, CM Bio-Gel A agarose column chromatography, and gel filtrations on Bio-Gel A-0.5m and Sephacryl S-200. The 13-glucosidase was a glycoprotein with native molecular weight of 340,000 and was composed of two subunits with molecular weights of about 165,000. The displayed optimal activity at 75°C and pH 4.5 and had a specific activity of 315 ,umol * min-' * mg of protein-' under these conditions. The purified ,-glucosidase was active against p-nitrophenyl-4-D-glucoside, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose, with Km values of 1.17, 1.00, 0.34, 0.36, 0.64, 0.68, and 1.65 mM, respectively. The enzyme activity was competitively inhibited by glucose (K' = 5.65 mM), while fructose, arabinose, galactose, mannose, and xylose (each at 56 mM) and sucrose and lactose (each at 29 mM) were not inhibitory. The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol (7.5%, vol/vol) stimulated the initial enzyme activity by 15%. Glucose production was enhanced by 7.9% when microcrystalline cellulose (2%, wt/vol) was treated for 48 h with a commercial preparation (5 U/ml) that was supplemented with the purified 0-glucosidase (0.21 U/ml) from A. pullulans.

Currently, over one billion gallons of ethanol is produced canase and cellobiohydrolase activities are often inhibited by annually in the United States, with approximately 95% derived cellobiose (6, 12, 38). Thus, the 3-glucosidase not only pro- from the fermentation of corn. With increased attention to duces glucose from cellobiose but also reduces cellobiose clean air and oxygenates for fuels, opportunities exist for inhibition, allowing the cellulolytic to function more expansion of the ethanol fuel industry. Lignocellulosic bio- efficiently. However, like ,3-, almost all ,3-glucosi- mass, particularly corn fiber, represents a renewable resource dases are subject to end product inhibition. Product inhibition that is available in sufficient quantities from the corn wet- and thermal inactivation of P-glucosidase constitute two major milling industry to serve as a low-cost feedstock. To lower the barriers to the development of enzymatic of cellu- costs associated with ethanol production from , ad- lose as a commercial process (38). There is an increasing vances in enzyme technology are necessary. demand for the development of a thermostable ,B-glucosidase Cellulose is a linear polymer of D-glucose units linked by for application in the conversion of cellulose to glucose for the 1,4-13-D-glucosidic bonds. The enzyme system for the conver- subsequent production of fuel ethanol. Thus, we sought to sion of cellulose to glucose comprises endo-1,4-p- discover a thermostable 3-glucosidase with superior proper- (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91), and P-glucosi- ties. dase (P-D-glucosidic glucohydrolase; EC 3.2.1.21). Cellulolytic The yeast-like Aureobasidium pullulans has been enzymes in conjunction with P-glucosidase act sequentially and recognized as an excellent producer of and xylano- cooperatively to degrade crystalline cellulose to glucose. En- lytic enzymes (17, 32). A number of industrial products from doglucanase acts in random fashion on the regions of low this organism have been characterized (7). We have found that crystallinity of the cellulosic fiber, whereas cellobiohydrolase a color variant strain ofA. pullulans produced an extracellular removes cellobiose (P-1,4 glucose dimer) units from the non- highly thermostable ,B-glucosidase activity which exhibited reducing end of the cellulosic chain. P-Glucosidase hydrolyzes half-lives of 72 h at 75°C and 24 h at 80°C and was optimally cellobiose and in some cases cellooligosaccharides to glucose. active at 80°C (unpublished results). In this paper, we report The ,B-glucosidase is generally responsible for the regulation of on the production, purification, and characteristics of this the entire cellulolytic process and is a rate-limiting factor enzyme. during enzymatic hydrolysis of cellulose, since both endoglu- MATERIALS AND METHODS * Corresponding author. Mailing address: USDA-ARS-NCAUR- FBR, 1815 N. University St., Peoria, IL 61604. Phone: (309) 681-6276. Materials. Cellobiose, microcrystalline cellulose (Sigmacell Fax: (309) 681-6686. Electronic mail address: bcsaha@heartland. type 50), all saccharides, all aryl-glycosides, salicin, molecular bradley.edu. weight markers for gel filtration, Schiffs reagent, and the t Permanent address: Department of Biochemistry, Michigan State glucose detection kit were purchased from Sigma Chemical University, East Lansing, MI 48824. Co., St. Louis, Mo. Molecular weight markers and precast gels 3774 VOL.V-GLUCOSIDASE60, 1994 FROM AUREOBASIDIUM PULLULANS 3775 for sodium dodecyl sulfate-polyacrylamide gel electrophoresis TABLE 1. Effect of carbon source on growth of and ,B-glucosidase (SDS-PAGE), CM Bio-Gel A agarose, and Bio-Gel A-0.5m production by A. pullulans NRRL Y-12974a were Calif. obtained from Bio-Rad Laboratories, Hercules, ,3-Glucosidase (mU/ml of Sephacryl S-200 was from Pharmacia LKB Biotechnology, Carbon source Growth culture broth) Piscataway, N.J. were prepared by a method (1%, wthvol) (A660) Total Extracellular described previously (10). Corn bran (Dietfiber NU 20085) was purchased from Lauhoff Grain Company, Danville, Ill. Cyto- Corn bran b 230 36 lase 123 was supplied by Genencor International, Rochester, Cellulose 74 8 N.Y. Yeast extract was from Difco Laboratories, Detroit, Xylan 72 12 Mich. A reversible amino column for high-pressure liquid Soluble starch 7.1 79 6 chromatography (HPLC) was purchased from Regis Chemical Pullulan 3.6 150 13 Co., Morton Grove, Ill. All other chemicals were purchased Cellobiose 8.0 120 10 Lactose 5.1 210 46 from Sigma Chemical Co. Sucrose 6.6 60 3 Organism and enzyme production. The color variant strain Maltose 5.7 41 9 NRRL Y-12974 of A. pullulans was obtained from the Agri- Glucose 6.1 59 5 cultural Research Service culture collection, National Center Xylose 7.4 56 8 for Agricultural Utilization Research, Peoria, Ill. The medium Arabinose 8.4 44 11 for seed culture and enzyme production contained the follow- Xylitol 7.9 120 21 per carbon source, ing ingredients (grams liter): 10; NaNO3, 2; a Values reported are averages from duplicate experiments for each substrate. MgSO4 * 7H2O, 0.5; NaCl, 0.5; FeSO4 * 7H20, 0.01; KH2PO4, Cultures were grown in 250-ml Erlenmeyer flasks containing 100 ml of medium 1; and yeast extract, 0.4. The initial pH of the medium was 5.0 at 28°C for 4 days. before autoclaving. A 250-ml Erlenmeyer flask containing 100 b_b, not determined. ml of medium with xylose as a carbon source was inoculated with a loopful of cells taken from a stock slant tube and incubated at 28°C on a rotary shaker (200 rpm) for 3 days. The (1.5 by 120 cm) preequilibrated with 50 mM acetate buffer, pH shake flasks (250-ml Erlenmeyer flask containing 100 ml of 6.0. The enzyme solution in 50 mM acetate buffer, pH 6.0, was medium) were inoculated with 2 ml of this culture and applied to the column and eluted with the same buffer. The cultivated on a rotary shaker (200 rpm) at 28°C. After 4 days, first active 3-glucosidase peak fractions (major peak) were the cells and unutilized insoluble substrates were removed pooled and concentrated by ultraffiltration (PM 10 membrane). from the culture broth by centrifugation (18,000 x g, 20 min). (iv) Sephacryl S-200 gel filtration. The P-glucosidase ob- The resulting supernatant solution was used as the crude tained after Bio-Gel A-0.5m gel filtration was subjected to a enzyme preparation. For the purification of 3-glucosidase, the second gel filtration on a Sephacryl S-200 column (1.5 by 120 organism was grown in 1-liter Erlenmeyer flasks containing 500 cm) preequilibrated with 50 mM acetate buffer, pH 6.0. The ml of medium with corn bran (2.5%, wt/vol) as a carbon enzyme was eluted with the same buffer. The first two active source. enzyme fractions were pooled, concentrated by ultrafiltration Enzyme assay. 1-Glucosidase activity was assayed- in a (PM 10 membrane) and dialyzed against 50 mM acetate buffer, reaction mixture (1 ml) containing 4 mM p-nitrophenyl-p3-D- pH 6.0. The dialyzed enzyme solution was used as purified glucoside (pNP,BG), 50 mM acetate buffer (pH 5.0), and ,B-glucosidase for subsequent studies. appropriately diluted enzyme solution. After incubation at Other methods. Protein was estimated by the method of 50°C for 30 min, the reaction was stopped by adding ice-cold Lowry et al. (21) with bovine serum albumin as the standard. 0.5 M Na2CO3 (1 ml), and the color formed was measured at Protein in the column effluents was monitored by measuring 405 nm. One unit of 3-glucosidase activity is defined as the A280. SDS-PAGE was performed with a 7.5% gel according to amount of enzyme which liberates 1 p,mol ofp-nitrophenol per the method of Laemmli (16). The gels were stained with min in the reaction mixture under these assay conditions. Coomassie brilliant blue R-250 for protein and with periodic Purification of 1-glucosidase. All purification steps were acid-Schiff's reagent for carbohydrate. The molecular weight of performed at 4°C, unless otherwise stated. the native enzyme was determined by gel filtration on a (i) Ammonium sulfate treatment. The culture broth (6 Bio-Gel A-0.Sm column, as described by Andrews (2), with liters) was concentrated with an Amicon cell (model 202; apoferritin (molecular weight, 443,000), sweet potato p-amy- Amicon, Inc., Beverly, Mass.) equipped with a PM 30 mem- lase (200,000), yeast alcohol dehydrogenase (150,000), bovine brane under nitrogen pressure of 20 lb/in2. No ,-glucosidase serum albumin (66,000), and ovalbumin (45,000) as standard activity was detected in the ultrafiltrate. The concentrated proteins. Glucose was estimated by the glucose oxidase-perox- broth (2 liters) was then treated with ammonium sulfate (80% idase-o-dianisidine method of Hugget and Nixon (11). The Km saturation) and left overnight. The precipitate was collected by and Vma values were determined by the double-reciprocal plot centrifugation at 48,000 x g for 30 min, dissolved in 50 mM method of Lineweaver and Burk (19) with the KINET software acetate buffer, pH 4.0, and dialyzed overnight against the same program. Sugar analysis was performed by HPLC on a revers- buffer. ible amino column (250 by 4.6 mm) with acetonitrile-water (ii) CM Bio-Gel A agarose column chromatography. The (70:30, vol/vol) as the eluent (flow rate, 2 ml/min) at room dialyzed enzyme solution was applied to a CM Bio-Gel A temperature (5). Peaks were detected by refractive index and agarose column (2.5 by 26 cm) preequilibrated with 50 mM were identified and quantitated by comparison with retention acetate buffer, pH 4.0. The column was washed extensively times of authentic saccharide standards. with the same buffer and eluted with 50 mM acetate buffer, pH 6.0. The ,B-glucosidase activity was eluted as a single peak. The RESULTS active fractions were pooled, concentrated by ultrafiltration, and dialyzed overnight against 50 mM acetate buffer, pH 6.0. Production of 13-glucosidase. The effect of carbon sources on (iii) Gel filtration on Bio-Gel A-0.5m. The 3-glucosidase was growth of and P-glucosidase production byA. pullulans NRRL further purified by gel filtration on a Bio-Gel A-0.5m column Y-12974 in shake flasks was investigated (Table 1). The 3776 SAHA ET AL. APPL. ENVIRON. MICROBIOL.

0.30

0.25 -200.0 K E 0.20 : -116.2 K -97.4 K 0 I4 0.15 -~~~~~~~~~~o - 66.2 K

0 3 0.10 :

-45.0 K 2 0.05 1 2 1 0.00 FIG. 2. SDS-PAGE of purified 3-glucosidase from A. pullulans. 0 24 48 72 96 1 20 1 44 168 The enzyme was electrophoresed at pH 8.3 on a 7.5% acrylamide gel Time (h) and stained with Coomassie brilliant blue R-250. Lane 1, purified 3-glucosidase; lane 2, molecular weight standards. The standards were FIG. 1. Time course of f-glucosidase production by A. pullulans myosin (200,000 [200.0 K]), f3-galactosidase (116,250), phosphorylase b NRRL Y-12974 grown on corn bran (1%, wt/vol) at 28°C. Symbols: O, (97,400), bovine serum albumin (66,200), and ovalbumin (45,000). pH; 0, total ,B-glucosidase activity; 0, extracellular 3-glucosidase activity. Values are averages from duplicate experiments.

brilliant blue (Fig. 2) or periodic acid-Schiffis reagent. The organism was grown in liquid culture with 1% (wt/vol) sub- ,-glucosidase was purified 129-fold with a 3.0% retention of strate at 28°C for 4 days. The organism grew well and produced total extracellular activity and 0.023% retention of total pro- ,-glucosidase activity on every substrate tested. It is interesting tein. The purified ,-glucosidase had a specific activity of 124 that the highest level of total j-glucosidase activity was pro- U/mg of protein (assayed at pH 5.0 and 50°C with pNPiG as duced in corn bran medium but not in cellobiose medium. the substrate). The specific activity of the purified enzyme Lactose was also a good carbon source for the production of under optimal conditions (at pH 4.5 and 75°C) was 315 U/mg ,B-glucosidase activity. Most of the f-glucosidase activity was of protein. cell associated, regardless of the carbon source used. The Physicochemical characteristics. The molecular weight of organism grew well on glucose and produced readily detectable the native 3-glucosidase estimated by gel filtration on Bio-Gel amounts of j-glucosidase. A time course of ,3-glucosidase A-0.5m was 340,000, but by SDS-PAGE analysis, the molecular production byA. pullulans grown on corn bran is shown in Fig. weight of the enzyme was about 165,000 (Fig. 2), suggesting 1. The ,3-glucosidase production increased gradually up to 4 that the ,B-glucosidase was a dimer composed of two subunits days, after which it remained constant. of equal molecular weight. Purification of 13-glucosidase. An extracellular ,B-glucosi- The thermostability and thermoactivity of the purified ,-glu- dase was purified to homogeneity from the culture filtrates of cosidase from A. pullulans are shown in Fig. 3. The purified A. pullulans grown on corn bran. A summary of the purification enzyme in 50 mM acetate buffer, pH 5.0 (0.03 U/ml; 0.229 procedures is presented in Table 2. About 50% of the j-glu- pug/ml), was fully stable at up to 60°C for 1 h in the presence of cosidase activity did not adsorb onto the CM Bio-Gel A bovine serum albumin (0.1%, wt/vol) and retained about 55% agarose column. The 3-glucosidase was separated into two activity at 80°C and 17% activity at 85°C. The enzyme was active enzyme peaks (one major and one minor) upon gel inactivated upon incubation for 1 h at 90°C. The purified filtration chromatography on Bio-Gel A-0.5m. The recovery of enzyme (4.7 ,ug/ml) retained its full activity at 4°C after storage the enzyme from Sephacryl S-200 gel filtration was poor in 50 mM acetate buffer, pH 6.0, for more than 4 months. The because only the first two active fractions were pooled. The enzyme exhibited maximum activity at 750C under the assay later fractions were found to be contaminated with j-xylosi- conditions used (Fig. 3). The enzyme was stable at pH 4.0 to dase activity. Upon SDS-PAGE of the purified 13-glucosidase, 6.5 (1 h at 500C), with 55% activity remaining at pH 3.0 and a single band was visualized when stained with Coomassie 77% activity remaining at pH 8.0 (Fig. 4). It displayed an optimum activity at pH 4.5, with 71 and 50% relative activities at pH 3.0 and 6.0, respectively (Fig. 4). TABLE 2. Purification of 3-glucosidase from A. pullulans Catalytic properties. The relative rates of hydrolysis of NRRL Y-12974 various substrates by the purified ,B-glucosidase are presented in Table 3. The enzyme hydrolyzed cellobiose and pNP3G Total Total Sp act Recver Purifi- was at Step protein activity (U/mg of cation effectively. Salicin hydrolyzed 15.2% of the level of (mg) (U) protein) (%)M (fold) hydrolysis of cellobiose. The purified enzyme had very little (<5%) or no activity on lactose, maltose, sucrose, and treha- Culture supernatant 1,600 1,500 1 100 1 lose. It also had no or very little (<5%) activity onp-nitrophe- Ammonium sulfate 600 830 1.4 54 1.4 nyl-at-D-glucoside, p-nitrophenyl-j3-D-xyloside, p-nitrophenyl-,3- CM Bio-Gel A agarose 54 250 5 16 5 and Bio-Gel A-0.5m 2.2 220 100 14 104 D-cellobioside, p-nitrophenyl-ot-L-arabinofuranoside, p-ni- Sephacryl S-200 0.37 46 124 3 129 trophenyl-13-D-glucuronide. p-Nitrophenyl-f-D-galactoside was hydrolyzed at 7.6% of the level of hydrolysis of pNP3G. The VOL.V-GLUCOSIDASE60, 1994 FROM AUREOBASIDIUM PULLULANS 3777

1 20 TABLE 3. Relative initial rates of hydrolysis of various substrates by purified 3-glucosidase from A. pullulans NRRL Y-12974 Relativc 1 00 Substrate Linkageglycosylof initialof hydro-rate group lysis (% )' 0>% 80 Saccharides X Cellobiose (29 mM) 3(1,4)Glc 1H) ._- Lactose (29 mM) ,B(1,4) 2.5 I) 60 Sucrose (29 mM) 13(1,2) 1.5 Maltose (28 mM) o(1,4)Glc 0.0 Trehalose (26 mM) o(41,1)Glc 4.1 40 Salicin (35 mM) ,BGlc 15.2 -a (1Cc, wt/vol) ,B(1,4)Glc 4.5 Aryl-glycosides 20 /8 pNPfG (4 mM) 1Glc 100 p-Nitrophenyl-ot-D-glucoside (4 mM) csGlc 1.2 2O[ I p-Nitrophenyl-P-D-xyloside (4 mM) fXyl 0.8 p-Nitrophenyl-P-D-galactoside (4 mM) ,BGal 7.6 p-Nitrophenyl-p-D-cellobioside (4 mM) ,BGlc 1.5 20 30 40 50 60 70 80 90 1 00 p-Nitrophenyl-u-L-arabinofuranoside (4 mM) oAra 0.3 Temperature (°C) p-Nitrophenyl-13-D-glucuronide (4 mM) ,BGlcA 0.3 FIG. 3. Effect of temperature on stability and activity of purified a Values reported are averages from duplicate experiments for each substrate. 3-glucosidase from A. pullulans. For measurements of stability (0), Sufficient enzyme was added to cause a linear release of product during the first the enzyme solution in acetate buffer (50 mM, pH 5.0) with 0.1% 15 min of reaction at pH 4.5 and 75°C. Depending on the type of substrate, activity was determined by measuring the release of glucose (glucose oxidase (wt/vol) bovine serum albumin was incubated for 1 h at various method) or ofp-nitrophenol (405 nm). The relative initial rate of hydrolysis of a temperatures, and then the residual enzyme activities were assayed. saccharide is expressed as a percentage of that obtained with cellobiose, and that For measurements of activity (0), the enzyme activity was assayed at of an aryl-glycoside is expressed as a percentage of that obtained with pNP,3G. various temperatures by the standard assay method. The enzyme was The relative rate of hydrolysis of pNPf3G was 90% of that of cellobiose. The used at 0.03 U/ml. enzyme was used at 36 mU/ml.

dependence of the rate of the enzymatic reaction on the ,umol - min-' - mg of protein-' for the hydrolysis of pNP3G substrate concentration at pH 4.5 and 75°C followed Michae- and cellobiose, respectively. Glucose was detected by HPLC lis-Menten kinetics. Reciprocal plots showed apparent Km analysis as the only product from hydrolysis of cellobiose (1%, values of 1.17 and 1.00 mM and Vmax values of 897 and 800 wt/vol) with the enzyme preparation (0.21 U/mI) after 24 h. The K,, values for hydrolysis of cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose at pH 4.5 and 75°C were 0.34, 0.36, 0.64, 0.68, and 1.65 mM, respectively. The 1 20 Vmax values for the hydrolysis of these substrates were 369, 271, 269, 180, and 325 pLmol - min'- mg- , respectively. The spec- ificity constants (Vm.I/K,..) for the hydrolysis of pNP3G, cello- 100 F biose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose were calculated as 767, 800, 1,085, 753, 420, 265, and 197, respectively. Substrate inhibition was observed 80 with pNP3G concentrations greater than 20 mM and with cellobiose concentrations greater than 175 mM (6%, wt/vol). The study of inhibition by glucose was performed with _)0 60 pNP,BG as the substrate. Glucose acted as a competitive >xa) inhibitor of pNP3G hydrolysis, with an inhibition constant (Ki) of 5.65 mM obtained at the intersection of the lines on the a 40 Dixon plot analysis. Galactose, mannose, arabinose, fructose, and xylose (each at 56 mM) and sucrose and lactose (each at 29 20 mM) did not inhibit the 3-glucosidase activity. The effect of certain inhibitors or activators on ,B-glucosidase activity was studied. The enzyme did not require Ca2+, Mg2+, un Mn2, or Co2+ (each at 5 mM) for activity. Enzyme activity 2 3 4 5 6 7 was not affected by EDTA (10 mM), dithiothreitol (10 mM) or pH p-chloromercuribenzoic acid (0.2 mM). Cyclodextrins (10 mM) did not inhibit the ,B-glucosidase activity. Ethanol had a FIG. 4. Effect of pH on stability and activity of purified 3-glucosi- stimulating effect on the initial P3-glucosidase activity. The dase from A. pullulans. For measurements of stability (0), the enzyme pNP,BG hydrolysis by the enzyme at pH 4.5 and 50°C was solutions in 50 mM buffer at various pH values were incubated for 1 h increased up to 15% (30-min at 7.5% at 50°C. After adjustment of pH, the residual activity was assayed by reaction) (vol/vol) the standard method. For measurements of activity (0), the enzyme ethanol, after which the activity dropped, probably because of activity was assayed by the standard assay method by changing the precipitation of the enzyme protein. HPLC analysis of the buffer to obtain the desired pH. Buffers used were acetate (pH 3.0 to reaction products of 3-glucosidase in the presence of 6% 6.0) and phosphate (pH 6.5 to 8.0). The enzyme was used at 0.03 U/ml. ethanol after 24 h indicated the formation of an additional 3778 SAHA ET AL. APPL. ENVIRON. MICROBIOL. unidentified peak. Ethanol at 6% (vol/vol) did not affect the was observed at 75°C and pH 4.5 (Fig. 3 and 4). Under inhibitory effect of glucose (56 mM) on P-glucosidase activity. identical assay conditions, the temperature for maximum ac- Microcrystalline cellulose (Sigmacell type 50; 2%, wt/vol) tivity of the purified ,3-glucosidase was lower than that (80°C) hydrolysis was performed at pH 4.5 and 50°C with a cellulase for the crude 3-glucosidase of A. pullulans. A similar finding preparation (Cytolase 123; 5 U/ml) supplemented with purified was reported with 3-glucosidase from Neocallimastix frontalis, P-glucosidase (0.21 U/ml) from A. pullulans. The reaction in which case the pure enzyme demonstrated an optimum products were analyzed by HPLC. After 48 h, there was a 7.9% temperature at 45°C whereas the crude enzyme showed opti- increase in the production of glucose by the cellulase prepa- mum activity at 55 to 600C (18). While the crude P3-glucosidase ration supplemented with ,B-glucosidase fromA. pullulans. The from Clostnidium stercorarium exhibited a half-life of 3 h at 3-glucosidase itself had no activity on microcrystalline cellu- 60°C, the purified enzyme was rapidly inactivated at this lose. Thus, the 3-glucosidase from A. pullulans had a synergis- temperature (4). The fungal f- had a broad range tic interaction with cellulase to increase the efficiency of of pH and temperature dependence, with optima between pH glucose production from cellulose. 3 and 7 and between 40 and 70°C (6). Thermostable 3-gluco- sidases reported to date include those purified from thermo- DISCUSSION philes such as thennocellum (1), Talaromyces em- ersonii (22), a Thernotoga sp. (31), Microbispora bispora (39), a This is the first report to our knowledge on the production, thermophilic cellulolytic anaerobe (Tp8) (29), an extremely purification, and properties of 3-glucosidase from a color thermophilic anaerobic strain (Wai21W.2) (27), and a hyper- variant strain ofA. pullulans. The production of 3-glucosidase thermophilic archaeon (Pyrococcus furiosus) (13). The most by A. pullulans depended strongly on the carbon sources thermostable 3-glucosidase from P. furiosus (half-lives of 85 h (Table 1). Lactose was the best carbon source for the produc- at 100°C and 13 h at 110°C) exhibited an optimum tempera- tion of extracellular 3-glucosidase. It is interesting that the ture at 102 to 105°C (13). Apparently, the thermophilic organism grew well on corn bran and on corn bran produced characteristic of the 3-glucosidase from the mesophilic yeast- the highest total 3-glucosidase activity (231 mU/ml of culture like fungusA. pullulans makes it suitable for use in commercial broth) among the carbon sources used. However, A. pullulans cellulose saccharification processes operating at elevated tem- produced ,B-glucosidase activity constitutively when grown in peratures. liquid medium containing a carbon source. The major portion The preferred substrates for 3-glucosidase from A. pullulans of the 13-glucosidase activity remained cell associated. The were cellobiose and pNP3G (Table 3), and this type of extracellular P-glucosidase was purified 129-fold from the corn 3-glucosidase is most common in cellulolytic microbes (6, 38). bran-grown cell-free culture broth by ammonium sulfate treat- The 3-glucosidase from P. furiosus hydrolyzed lactose very well ment, CM Bio-Gel A agarose column chromatography, and gel (13). Although A. pullulans produced very high levels of filtrations on Bio-Gel A-0.5m and Sephacryl S-200 (Table 2). ,B-glucosidase when grown on lactose (Table 1), no significant On the basis of the assay of 3-glucosidase activity in the activity was found associated with the extracellular or column applicate and washings, about 50% of the 3-glucosi- cell-associated ,-glucosidase. It is interesting that the 3-gluco- dase activity was not adsorbed onto the CM Bio-Gel A agarose sidase from A. pullulans exhibited minimal hydrolysis of p-ni- column. The unadsorbed fraction did not adsorb onto another trophenyl-3-D-cellobioside (Table 3), while it readily hydro- CM Bio-Gel A agarose column preequilibrated at pH 4.0. The lyzed cellooligosaccharides. The enzyme had very little adsorbed 3-glucosidase was separated into two active enzyme 3-xylosidase, L-arabinofuranosidase, and 3-glucuronidase ac- peaks by Bio-Gel A-0.5m gel filtration. These results indicate tivity (less than 1% of that of 3-glucosidase) (Table 3). that the enzyme preparation from A. pullulans contains multi- However, it hydrolyzed p-nitrophenyl-3-D-galactoside at 7.6% ple forms of ,-glucosidase. All these forms of 3-glucosidase of the level of hydrolysis of pNP3G, although it did not could hydrolyze cellobiose, as was evident from the product hydrolyze lactose. According to the criterion of substrate analysis by HPLC. Only the initial two fractions that contained specificity, the P3-glucosidase from A. pullulans belongs to the ,B-glucosidase activity were collected after Sephacryl S-200 gel group of enzymes which hydrolyze both aryl-3-D-glucosides filtration because the latter fractions were contaminated with and cellooligosaccharides (25). Substrate inhibition by cellobi- 3-xylosidase activity. The specific activity of 3-glucosidase from ose is a common property of 3-glucosidases from A. pullulans was 315 U/mg of protein at pH 4.5 and 75°C. The spp. and other microorganisms (33, 34). The 3-glucosidase specific activities of purified 3-glucosidases from various mi- from A. pullulans was not inhibited by up to 20 mM pNP,BG or croorganisms examined by other researchers varied from 5 to 175 mM (6%, wt/vol) cellobiose in the reaction mixture. The 979 U/mg of protein (4, 9, 15, 18, 37). inhibition by glucose, which is a common characteristic of The P-glucosidase from A. pullulans exhibited a molecular ,B-glucosidases (8, 14, 20, 26, 38) although there are exceptions mass of 340,000 Da, with two similar subunits with molecular (1, 9, 24, 39), is an important constraint for industrial use of masses of 165,000 Da (Fig. 2). Work by Rodionova et al. (30) this enzyme. Most of the ,B-glucosidases studied were compet- indicated that the molecular mass of 3-glucosidase from itively inhibited by glucose. Glucose inhibited the 3-glucosi- Trichoderma longibrachiatum was about 350,000 Da and that it dase-catalyzed reaction of cellulase of in a contained at least two subunits (30). The 3-glucosidase from mixed inhibition pattern with a competitive character (23). The Candida pelliculosa var. acetaetherius was 360,000 Da, but it 3-glucosidase of A. pullulans was competitively inhibited by was a tetramer with each subunit 90,000 Da (14). The ,B-glu- glucose, with an inhibition constant (Ki) of 5.65 mM. The cosidase from Piromyces sp. strain E2 was a monomer with a glucose inhibition of 3-glucosidase could probably be over- molecular mass of 45,000 Da (34). The 3-glucosidase of come by employing a combined saccharification and fermen- Botryodiplodia theobromae Pat. (350,000 to 380,000 Da) con- tation process using a glucose fermenting organism. None of sisted of eight subunits (35). Thus, there is considerable the metal ions tested stimulated or inhibited ,-glucosidase diversity in enzyme structure for different microbial ,-glucosi- activity. In contrast to most ,-glucosidase activities, the 3-glu- dases. Periodic acid-Schiff's reagent staining indicated that the cosidase activity from A. pullulans was not affected by the 3-glucosidase from A. pullulans might be glycosylated. The thiol-specific inhibitor (p-chloromercuribenzoic acid). This maximal activity of the purified ,B-glucosidase fromA. pullulans suggests that thiol groups were not essential for 13-glucosidase VOL.V-GLUCOSIDASE60, 1994 FROM AUREOBASIDIUM PULLULANS 3779 activity from A. pullulans. The 3-glucosidase from P. furiosus etaetherius. Agric. Biol. Chem. 49:779-784. was also unaffected by thiol-specific inhibitors (13). 15. Kwon, K.-S., H. G. Kang, and Y. C. Hah. 1992. Purification and Some 13-glucosidases could preferentially utilize alcohols characterization of two extracellular 3-glucosidases from Aspergil- rather than water as for the lus nidulans. FEMS Microbiol. Lett. 97:149-154. acceptors glycosyl moiety during 16. Laemmli, U. K. 1970. Cleavage of structural proteins during the catalysis, resulting in elevated reaction rates (28, 31, 36). The assembly of the head of bacteriophage T4. Nature (London) 3-glucosidase of Dekkera intermedia was also activated by 2 M 227:680-685. ethanol with pNP,BG as the substrate, suggesting that ethanol 17. Leathers, T. D. 1986. Color variants of Aureobasidium pullulans increases the rate of hydrolysis of pNP,BG by acting as an overproduce with extremely high specific activity. Appl. acceptor molecule for the intermediary glycosyl cation (3). The Environ. Microbiol. 52:1026-1030. initial activity of P-glucosidase from A. pullulans was stimu- 18. Li, X., and R. E. Calza. 1991. Purification and characterization lated by ethanol. of an extracellular 1-glucosidase from the rumen fungus Neo- As expected, the ,B-glucosidase from A. pullulans exhibited callimastix frontalis EB188. Enzyme Microb. Technol. 13:622- interaction with cellulase to increase the 628. synergistic efficiency 19. Lineweaver, H., and D. Burk. 1934. The determination of enzyme of glucose production from cellulose by converting cellobiose dissociation constants. J. Am. Chem. Soc. 56:658-666. to glucose. This 3-glucosidase may have utility in enzymatic 20. Lo, A. C., J.-R. Barbier, and G. E. Willick. 1990. Kinetics and hydrolysis of cellulose from corn fiber and other cellulosic specificities of two closely related 3-glucosidases secreted by biomass for the subsequent production of ethanol fuel. Schizophyllum commune. Eur. J. Biochem. 192:175-181. The high activity of the ,B-glucosidase from A. pullulans on 21. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. cellobiose, its ability to hydrolyze a variety of cellooligosaccha- 1951. Protein measurement with the Folin phenol reagent. J. Biol. rides, its high substrate tolerance, its nondependence on metal Chem. 193:265-275. ions or thiol compounds, and its high thermoactivity make the 22. McHale, A., and M. P. Coughlan. 1981. The cellulolytic system of a suitable candidate for in the Talaromyces emersonii. Purification and characterization of the enzyme application enzymatic extracellular and intracellular ,-glucosidases. Biochim. Biophys. hydrolysis of cellulose to glucose. Acta 662:152-159. 23. Montero, M., and A. 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