PRODUCTION, PURIFICATION, AND PROPERTIES OF A THERMOSTABLE BETA-GLUCOSIDASE FROM A COLOR VARIANT STRAIN OF AUREOBASIDIUM-PULLULANS

A color variant strain of Aureobasidium pullulans (NRRL Y-12974) produ ced beta-glucosidase activity when grown in liquid culture on a variet y of carbon sources, such as cellobiose, xylose, arabinose, lactose, s ucrose, maltose, glucose, xylitol, xylan, cellulose, starch, and pullu lan. An extracellular beta-glucosidase was purified 129-fold to homoge neity from the cell-free culture broth of the organism grown on corn b ran. The purification protocol included ammonium sulfate treatment, CM Bio-Gel A agarose column chromatography, and gel filtrations on Bio-G el A-0.5m and Sephacryl S-200. The beta-glucosidase was a glycoprotein with native molecular weight of 340,000 and was composed of two subun its with molecular weights of about 165,000. The enzyme displayed opti mal activity at 75 degrees C and pH 4.5 and had a specific activity of 315 mu mol.min(-1).mg of protein(-1) under these conditions. The puri fied beta-glucosidase was active against p-nitrophenyl-beta-D-glucosid e, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose , and celloheptaose, with K-m 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-i = 5.65 mM), while fructose, arabinose, gala ctose, mannose, and xylose (each at 56 mM) and sucrose and lactose (ea ch at 29 mM) were not inhibitory. The enzyme did not require a metal i on for activity, and its activity was not affected by p-chloromercurib enzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol (7. 5%, vol/vol) stimulated the initial enzyme activity by 15%. Glucose pr oduction was enhanced by 7.9% when microcrystalline cellulose (2%, wt/ vol) was treated for 48 h with a commercial cellulase preparation (5 U /ml) that was supplemented with the purified beta-glucosidase (0.21 U/ ml) from A. pullulans.