PROPERTIES OF AN INTRACELLULAR BETA-GLUCOSIDASE PURIFIED FROM THE CELLOBIOSE-FERMENTING YEAST CANDIDA-WICKERHAMII

An intracellular beta-glucosidase was isolated from the cellobiose-fer menting yeast, Candida wickerhamii. Production of the enzyme was stimu lated under aerobic growth, with the highest level of production in a medium containing cellobiose as a carbohydrate source. The molecular m ass of the purified protein was approximately 94 kDa. It appeared to e xist as a dimeric structure with a native molecular mass of about 180 kDa. The optimal pH ranged from 6.0 to 6.5 with p-nitrophenyl beta-D-g lucopyranoside (NpGlc) as a substrate. The optimal temperature for sho rt-term (15-min) assays was 35 degrees C, while temperature-stability analysis revealed that the enzyme was labile at temperatures of 28 deg rees C and above. Using NpGlc as a substrate, the enzyme was estimated to have a K-m of 0.28 mM and a V-max of 525 mu mol product min(-1) mg protein(-1). Similar to the extracellular beta-glucosidase produced b y C. wickerhamii, this enzyme resisted end-product inhibition by gluco se, retaining 58% of its activity at 100 mM glucose. The activity of t he enzyme was highest against aryl beta-1,4-glucosides. However, p-nit rophenyl xylopyranoside, lactose, cellobiose, and trehalose also serve d as substrates for the purified protein. Activity of the enzyme was s timulated by long-chain n-alkanols and inhibited by ethanol, 2-propano l, and 2-butanol. The amino acid sequence, obtained by Edman degradati on analysis, suggests that this beta-glucosidase is related to the fam ily-3 glycosyl hydrolases.