Purification and characterization of the extracellular β-glucosidase produced by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 produced a cell-bound β-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The β-glucosidase contained approximately 30.5% ( w w ) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The K m of the purified β-glucosidase was 6.74 × 10 −2 m for cellobiose and 4.17 × 10 −3 m for p-nitrophenyl-β- d-glucopyranoside. Glucose did not appear to inhibit the enzyme.