Purification of an exo-β-D-glucanase from cell-free extracts of Candida utilis

Abstract

.beta.-Glucanase present in cell-free extracts from C. utilis was isolated and purified 562-fold by procedures that include adsorption on DEAE-Sephadex A-50 and filtration through columns of Sephadex G-50, G-100 and G-200, Bio-Gel P-10, and Concanavalin A-Sepharose 4B. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (s20,w [Svedberg coefficient, 20.degree. C in water] = 1.74 S). The enzyme behaved as an acidic glycoprotein (pI [isoelectric point] 4.1) with 68% carbohydrate and a high content of acidic amino acids. The estimated MW was 20,000 from gel filtration and polyacrylamide-gel electrophoresis and 36,000 from sedimentation experiments. Studies on the hydrolysis of different substrates showed that the enzyme is an unspecific .beta.-glucanase able to break down both (1 .fwdarw. 3)-.beta.- and (1 .fwdarw. 6)-.beta.-linkages by an exo-splitting mechanism. Glucono-.delta.-lactone, Zn2+ and Hg2+ inhibited the enzyme activity.