Purification and characterization of an α-glucosidase from Saccharomyces carlsbergensis

Abstract

.alpha.-Glucosidase (EC 3.2.1.20) was purified to homogeneity from logarithmically growing cells of S. carlsbergensis. The purification involved ammonium sulfate fractionation, Sephadex G-100 chromatography, DEAE-cellulose chromatography and hydroxylapatite chromatography. This procedure gave a preparation judged to be greater than 98% pure by Na-DodSO4[sodium dodecylsulfate]-polyacrylamide gel electrophoresis. The enzyme was shown to be a monomer of 63,000 daltons by gel filtration on Sephacryl S-200 under native conditions and by polyacrylamide gel electrophoresis under denaturing conditions. The Km values of the enzyme for the substrates maltose and p-nitrophenyl .alpha.-D-glucoside were 1.66 .times. 10-2 and 3.1 .times. 10-4 M, respectively. The corresponding Vmax value for maltose was 44.8 .times. 10-6 mol min-1 mg-1 and that for p-nitrophenyl .alpha.-D-glucoside was 134 .times. 10-6 mol min-1 mg-1. The pH optimum for the purified enzyme was between pH 6.7 and 6.8. The enzyme has an absolute anomeric specificity for .alpha.-glycosidic linkages and appears to recognize a glucosyl residue in .alpha. linkage on the nonreducing end of its substrate. For the strain used in this study, which carries the MAL 6 locus, only a single form of the enzyme was detected.