Amylolytic enzymes produced by a color variant strain ofAureobasidium pullulans

Abstract

A color variant strain ofAureobasidium pullulans (NRRL Y-12974) produced amylase and α-glucosidase activities when grown at 28°C for 4 days in liquid culture on a wide variety of carbon sources such as starch, pullulan, glucose, maltose, cyclodextrins, sucrose, xylose, and xylan. An α-glucosidase was separated by Q-Sepharose adsorption from the cell-free culture broth and partially purified by hydroxylapatite and octyl-Sepharose chromatography. After ammonium sulfate treatment of the culture supernatant (obtained after Q-Sepharose adsorption), the amylase fraction was separated into three active fractions by hydroxylapatite column chromatography, which were identified as α-amylase, glucoamylase A, and glucoamylase B. The glucoamylase A was further purified by octyl-Sepharose column chromatography. The pH optima for the action of α-amylase, glucoamylase A, glucoamylase B, and α-glucosidase were 5.0, 4.5, 4.0–4.5, and 4.5, respectively. The α-amylase and glucoamylase B were fully stable at pH 3.0–6.0, glucoamylase A at pH 4.5–5.5, and α-glucosidase at pH 3.5–7.0 for 1 h at 50°C. The optimum temperatures for the action of these enzymes were 55°, 50°–60°, 65°, and 65°C, respectively. The α-amylase, glucoamylase A, and glucoamylase B were adsorbed onto raw corn starch and degraded it. Glucoamylase B readily cleaved pullulan. The α-glucosidase was not adsorbed onto raw starch and did not degrade it at all. It hydrolyzed both α-1,4 and α-1,6 linkages in oligosaccharides. All four enzymes did not require any metal ion for activity and were inhibited by cyclodextrins (α-and β-, 10mm).