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Appl Environ Microbiol. 1985 December; 50(6): 1474-1482
Copyright © 1985, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of Extracellular Amylolytic Enzymes from the Yeast Filobasidium capsuligenum

Laboratory of Industrial Microbiology and Biochemistry, University of Leuven, B-3030 Leuven, Belgium

ABSTRACT

The extracellular amylolytic system of Filobasidium capsuligenum consisted of an -amylase (1,4--D-glucan glucanhydrolase, EC 3.2.1.1) and two forms of glucoamylase (1,4--D-glucan glucohydrolase, EC 3.2.1.3). The enzymes were purified by ammonium sulfate fractionation, repeated ion-exchange chromatography (DEAE-Sephadex A-50), and gel filtration (Sephadex G-25, Sephadex G-100 sf). -Amylase had an optimum pH of 5.6 and an optimum temperature of 50°C but was rapidly inactivated at higher temperature. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 64,000. An acarbose concentration of 20 µg/ml was required for 50% inhibition of the -amylase. Both glucoamylases are glycoproteins of identical molecular weight (60,000) and produce only glucose by exohydrolysis. The debranching activity of the glucoamylases was evidenced with substrates containing -1,6 linkages. The pH optima were 5.0 to 5.6 for glucoamylase I and 4.8 to 5.3 for glucoamylase II. Glucoamylase I had a higher optimum temperature (55°C) than glucoamylase II (50°C) and was also more resistant to thermal inactivation. Only low acarbose concentrations (<0.1 µg/ml) were required to reduce the activity of the glucoamylases by 50%.

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