Purification and Characterization of a Minor Glucoamylase from Aspergillus saitoi

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Abstract
1. From a digestive produced from Aspergillus saitoi, a minor glucoamylase[EC 3.2.1.3] named Gluc M2 was purified in a yield of 67percnt;, besides a major glucoamylase named Gluc M1 (M.W. 90,000) which was previously isolated in a yield of 21%. The purified enzyme was proved homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, ultracentrifugation, immunodiffusion and also from the absence of the glycosidase activities detected in the crude extract. 2. The pH optimum of Gluc M2 was 4.5 with soluble starch as a substrate. The enzyme was stable between pH 2.5 and 7.5 and retained full activity at temperatures up to 50°C. Gluc M2 was denatured almost completely with 5M guanidine hydrochloride but only partially denatured with 8M urea. 3. Gluc M2 was a glycoprotein containing 0.55% glucosamine and 12% neutral sugar which consisted of a large amount of mannose and small amounts of galactose and glucose. The molecular weight of the enzyme was estimated to be about 70,000 by SDS-polyacrylamide gel electrophoresis and its amino acid and sugar compositions. 4. Although differing in both amino acid and sugar compositions and also in molecular weight, Gluc M2 and Gluc M1 had almost identical isoelectric points and showed a common antigenicity in irnmunodiffusion. The N-terminal amino acid sequences of both enzymes were the same: H2N·Ala-Val-Ile-Val-. 5. The kinetic parameters, Km and Vm of Gluc M2 for the low-molecular-weight substrates were similar to those of Gluc M1 In contrast, both Km and Vm, values of Gluc M2 for the high-molecular-weight substrates were consistently larger than those of Gluc M1, the difference was most pronounced when glycogen was the substrate and less pronounced when amylopectin was the substrate.

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