Glucoamylase frgm Tricli.oderma viride

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  • OKADA Gentaro
    Department of Biology , Faculty of Education, Shizuoka University

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  • ?EＴｒｉｃｈｏｄｅｒｍａ　ｖｉｒｉｄｅ?Fの生産するグルコアミラーゼ
  • Trichoderma viride ノ セイサンスル グルコアミラーゼ ニカ

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Abstract

The major amylolytic enzyme present in Meicelase, a commercial crude cellulase preparation from Trichoderma viride; was purified by consecutive column chromatography, and characterized as a glucoamylase [EC 3.2.1.3]. The specific activity was brought to 18 .3 units of soluble starch-saccharif ying activity/mg of enzyme protein, and the enzyme showed a single band on polyacrylamide gel disc electrophoresis. Some properties of the purified glucoamylase were investigated. The molecular weight of the enzyme was estimated to be about 75, 000 on the basis of SDS polyacrylamide gel electrophoresis. The optimum pH and the optimum temperature for the activity of the enzyme were pH 5.0-5.5 and 60°C, respectively. The enzyme was stable over the range pH 5.0-7.0 at 4°C and was completely inactivated by heating at 90°C for 10 min. Hg2+ completely inhibited the enzyme, but other metal ions tested had little effect on the activity at the concentration of ions used (1 mM) . The action of the enzyme on glycogen, amylopectin, soluble starch, short chain amylose (DP =17.3), maltose, isomaltose and panose was studied. Glucose was the sole hydrolysis product found in digests of these substrates. At the same substrate concentration (0.075%, w/v) and enzyme concentration, the relative initial rates of glucose production from amylopectin, soluble starch, short chain amylose and maltose were about in the proportions 8 : 8 : 8 : 1. The Km and Vmax values at 30°C and pH 5.0 were calculated for the enzyme acting on glycogen, amylopectin, amylose, soluble starch, short chain amylose and maltose. By using the authentic anomers of D-glucopyranose under conditions limiting mutarotation, it was found that the condensation reactions catalyzed by glucoamylase require a donor substrate of specific configuration. The purified enzyme, which hydrolyzes amylaceous substrates to β-D-glucopyranose, was found to catalyze the rapid synthesis of maltose and isomaltose specifically from β-D-glucopyranose. The configurational inversion accompanying the condensations indicates that the D-glucopyranosyl portion of β-D-glucopyranose is interchanged with hydrogen at the C4 or C6 carbinol site of a second D-glucose molecule (glucosyl transfer).

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