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KMID : 0380219850180030222
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Journal of Biochemistry and Molecular Biology
1985 Volume.18 No. 3 p.222 ~ p.232
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Purification and Characterization of Glucoamylase from Aspergillus shirousamii
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Na Byung-Gook
Yang Chul-Hak
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Abstract
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Glucoamylase ($\alpha$-1,4 glucan glucohydrolase, EC 3.2.1.3) of Aspergillus shirousamii was purified using ammonium sulfate fractionation, DEAE-Sephacel chromatography, affinity chromatography on Concanavalin-A Sepharose 4B and isoelectric focusing. The purification achieved was 19.8 fold from crude extract with a yield of 47.5%. Its molecular weight was estimated to be about 89,000 by Sephadex G-200 gel filtration and SDS-polyacrylamide gel electrophoresis. The pI value of the enzyme was 3.4. The enzyme was stable below $55^{\circ}C$ and retained almost full activity after 1 h. But, it was very labile over $70{\circ}C$. At this temperature, it lost almost all activity witin 10 min. Maximum hydrolysis was occured at $65^{\circ}C$ for 15 min and $60^{\circ}C$ for 60 min, respectively. The enzyme was most stable at pH 4.5, and was more stable in the acidic region than in neutral region. The pH optimum of the enzyme was 4.5 with p-nitrophenyl $\alpha$-D-glucopyranoside as a substrate. The enzyme activity was inhibited by $Hg^{++}$, $Ca^{++}$ markedly increased the heat stability of the enzyme, but it did not affect on the enzyme activity. Most of the anions tested showed the inhibition effect on the enzyme activity. Among these, $B_4{O_7}^{-2}$, $As{O_2}^-$ and $S{O_3}^{-2}$ inhibited remarkably up to 94%, 95% and 88%, respectively. In kinetic studies, the enzyme hydrolyzed maltotriose more than twice of the rate for maltose. Isomaltose and p-nitrophenyl $\alpha$-D-glucopyranoside showed much lower relative rate of hydrolysis than maltose, 0.15 and 0.066, respectively. For the highmolecular-weight substrates, amylopectin showed the highest and dextrin showed the lowest relative rate of hydrolysis, respectively.
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KEYWORD
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