Cloning, Sequencing and Heterologous Expression of the Gene Encoding Glucoamylase from Clostridium thermoamylolyticum and Biochemical Characterization of the Recombinant Enzyme

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Clostridium thermoamylolyticum glucoamylase gene was overexpressed in Escherichia coli cells. This glucoamylase gene consisted of 2133 bp that encoded a 710-amino-acid protein with a molecular mass of 79,920 Da. The glucoamylase fell into glycoside hydrolase family 15, showing 84% identity and 90% similarity to an amino acid sequence of Clostridium sp. G0005 glucoamylase, and showing 82% identity and 87% similarity to that of Thermoanaerobacterium thermosaccharolyticum glucoamylase. The corresponding sequence to the mature protein was placed under the control of the T7 promoter as a strong and constitutive promoter. The recombinant glucoamylase was purified by a Ni-NTA column. The molecular mass of the mature glucoamylase was 77 kDa by SDS-PAGE, and it was purified 10-fold with a recovery of 65%. The specific activity was determined to be 1.8 U/mg for maltose. The value of Km for maltose was determined to be 5.4 mM, and the k0 was 7.1 s-1. The optimum pH of the enzyme was determined to be 4.5, and more than 80% of the enzyme activity remained between pH 3.5 and 9.0. The optimum temperature was 65°C, and more than 80% of the enzyme activity remained up to 65°C.
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