Cloning, sequencing and heterologous expression of the gene encoding glucoamylase from Clostridium thermoamylolyticum and biochemical characterization of the recombinant enzyme

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. GO005 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 K subm for maltose was determined to be 5.4 mM, and the k sub0 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 deg C, and more than 80 % of the enzyme activity remained up to 65 deg C.