Breeding and Incubation of Recombinant Escherichia coli Having Overexpression System of Cloned Gene for Effective Production of Glucoamylase

Abstract

In order to produce a glucoamylase rapidly and efficiently, the breeding and incubation of recombinant Escherichia coli having a genetically engineered overexpression system were investigated experimentally. The recombinat E. coli BL 21 (DE 3) [pET-12-STA 1] having an overexpression system with a recombinant plasmid (pET-12-STA 1) was constructed by inserting a STA1 gene (a glucoamylase gene) into an overexpression vecter (pET-12). Though the conventional recombinant E. coli JM 109 [pET-12-STA 1] synthesized only a little glucoamylase, the recombinant E. coli BL 21 (DE 3) [pET-12 -STA 1] produced about 3U/ml of glucoamylase. The optimal conditions for producing the glucoamylase were determined by changing the carbon sources, pH, and the metal ions in the culture medium. In a M 9 minimal medium using glucose as a carbon source at pH 7, the recombinant E. coli produced a large amount of glucoamylase. Furthermore, the addition of sodium ions to the culture medium enhanced not only the production but also the secretion of glucoamylase. The maximum extracellular glucoamylase activity obtained in this work was 6.6U/ml and this value was much higher than that secreted in the conventional LB medium, i. e. 0.002U/ml.