Breeding and Incubation of Recombnant Escherichia coli Having Overexpression System of cloned Gene for Effective Production of Glucoamylase

摘要

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 ony a little glucoamylase, the recombinant E., coli BL 21 (DE 3) [PET-12-STA 1] produced about 3 U/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 glucoaylase. The maximum extracellular glucoamylase activity obtained in this work was 6.6 U/ml and this vale was much higher than that secreted in the conventional LB medium, i.e. 0.002 U/ml.