PRODUCTION OF THERMOPHILIC ALPHA-AMYLASE USING IMMOBILIZED TRANSFORMED ESCHERICHIA-COLI BY ADDITION OF GLYCINE

Efficient production of thermophilic alpha-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with kappa-carrageenan by the addition of glycine. The effects of glycine, the concentrations of kappa-carrageenan and KCl on the production of the enzyme as well as the stability of plasmid pH1301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of alpha-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term "release", while diffusion-out from gel beads is referred to by the term "liberate".) Concentrations of KCl and immobilizing support significantly affected on the liberation of alpha-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCl, which is a hardening agent for carrageenan. Although the viability of cells and alpha-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of alpha-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.