Process optimization of alpha-amylase production by recombinant Bacillus subtilis using immobilization systems

Recombinant microorganisms have a great potential in the production of valuable proteins. Bacillus subtilis harboring the multicopy plasmid pC194 possessing an {dollar}alpha{dollar}-amylase gene and chloramphenicol resistance was used for the optimization of recombinant protein production.; Growth and sporulation characteristics of this strain were different from the untransformed strain. A greater tendency to lyse after stationary phase and a tendency to shed the amylase gene on sporulation leading to little amylase activity in the resulting were the unique features of this strain.; To evolve a strategy to optimize amylase production in recombinant cells, the regulatory mechanisms of gene expression were studied. Catabolite repression was not a dominating mechanism, unlike in untransformed strains where it inhibits the temporal activation of amylase. Temporal activation was induced by presenting conditions that favor initiation of sporulation, as both are triggered by similar conditions. Temporal activation was not inhibited by catabolite repression. Temporal turn off occurred later than that in untransformed strains.; Based on these results, a three-stage immobilized culture system was developed, where calcium alginate immobilized recombinant Bacillus subtilis was grown to high cell densities, starved by limited supply of nutrients to induce temporal activation, and then provided with enough nutrients to produce the amylase. An optimal operating strategy was evolved, both with respect to the nature and amount of nutrient that has to be supplied, as well as the duration of the various stages.; Maltose uptake rates were analysed in free cell cultures and immobilized cultures. The free cells had a higher rate of uptake compared to immobilized cells, and the uptake rates were higher in the starvation stage compared to the growth or production stages in three stage cultures.; A modified hollow fiber reactor was fabricated, and evaluated. A thin coating of alginate on the shell side of the hollow fiber was used to entrap the recombinant cells. Preliminary experiments indicated a good potential for its use in the production of valuable recombinant proteins, as the plasmid stabilities were comparable or higher than the bead immobilization system, with minimal cell leakage. In addition this system exhibited higher productivities on a per volume reactor basis.