Extracellular production of a hybrid beta-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors.

Abstract

Cultivation conditions for the extracellular production of a hybrid beta-glucanase from Bacillus were established by using Escherichia coli JM 109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid beta-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of beta-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of beta-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of beta-glucanase [E. coli JM 109(pLF3)] was tested on the fermenter scale.