Optimization of Bacillus intermedius glutamyl endopeptidase production by recombinant strain of Bacillus subtilis and localization of glutamyl endopeptidase in Bacillus subtilis cells

Abstract

The biosynthesis of glutamyl endopeptidase from Bacillus intermedius 3-19 in recombinant strain of Bacillus subtilis has been investigated. The composition of culture medium, which yielded the maximum glutamyl endopeptidase production by B. subtilis strain, was developed, employing response surface methodology. The pathways of regulation of glutamyl endopeptidase synthesis in recombinant strain in general were found to be similar to those of other serine proteinases and of glutamyl endopeptidase in B. intermedius. Biosynthesis of glutamyl endopeptidase by recombinant strain was suppressed by easily metabolizable carbon sources. Ions of Ca2+(2mM), Mg2+ (1mM), and Co2+ (5mM) stimulated production of the proteinase by B. subtilis. In case of Co2+ ions strong stimulating effect (up to 400%) possibly was due to the release of the membrane-bound enzyme into the culture liquid, according to the mechanism described earlier for B. intermedius. The addition of Fe2+, Zn2+, and Cu2+ to the medium at concentrations of 1 to 10mM led to the gradual decrease in proteinase production by B. subtilis. This study has demonstrated a requirement by recombinant strain for excess carbon, nitrogen, and inorganic phosphate for active glutamyl endopeptidase production. In contrast with B. intermedius, for the maximum yield of endopeptidase by B. subtilis the presence in the culture medium of yeast extract at concentration of 2% and one of the organic substrates of proteinase - casein or gelatin (1%) was found to be necessary. Our study has revealed the changes in the pathways of secretion of glutamyl endopeptidase of B. intermedius by B. subtilis cells, expressing the gene for glutamyl endopeptidase from the plasmids: the part of the enzyme (2-5%) remained bound to the cell wall. © 2002 Elsevier Science Inc. All rights reserved.