In Fig results of this experiment are shown
In Fig. 1 results of this experiment are shown as the contour plots of endopeptidase activity. Optimal for the enzyme production by this strain concentrations of peptone and inorganic phosphate were found to be 24 and 0.3g/l, respectively. For specific activity, the optima close to those for glutamyl endopeptidase activity have been observed. For B. subtilis strain carrying pV plasmid, the optimal concentrations of peptone and inorganic phosphate were earlier found to be 17.5 and 0.36g/l, respectively . Thus, for effective biosynthesis of glutamyl endopeptidase by both recombinant strains, significantly higher degree of maintenance of the inorganic phosphate in the medium was necessary than that for glutamyl endopeptidase synthesis by native B. intermedius strain . To reach a higher yield of endopeptidase production by B. subtilis cells, we used yeast extract as a source of nitrogen, microelements, and vitamins (Fig. 2). The addition of yeast extract into the medium essentially stimulated endopeptidase production. The optimal concentration of yeast extract for glutamyl endopeptidase production was found to be 2%. Therefore, the medium containing 2% of yeast extract was used for cultivation of B. subtilis AJ73 Δ58.21. For B. intermedius and B. subtilis pV, the combination of organic (peptone) and inorganic (NH4Cl) nitrogen sources in the medium has been reported to be favorable for the growth of direct renin inhibitors and glutamyl endopeptidase production , . The effect of nitrogen concentration on proteinase synthesis by recombinant strain carrying Δ58.21 plasmid was examined in the medium containing NH4Cl (Fig. 3). In contrast with the data obtained earlier, the presence of NH4+ at concentrations of 1 to 5mM in the medium for B. subtilis AJ73 Δ58.21 did not change the efficiency of endopeptidase biosynthesis by this strain. Most probably, this strain did not need the additional nitrogen source for active glutamyl endopeptidase production because the medium for its cultivation contained high concentration of yeast extract. Fig. 4 (a,b,c) describe the mechanism of growth of B. subtilis AJ73 Δ58.21 and of glutamyl endopeptidase biosynthesis by this strain, using the elaborated culture media in comparison with those of B. intermedius and B. subtilis AJ73 pV. Glutamyl endopeptidase appeared in the culture liquid of both B. subtilis strains at the stage of slowing down of the growth of the culture. Proteinase activity reached a maximum at the late stationary phase of growth. The specific rate of proteinase formation increased in the period, when the specific rate of growth decreased. Thus, the mechanism of glutamyl endopeptidase biosynthesis in recombinant B. subtilis strains is analogous to that in B. intermedius. But it must be stressed that glutamyl endopeptidase activity of recombinant cells reached its maximum much later than in the case of the native strain: at the 20th hour of cultivation for the strain carrying plasmid Δ58.21, at 22nd hour for the strain with a bigger plasmid pV. Besides, glutamyl endopeptidase appeared in the culture liquid of B. subtilis strains later than in the culture liquid of B. intermedius. This can be due to the changes in metabolism of the cells, expressing foreign genes from the plasmids. A number of workers reported the repression of biosynthesis of different serine preoteinases with glucose and the related substrates , . Repression of B. intermedius glutamyl endopeptidase biosynthesis by easily metabolizable carbon sources has been demonstrated as well , . On the other hand, the presence of glucose sometimes happened to be favorable for proteinase biosynthesis . We analyzed the effect of a set of monosaccharides (glucose, galactose) and disaccharides (sucrose, maltose, lactose) on the growth and glutamyl endopeptidase production of B. subtilis AJ73 Δ58.21 strain. In all cases, the addition of carbohydrates into the culture medium strongly stimulated growth of B. subtilis, while suppressing the biosynthesis of proteinase. So, the biosynthesis of glutamyl endopeptidase by recombinant strain was suppressed by easily metabolizable carbon sources in a similar manner to glutamyl endopeptidase synthesis by B. intermedius.