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  • Metal ions are known not to

    2019-08-22

    Metal ions are known not to be necessary for catalytic activity of serine proteinases. Nevertheless, Co2+ and Ca2+ ions were found to stabilize glutamyl endopeptidase molecule [1], [7], [19]. In our previous study, we have described significant stimulating effect of Ca2+, Mg2+, and Co2+ on biosynthesis of glutamyl endopeptidase of B. intermedius[8], [10]. In the present work we investigated the effect of divalent metal ions on the biosynthesis of glutamyl endopeptidase by recombinant strain of B. subtilis, carrying the complete gene for glutamyl endopeptidase on Δ58.21 plasmid (Fig. 5). Ca2+ ions, being present in the medium at concentration of 2mM, caused an increase of 25% in the enzyme production (the initial medium contained 1mM of Ca2+). Increase in Ca2+ concentration resulted in a decrease of endopeptidase production as in the case of B. intermedius strain [8]. Studying the effect of Mg2+ on biosynthesis of glutamyl endopeptidase by recombinant strain, maximal production of the enzyme was observed at 1mM of Mg2+. The Mn2+ ions failed to affect endopeptidase biosynthesis by this strain. The addition into the medium of Fe2+, Zn2+, and Cu2+ at concentrations of 1 to 10mM led to the gradual decrease in glutamyl endopeptidase production. As for the presence of Co2+ in the culture medium, it methylphenol caused strong stimulating effect on the production of the enzyme, enhancing glutamyl endopeptidase specific activity four-fold. Maximal level of glutamyl endopeptidase production by B. subtilis (400%) was indicated at 5mM of Co2+. Simultaneously the presence of Co2+ ions in the medium inhibited the growth of recombinant cells. As stated by Sharipova et al. for B. intermedius 3-19[20], CoCl2 promoted the release of the membrane-bound enzyme and formation of extracellular protein. Thus, the increase of the extent of glutamyl endopeptidase production was not the result of stimulation of biosynthesis process. It is likely that for both recombinant B. subtilis strains, the same mechanism of action of Co2+ takes place. Up to now, many evidences are available in the literature regarding the role of complicated organic substrates in the biosynthesis of catabolic enzymes [12], [21], [22]. We have shown previously the absence of any measurable effect of organic substrates, such as casein, gelatin, and hemoglobin, on the intensity of glutamyl endopeptidase biosynthesis of B. intermedius[8]. In contrast, strong stimulating effect of casein and gelatine on the synthesis of endopeptidase by B. subtilis AJ73 pV was reported [10]. Now the effect of the above-mentioned substrates on the biosynthesis of glutamyl endopeptidase by recombinant strain carrying Δ58.21 was investigated. Casein, gelatin, and hemoglobin were added to the culture medium at concentrations of 0.5 to 2%. In the presence of any concentrations of hemoglobin glutamyl endopeptidase activity as well the productivity of B. subtilis cells did not change. When the cells were cultivated in the medium, containing casein or gelatin, a strong stimulating effect on proteinase synthesis was determined (Fig. 6). In case of B. subtilis strain carrying plasmid Δ58.21, the addition of 1% of casein enhanced the enzyme production for 40%. The addition of 1% of gelatin led to 1.5-fold increase in specific activity of glutamyl endopeptidase by this strain. Stimulating endopeptidase production, these organic substrates did not affect the effectiveness of growth of both recombinant strains. Thus, the response of glutamyl endopeptidase synthesis of B. subtilis recombinant strains to the presence of complicated organic substrates in culture medium differs from that of the native B. intermedius strain. In contrast with B. intermedius, the biosynthesis of glutamyl endopeptidase by both B. subtilis strains was found to be activated by casein and, in more extent, by gelatin. Due to the higher stimulating effect of the gelatin, it was decided to be included into the optimal medium for cultivation of B. subtilis cells.