In the previous study glutamyl endopeptidase of
In the previous study glutamyl endopeptidase of B. intermedius has been reported to be distributed in the NHS-SS-Biotin of B. intermedius as follows: 90% of synthesized enzyme has been detected in the culture liquid, 9.3% of glutamyl endopeptidase—in the fraction of membrane proteins . The distribution of glutamyl endopeptidase in the cells of both recombinant strains in comparison with that in B. intermedius cells is presented in Table 2. The location of glutamyl endopeptidase of B. intermedius in recombinant B. subtilis strains happened to be different from native strain. Catalytic activity was not observed in cytoplasm of B. subtilis at all. At the same time, the relative maintenance of membrane-bound glutamyl endopeptidase in the cells of B. subtilis AJ73 Δ58.21 increased in comparison with that of native B. intermedius strain. In contrast with native strain, glutamyl endopeptidase was detected in the cell wall of both recombinant strains: 2% from the total amount of the enzyme in case of B. subtilis AJ73 pV and 5% in case of B. subtilis AJ73 Δ58.21. This finding supported the idea that in recombinant strains glutamyl endopeptidase was also released from membrane to the medium, but partly remained bound to the cell wall. In Table 3 the results of study of proteolytic activity against specific substrate for subtilisin (Z-Ala-Ala-Leu-pNA) are shown. Subtilisin was found in the membrane fraction of B. subtilis cells, suggesting that the recipient for recombinant plasmids strain B. subtilis AJ73 produced into the culture medium its own subtilisin.
Thus, 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 remained bound to the cell wall.
At the same time, in both recombinant strains of B. subtilis carrying the complete gene for glutamyl endopeptidase on plasmids, the pathways of regulation of glutamyl endopeptidase synthesis in general were shown to be similar to those of other serine proteinases and of glutamyl endopeptidase in B. intermedius 3-19. 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+ into the medium at concentrations of 1 to 10mM led to the gradual decrease in glutamyl endopeptidase production by B. subtilis AJ73 Δ58.21. This study has demonstrated a requirement by recombinant strains 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 strains 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%), were found to be necessary.
The composition of culture media for effective production of glutamyl endopeptidase by recombinant strains of B. subtilis developed on the base of the results of this work in comparison with that for B. intermedius 3-19 are summarized in Table 4.