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    2019-09-11


    Conflict of interest
    Acknowledgements We wish to thank Arantza Pérez (University of the Basque Country) for her technical contribution to this study. This work was supported by grants from the Jesús Gangoiti-Barrera Foundation, Gobierno Vasco (GIC07/84), MEC () and SAIOTEK (SA-2008/00046).
    Introduction Glutamyl endopeptidases (Glu, Asp-specific proteases; EC 3.4.21.19) constitute a new, recently discovered subfamily within chymotrypsin family of serine proteases. These enzymes possess narrow substrate specificity and split only the peptide bonds formed by α-carboxyl groups of glutamyc and aspartic Alfacalcidol [1]. Glutamyl endopeptidases have been isolated from Staphylococcus, Streptomycetes and Bacilli[2], [3], [4]. These are secreted proteins of 18–29kDa, their pI varying in a wide range of pH values. Enzymatic properties of glutamyl endopeptidases are thoroughly studied [5]. The most known commercially available representative of Glu, Asp-specific proteases is V8 proteinase from Staphylococcus aureus[6]. So, structure and functions of bacterial glutamyl endopeptidases are well enough studied, whereas their biological role is still unclear and too little is currently known about the mechanisms of regulation of biosynthesis of these enzymes. Thus, further research of the biosynthesis of bacterial glutamyl endopeptidases would be desirable. Recently we isolated and characterized glutamyl endopeptidase from streptomycin-resistant strain Bacillus intermedius 3-19[7]. The pathways of its biosynthesis regulation and the location of the enzyme in B. intermedius cells were described [8]. The gene encoding for glutamyl endopeptidase of B. intermedius 3-19 was cloned in B. subtilis. Two recombinant plasmids have been obtained: pV and Δ58.21, differing in the size of DNA from B. intermedius 3-19 chromosome [9]. Earlier we studied the character of biosynthesis of glutamyl endopeptidase from B. intermedius in B. subtilis strain carrying plasmid pV. Plasmid pV contains 6.2kb fragment from B. intermedius chromosomal DNA [10]. In this fragment some additional regions were present besides the gene for glutamyl endopeptidase. In contrast, the plasmid Δ58.21 is 2.6kb in size and contains strictly the gene for glutamyl endopeptidase. The differences in the character of biosynthesis of glutamyl endopeptidase by two recombinant strains obtained could suggest the presence of regulatory elements in B. intermedius chromosome DNA fragment included into pV plasmid. The objective of the present work was to optimize the culture medium for maximum yields of glutamyl endopeptidase by recombinant B. subtilis AJ73 strain carrying Δ58.21 plasmid, to study the pathways of biosynthesis of B. intermedius glutamyl endopeptidase in the recombinant strain and the location of the enzyme in B. subtilis cells.
    Materials and methods
    Results and discussion The presence of recombinant plasmids is shown to affect the level of individual metabolites in the cell and lead to the total changes in cell metabolism [14], [15]. The cells carrying plasmids spend the part of their resources, which are normally used for the growth and reproduction, for the replication of heterologous plasmids and expression of the foreign genes [16]. This results in the increased requirements for nutrients by recombinant cells. Therefore, recombinant strains usually are cultivated on the culture media enriched with the sources of phosphate, nitrogen, microelements, and vitamins [11]. The first step of our study was to work out the medium which would yield the maximum glutamyl endopeptidase production by B. subtilis AJ73 Δ58.21 strain, since it showed slight growth and enzyme production on the initial culture medium. As an efficient technique for optimization of culture medium, we have employed the response surface methodology [13]. To find the optimum conditions for glutamyl endopeptidase production by B. subtilis, the surface response for the enzyme activity as a function of the selected key factors had to be determined. In our experiments, peptone (X1) and inorganic phosphate (X2) were selected as key factors. The experiments were organized in B2 factorial design when two factors varied on three levels. The different levels of each factor in the form of both coded values and natural values and the measured response for each test (biomass, glutamyl endopeptidase activity, and specific glutamyl endopeptidase activity) for B. subtilis AJ73 Δ58.21 are described in Table 1. The regression equation obtained for glutamyl endopeptidase activity was expressed as follows (with confidence probability of 95%):where Y is the measured response (peptidase activity) and X1 and X2 are coded independent variables.