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Sharipova M. - One of the best experts on this subject based on the ideXlab platform.

  • Salt stress induction of Glutamyl Endopeptidase biosynthesis in Bacillus intermedius
    2020
    Co-Authors: Gabdrakhmanova L., Sharipova M., Balaban N., Kostrov S.
    Abstract:

    Bacteria from the genus Bacillus have evolved complicated regulatory networks to be protected from various environmental stresses, including sudden increase in salinity. Among these regulatory mechanisms is the DegS-DegU signal transduction system, which controls degradative enzyme synthesis and is involved in sensing salt stress in Bacillus subtilis. We report the study of biosynthesis regulation of Bacillus intermedius Glutamyl Endopeptidase under salt stress conditions. Salt stress during growth in medium containing 1-2.5 M NaCl, KCl or disodium succinate leads to the induction of Glutamyl Endopeptidase. Analysis of the regulatory region of the gene for B. intermedius Glutamyl Endopeptidase revealed the presence of a tentative target sequence for DegU control, AGATN10TTGAG. For the expression of the Glutamyl Endopeptidase gene, functional DegU protein is required. Thus, we suggest that expression of the gene for B. intermedius Glutamyl Endopeptidase may be controlled by a regulatory system analogous to DegS-DegU two-component system in B. subtilis. © 2005 Elsevier GmbH. All rights reserved

  • Optimization of cultivation medium for the production of Bacillus intermedius 3-19 Glutamyl Endopeptidase | Optimizatsiia sredy kul'tivirovaniia dlia produktsii glutamiléndopeptidazy Bacillus intermedius 3-19.
    2020
    Co-Authors: Gabdrakhmanova L., Sharipova M., Balaban N., Sokolova E., Rudenskaia G.
    Abstract:

    The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of Glutamyl Endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of Glutamyl Endopeptidase

  • Isolation and characterisation of Bacillus amyloliquefaciens H2 Glutamyl Endopeptidase that is secreted in stationary phase of culture growth
    2020
    Co-Authors: Balaban N., Mardanova A., Shamsutdinov T., Malikova L., Ilinskay O., Rudenskay G., Sharipova M.
    Abstract:

    The culture broth of Bacillus amyloliquefaciens H2 was used for isolation by chromatography on CM-cellulose and MonoS columns of proteinase that are secreted in early and late stationary phases of culture growth. Based on their inhibition by specific serine proteinase inhibitors and their action on specific chromogenic substrates and on oxidised B-chain of insulin, the enzymes were classified as Glutamyl Endopeptidases. The molecular weight of secreted enzyme is 26.5 kDa. We show that although the enzyme is secreted in different phases of culture growth, both enzyme fractions have similar enzymatic properties and amino acid content, but they were distinguished in

  • Selection of cultivation medium for production of late stationary phase serine proteinases from Bacillus intermedius
    2020
    Co-Authors: Balaban N., Gabdrakhmanova L., Sharipova M., Sokolova E., Mardanova A., Malikova L., Rudenskaya G.
    Abstract:

    B. intermedius have been shown previously to secrete two serine proteinases: Glutamyl Endopeptidase 2 and subtilisin 2 during the late stationary phase, with maximal levels of the enzymes activities recorded at the 40th and 44th hours of growth, respectively. In the current study, we analyzed the impact of various culture medium components on biosynthesis of these proteinases. Yeast extract and gelatin did not stimulate the enzymes biosynthesis. However, on the medium containing 0.1% casein subtilisin 2 production increased to reach 140%. Biosynthesis of both serine proteinases, produced by B. intermedius at the late stationary phase, were found to be inhibited by individual amino acids, and to be insensitive to catabolite repression. In order to maximise enzyme production, the presence of Ca 2+ and Mg2+ at concentration of 5 mM was shown to be necessary. Based on the results of this work, the composition of a complex culture media for the effective production of late stationary phase proteinases by B. intermedius was developed

  • Screening of Heterologous Signal Peptides for Optimization of the LIKE-Expression System
    2020
    Co-Authors: Tikhonova A., Toymentseva A., Sharipova M.
    Abstract:

    © 2016, Springer Science+Business Media New York.The LIKE-expression system was optimized and used for the production of serine proteinases: subtilisin-like proteinase (AprBp) and Glutamyl Endopeptidase (GseBp) from Bacillus pumilus. Genes of these enzymes were amplified from genomic DNA of the B. pumilus strain 3-19 and cloned into the LIKE-expression system under the control of the PliaI antibiotic-inducible promoter. Two parameters were investigated to increase the yield of secretory enzymes: heterologous signal peptides from B. megaterium (SPPac, SPYngk) and B. subtilis host strain, which is deficient in two major extracellular proteinases (nprE, aprE). Increased production of GseBp with recombinant SPYngk was achieved after 12 h of growth while increased production of AprBp with its own signal peptide after adding bacitracin was achieved after 20 h of growth. These results suggest that optimized LIKE-expression system can be used for heterologous secretory protein production in B. subtilis

I. B. Leshchinskaya - One of the best experts on this subject based on the ideXlab platform.

  • peculiarities of the biosynthesis of bacillus intermedius Glutamyl Endopeptidase in recombinant bacillus subtilis cells during the stationary growth phase
    Microbiology, 2005
    Co-Authors: I. B. Chastukhina, L. A. Gabdrakhmanova, Galina N. Rudenskaya, M. R. Sharipova, N. P. Balaban, S V Kostrov, I. B. Leshchinskaya
    Abstract:

    We studied the biosynthesis of bacillus intermedius Glutamyl Endopeptidase in the recombinant bacillus subtilis strain AJ73 Δ58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase and found that the nutritional requirements for Glutamyl Endopeptidase synthesis were different in the stationary phase and the growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During the final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of Glutamyl Endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.

  • Salt stress induction of Glutamyl Endopeptidase biosynthesis in Bacillus intermedius.
    Microbiological research, 2005
    Co-Authors: L. A. Gabdrakhmanova, Nelly P. Balaban, Margarita R. Sharipova, Sergey V. Kostrov, Innokentii Vishniakov, I. B. Leshchinskaya
    Abstract:

    Summary Bacteria from the genus Bacillus have evolved complicated regulatory networks to be protected from various environmental stresses, including sudden increase in salinity. Among these regulatory mechanisms is the DegS–DegU signal transduction system, which controls degradative enzyme synthesis and is involved in sensing salt stress in Bacillus subtilis . We report the study of biosynthesis regulation of Bacillus intermedius Glutamyl Endopeptidase under salt stress conditions. Salt stress during growth in medium containing 1–2.5 M NaCl, KCl or disodium succinate leads to the induction of Glutamyl Endopeptidase. Analysis of the regulatory region of the gene for B. intermedius Glutamyl Endopeptidase revealed the presence of a tentative target sequence for DegU control, AGATN 10 TTGAG. For the expression of the Glutamyl Endopeptidase gene, functional DegU protein is required. Thus, we suggest that expression of the gene for B. intermedius Glutamyl Endopeptidase may be controlled by a regulatory system analogous to DegS–DegU two-component system in B. subtilis .

  • Optimization of cultivation medium for the production of Bacillus intermedius 3-19 Glutamyl Endopeptidase
    Mikrobiologiia, 2002
    Co-Authors: L. A. Gabdrakhmanova, Nelly P. Balaban, Margarita R. Sharipova, Yu. S. Tokmakova, E. A. Sokolova, Galina N. Rudenskaya, I. B. Leshchinskaya
    Abstract:

    The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of Glutamyl Endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of Glutamyl Endopeptidase.

  • Optimization of Bacillus intermedius Glutamyl Endopeptidase production by recombinant strain of Bacillus subtilis and localization of Glutamyl Endopeptidase in Bacillus subtilis cells
    Enzyme and Microbial Technology, 2002
    Co-Authors: L. A. Gabdrakhmanova, Nelly P. Balaban, Margarita R. Sharipova, Galina N. Rudenskaya, Tatiana V. Akimkina, Sergey V. Kostrov, I. B. Leshchinskaya
    Abstract:

    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 Ca 2+ (2 mM), Mg 2+ (1 mM), and Co 2+ (5 mM) stimulated production of the proteinase by B. subtilis . In case of Co 2+ 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 Fe 2+ , Zn 2+ , and Cu 2+ to the medium at concentrations of 1 to 10 mM 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.

  • optimized medium for the efficient production of bacillus intermedius Glutamyl Endopeptidase by the recombinant bacillus subtilis strain aj73
    Microbiology, 2000
    Co-Authors: L. A. Gabdrakhmanova, Galina N. Rudenskaya, Tatiana V. Akimkina, Sergey V. Kostrov, Eugene V. Shakirov, M. R. Sharipova, N. P. Balaban, I. B. Leshchinskaya
    Abstract:

    A nutrient medium was elaborated for the efficient production of Glutamyl Endopeptidase by the recombinantBacillus subtilis strain AJ73 bearing theBacillus intermedius 3–19 Glutamyl Endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient Glutamyl Endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of Glutamyl Endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of Glutamyl Endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+.

Rudenskaya G. - One of the best experts on this subject based on the ideXlab platform.

  • Optimization of cultivation medium for the production of Bacillus intermedius 3-19 Glutamyl Endopeptidase
    2020
    Co-Authors: Gabdrakhmanova L., Sharipova M., Balaban N., Sokolova E., Tokmakova Y., Rudenskaya G.
    Abstract:

    The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of Glutamyl Endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of Glutamyl Endopeptidase. © 2002 MAIK "Nauka/Interperiodica"

  • Peculiarities of the biosynthesis of Bacillus intermedius Glutamyl Endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase
    2020
    Co-Authors: Sharipova M., Gabdrakhmanova L., Balaban N., Kostrov S., Rudenskaya G.
    Abstract:

    We studied the biosynthesis of Bacillus intermedius Glutamyl Endopeptidase in the recombinant Bacillus subtilis strain AJ73 Δ58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for Glutamyl Endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of Glutamyl Endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different

  • Membrane-bound forms of serine proteases in Bacillus intermedius
    2020
    Co-Authors: Sharipova M., Gabdrakhmanova L., Balaban N., Mardanova A., Shilova M., Rudenskaya G.
    Abstract:

    Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of Glutamyl Endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes

  • Synthesis and secretion of proteinases by Bacillus intermedius in the late stages of sporulation
    2020
    Co-Authors: Balaban N., Gabdrakhmanova L., Sharipova M., Sokolova E., Tokmakova Y., Mardanova A., Rudenskaya G.
    Abstract:

    In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, Glutamyl Endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating β-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites

  • Crystal growth and preliminary X-ray study of glutamic acid specific serine protease from Bacillus intermedius
    2020
    Co-Authors: Blagova E., Balaban N., Rudenskaya G., Shakirov E.
    Abstract:

    The glutamic acid specific protease (Glutamyl-Endopeptidase) from Bacillus intermedius, strain 3-19, was isolated and purified using ion exchange chromatography on CM-cellulose and Mono-S FPLC column. The conditions for crystallization of the enzyme have been discussed. The crystals of enzyme were grown using hanging-drop vapor-diffusion technique. Crystals belong to the space group C2 with unit cell parameters of a = 61.62 Å, b = 55.84 Å, c = 60.40 Å, β = 117.6° X-ray diffraction data to 1.68 Å resolution were collected using synchrotron radiation (EMBL, Hamburg) and an imaging plate scanner. © 1999 Elsevier Science B.V. All rights reserved

Galina N. Rudenskaya - One of the best experts on this subject based on the ideXlab platform.

  • properties of the bacillus pumilus Glutamyl Endopeptidase at different growth stages of its recombinant strain
    Russian Journal of Bioorganic Chemistry, 2013
    Co-Authors: N. P. Balaban, Galina N. Rudenskaya, T. R. Shamsutdinov, A. M. Mardanova, Yu V Danilova, A M Cheremin, M. R. Sharipova
    Abstract:

    The Bacillus pumilus 3–19 Glutamyl peptidase (EC 3.4.21.19) was isolated from the culture medium of the B. subtilis recombinant strain at the following stages of the bacillus growth: a decelerating growth phase and a stationary growth phase. The action of the purified preparations of the enzyme on different phases of its growth was studied on the insulin B-chain and various protein and peptide substrates. Physicochemical properties of the enzyme were compared for different phases of its growth. The Glutamyl Endopeptidase preparations differed in their catalytic characteristics and their sensitivity to the metal cations.

  • The expression of Bacillus intermedius Glutamyl Endopeptidase gene in Bacillus subtilis recombinant strains
    Molecular Biology Reports, 2007
    Co-Authors: M. R. Sharipova, Galina N. Rudenskaya, Ilya V. Demidyuk, E. I. Shagimardanova, I. B. Chastukhina, T. R. Shamsutdinov, N. P. Balaban, A. M. Mardanova, Sergey V. Kostrov
    Abstract:

    The gene encoding for B. intermedius Glutamyl Endopeptidase ( gseBi ) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of Endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius Glutamyl Endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on Endopeptidase expression. Activity of Glutamyl Endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius Glutamyl Endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.

  • peculiarities of the biosynthesis of bacillus intermedius Glutamyl Endopeptidase in recombinant bacillus subtilis cells during the stationary growth phase
    Microbiology, 2005
    Co-Authors: I. B. Chastukhina, L. A. Gabdrakhmanova, Galina N. Rudenskaya, M. R. Sharipova, N. P. Balaban, S V Kostrov, I. B. Leshchinskaya
    Abstract:

    We studied the biosynthesis of bacillus intermedius Glutamyl Endopeptidase in the recombinant bacillus subtilis strain AJ73 Δ58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase and found that the nutritional requirements for Glutamyl Endopeptidase synthesis were different in the stationary phase and the growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During the final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of Glutamyl Endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.

  • The crystal structure of Glutamyl Endopeptidase from Bacillus intermedius reveals a structural link between zymogen activation and charge compensation
    Biochemistry, 2004
    Co-Authors: Rob Meijers, Galina G. Chestukhina, Galina N. Rudenskaya, Tatiana V. Akimkina, Sergey V. Kostrov, Elena Blagova, Vladimir M. Levdikov, Victor S. Lamzin, Inna P. Kuranova
    Abstract:

    Extracellular Glutamyl Endopeptidase from Bacillus intermedius (BIEP) is a chymotrypsin-like serine protease which cleaves the peptide bond on the carboxyl side of glutamic acid. Its three-dimensional structure was determined for C222(1) and C2 crystal forms of BIEP to 1.5 and 1.75 A resolution, respectively. The topology of BIEP diverges from the most common chymotrypsin architecture, because one of the domains consists of a beta-sandwich consisting of two antiparallel beta-sheets and two helices. In the C2 crystals, a 2-methyl-2,4-pentanediol (MPD) molecule was found in the substrate binding site, mimicking a glutamic acid. This enabled the identification of the residues involved in the substrate recognition. The presence of the MPD molecule causes a change in the active site; the interaction between two catalytic residues (His47 and Ser171) is disrupted. The N-terminal end of the enzyme is involved in the formation of the substrate binding pocket. This indicates a direct relation between zymogen activation and substrate charge compensation.

  • Optimization of cultivation medium for the production of Bacillus intermedius 3-19 Glutamyl Endopeptidase
    Mikrobiologiia, 2002
    Co-Authors: L. A. Gabdrakhmanova, Nelly P. Balaban, Margarita R. Sharipova, Yu. S. Tokmakova, E. A. Sokolova, Galina N. Rudenskaya, I. B. Leshchinskaya
    Abstract:

    The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of Glutamyl Endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of Glutamyl Endopeptidase.

M. R. Sharipova - One of the best experts on this subject based on the ideXlab platform.

  • properties of the bacillus pumilus Glutamyl Endopeptidase at different growth stages of its recombinant strain
    Russian Journal of Bioorganic Chemistry, 2013
    Co-Authors: N. P. Balaban, Galina N. Rudenskaya, T. R. Shamsutdinov, A. M. Mardanova, Yu V Danilova, A M Cheremin, M. R. Sharipova
    Abstract:

    The Bacillus pumilus 3–19 Glutamyl peptidase (EC 3.4.21.19) was isolated from the culture medium of the B. subtilis recombinant strain at the following stages of the bacillus growth: a decelerating growth phase and a stationary growth phase. The action of the purified preparations of the enzyme on different phases of its growth was studied on the insulin B-chain and various protein and peptide substrates. Physicochemical properties of the enzyme were compared for different phases of its growth. The Glutamyl Endopeptidase preparations differed in their catalytic characteristics and their sensitivity to the metal cations.

  • isolation and characterisation ofbacillus amyloliquefaciens h2 Glutamyl Endopeptidase that is secreted in stationary phase of culture growth
    Annals of Microbiology, 2008
    Co-Authors: N. P. Balaban, A. M. Mardanova, Liliya A Malikova, Talgat R Shamsutdinov, Olga N Ilinskay, Galina N Rudenskay, M. R. Sharipova
    Abstract:

    The culture broth ofBacillus amyloliquefaciens H2 was used for isolation by chromatography on CM-cellulose and MonoS columns of proteinase that are secreted in early and late stationary phases of culture growth. Based on their inhibition by specific serine proteinase inhibitors and their action on specific chromogenic substrates and on oxidised B-chain of insulin, the enzymes were classified as Glutamyl Endopeptidases. The molecular weight of secreted enzyme is 26.5 kDa. We show that although the enzyme is secreted in different phases of culture growth, both enzyme fractions have similar enzymatic properties and amino acid content, but they were distinguished in.

  • isolation and characteristics of bacillus intermedius Glutamyl Endopeptidase secreted by a recombinant strain of bacillus subtilis at various growth phases
    Russian Journal of Bioorganic Chemistry, 2008
    Co-Authors: T. R. Shamsutdinov, N. P. Balaban, A. M. Mardanova, Yu V Danilova, G N Rudeskaya, M. R. Sharipova
    Abstract:

    The recombinant strain of Bacillus subtilis bearing B. intermedius Glutamyl Endopeptidase gene in multicopy plasmid delta58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca+ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied.

  • Heterologous expression of Bacillus intermedius gene of Glutamyl Endopeptidase in Bacillus subtilis strains defective in regulatory proteins
    Microbiology, 2007
    Co-Authors: E. I. Shagimardanova, I. B. Chastukhina, T. R. Shamsutdinov, N. P. Balaban, A. M. Mardanova, S V Kostrov, M. R. Sharipova
    Abstract:

    Expression of the gene of Glutamyl Endopeptidase from Bacillus intermedius ( gseBi ) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5–5-fold decrease in Glutamyl Endopeptidase activity. It has been concluded that expression of the B. intermedius Glutamyl Endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.

  • The expression of Bacillus intermedius Glutamyl Endopeptidase gene in Bacillus subtilis recombinant strains
    Molecular Biology Reports, 2007
    Co-Authors: M. R. Sharipova, Galina N. Rudenskaya, Ilya V. Demidyuk, E. I. Shagimardanova, I. B. Chastukhina, T. R. Shamsutdinov, N. P. Balaban, A. M. Mardanova, Sergey V. Kostrov
    Abstract:

    The gene encoding for B. intermedius Glutamyl Endopeptidase ( gseBi ) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of Endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius Glutamyl Endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on Endopeptidase expression. Activity of Glutamyl Endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius Glutamyl Endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.