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1.
Biochemistry (Mosc) ; 81(8): 785-93, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27677548

ABSTRACT

ß-Propeller phytases of Bacillus are unique highly conservative and highly specific enzymes capable of cleaving insoluble phytate compounds. In this review, we analyzed data on the properties of these enzymes, their differences from other phytases, and their unique spatial structures and substrate specificities. We considered influences of different factors on the catalytic activity and thermostability of these enzymes. There are few data on the hydrolysis mechanism of these enzymes, which makes it difficult to analyze their mechanism of action and their final products. We analyzed the available data on hydrolysis by ß-propeller phytases of calcium complexes with myo-inositol hexakisphosphate.


Subject(s)
6-Phytase , Bacillus/enzymology , Bacterial Proteins , 6-Phytase/chemistry , 6-Phytase/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Enzyme Stability/physiology , Hydrolysis , Phytic Acid/chemistry , Phytic Acid/metabolism , Substrate Specificity/physiology
2.
Biochemistry (Mosc) ; 81(8): 884-91, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27677556

ABSTRACT

Features of gene expression of the secreted Bacillus pumilus metalloendopeptidase belonging to the adamalysin/reprolysin family were investigated. In the regulatory region of the gene, we identified hypothetical binding sites for transcription factors CcpA and TnrA. We found that the expression of the metalloendopeptidase gene is controlled by mechanisms of carbon and nitrogen catabolite repression. In experiments involving nitrogen metabolism regulatory protein mutant strains, we found that the control of the metalloendopeptidase gene expression involves proteins of ammonium transport GlnK and AmtB interacting with the TnrA-regulator.


Subject(s)
Bacillus pumilus/enzymology , Bacterial Proteins/biosynthesis , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Metalloendopeptidases/biosynthesis , Bacillus pumilus/genetics , Bacterial Proteins/genetics , Metalloendopeptidases/genetics , Nitrogen/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Bioorg Khim ; 39(1): 46-54, 2013.
Article in Russian | MEDLINE | ID: mdl-23844506

ABSTRACT

Bacillus pumilus 3-19 glutamylendopeptidase has been isolated from culture liquid of Bacillus subtilis recombinant strain on different growth stages: growth retardation (early enzyme) and stationary phase (late enzyme). The effect of purified proteinase of different growth stages on insulin beta-chain, protein and oligopeptide substrates has been studied. Comparative study of physicochemical properties of early and late proteinases was carried out. Two protein fractions were different in catalytic characteristics and demonstrated different sensitivity to the presence of metal cations.


Subject(s)
Bacillus/enzymology , Serine Endopeptidases/isolation & purification , Subtilisin/isolation & purification , Amino Acid Sequence , Bacillus/genetics , Bacillus/growth & development , Catalysis , Cations/chemistry , Insulin/chemistry , Metals/chemistry , Molecular Sequence Data , Serine Endopeptidases/metabolism , Substrate Specificity , Subtilisin/genetics , Subtilisin/metabolism
4.
Bioorg Khim ; 39(5): 552-7, 2013.
Article in Russian | MEDLINE | ID: mdl-25702412

ABSTRACT

Here wediscuss known properties of metzincin metalloproteinases, their structure, physiological roles in the cell and potential medical uses. We also present results describing a novel extracellular metzincin metalloproteinase from Bacillus pumilus with a unique combination of properties typical for both astacins and adamalysins.


Subject(s)
Bacillus/enzymology , Metalloproteases/chemistry , Protein Structure, Tertiary , Zinc/metabolism , Amino Acid Sequence , Humans , Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Metalloproteases/metabolism , Metalloproteases/therapeutic use , Protein Conformation
5.
Bioorg Khim ; 39(4): 430-6, 2013.
Article in Russian | MEDLINE | ID: mdl-24707724

ABSTRACT

Bacillus ginsengihumi phytase has been firstly isolated and studied from the recombinant Escherichia coli strain cellular lysates. The enzyme was obtained from the cellular lysate, purified till homogeneous condition, primary structure was determined. It's concluded that phytase relates to beta-propeller class of phosphatases. The molecular weight of the protein was 41 kDa, pI was 4.8. Some physical and chemical properties of the enzyme were studied.


Subject(s)
6-Phytase , Bacillus/enzymology , Escherichia coli/enzymology , 6-Phytase/chemistry , 6-Phytase/isolation & purification , Amino Acid Sequence , Enzyme Stability , Hydrogen-Ion Concentration , Substrate Specificity , Temperature
6.
Bioorg Khim ; 38(4): 439-48, 2012.
Article in Russian | MEDLINE | ID: mdl-23189558

ABSTRACT

Heterologous gene expression of extracellular minor metalloendopeptidase of Bacillus pumilus 3-19 in protease-deficient B. subtilis strain has been studied. The fraction of enzyme in total pool of B. pumilus 3-19 secreted proteases composes less than 8%. The enzyme was isolated from culture liquid of recombinant strain, its primary structure was determined, physicochemical properties were investigated. It was concluded that secreted metallo endopeptidase of B. pumilus 3-19 represents the first prokaryotic homolog of eukaryotic adamalysin/reprolysin protein family.


Subject(s)
Bacillus/enzymology , Metalloendopeptidases/chemistry , Metalloendopeptidases/isolation & purification , Amino Acid Sequence , Enzyme Stability , Kinetics , Molecular Sequence Data , Prokaryotic Cells , Protein Structure, Tertiary
7.
Bioorg Khim ; 38(2): 234-41, 2012.
Article in Russian | MEDLINE | ID: mdl-22792728

ABSTRACT

A protease secreted in Bacillus pumilus KMM 62 culture liquid on different growth stages was isolated using ion-exchange chromatography. On the basis of pattern of specific chromogenic substrates hydrolysis and inhibitory analysis the protease was classified as subtilisin like serine protease. The molecular weight ofprotease is 31 kDa. Proteolytic activity towards Z-Ala-Ala-Leu-pNa substrate was maximal at pH 8-8.5. The optimal temperature for proteolytic activity was observed at a temperature of 30 degrees C, and the protein was stable within the pH range of 7.5-10.0. Bacillus pumilus KMM 62 subtilisin like serine protease was shown to have thrombolytic activity.


Subject(s)
Bacillus/enzymology , Bacillus/growth & development , Bacterial Proteins/metabolism , Proteolysis , Subtilisins/metabolism , Peptides/chemistry , Substrate Specificity
8.
Biochemistry (Mosc) ; 77(2): 119-27, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22348470

ABSTRACT

In this review the main families of endopeptidases belonging to the clan of metzincins of zinc-dependent metalloproteinases in organisms of wide evolutional range from bacteria to mammals are considered. The data on classification, physicochemical properties, substrate specificity, and structural features of this group of enzymes are given. The activation mechanisms of metzincins, the role of these proteins in organisms, and their participation in various physiological processes are discussed.


Subject(s)
Metalloendopeptidases/chemistry , Amino Acid Motifs , Binding Sites , Catalytic Domain , Metalloendopeptidases/classification , Metalloendopeptidases/metabolism , Zinc/chemistry , Zinc/metabolism
10.
Biochemistry (Mosc) ; 75(10): 1294-301, 2010 Oct.
Article in English | MEDLINE | ID: mdl-21166648

ABSTRACT

A novel zinc-dependent metalloendopeptidase of Bacillus intermedius (MprBi) was purified from the culture medium of a recombinant strain of Bacillus subtilis. The amino acid sequence of the homogeneous protein was determined using MALDI-TOF mass spectrometry. The sequence of the first ten residues from the N-terminus of the mature protein is ASTGSQKVTV. Physicochemical properties of the enzyme and its substrate specificity have been studied. The molecular weight of the metalloproteinase constitutes 19 kDa, the K(m) and k(cat) values are 0.06 mM and 1210 sec⁻¹, respectively, and the pI value is 5.4. The effect of different inhibitors and metal ions on the enzyme activity has been studied. Based on the analysis of the amino acid sequence of the active site motif and the Met-turn together with the enzyme characteristics, the novel bacterial metalloproteinase MprBi is identified as a metzincin clan adamalysin/reprolysin-like metalloprotease.


Subject(s)
Bacillus/enzymology , Bacterial Proteins/chemistry , Metalloproteases/chemistry , Zinc , Amino Acid Sequence , Bacillus/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Metalloproteases/genetics , Metalloproteases/isolation & purification , Metalloproteases/metabolism , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity
11.
Biochemistry (Mosc) ; 74(3): 308-15, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19364326

ABSTRACT

Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers.


Subject(s)
Bacillus/enzymology , Bacterial Proteins/metabolism , Recombinant Proteins/metabolism , Subtilisin/metabolism , Amino Acid Sequence , Bacillus/genetics , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Calcium/pharmacology , Catalysis/drug effects , Copper/pharmacology , Ethanol/pharmacology , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration , Magnesium/pharmacology , Manganese/pharmacology , Molecular Sequence Data , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid , Subtilisin/genetics , Temperature
12.
Bioorg Khim ; 34(3): 322-6, 2008.
Article in Russian | MEDLINE | ID: mdl-18672679

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.


Subject(s)
Bacillus/enzymology , Serine Endopeptidases/isolation & purification , Bacillus/growth & development , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Electrophoresis, Polyacrylamide Gel , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity
13.
Mol Biol (Mosk) ; 42(1): 117-22, 2008.
Article in Russian | MEDLINE | ID: mdl-18389628

ABSTRACT

The translation initiation site in the extracellular serine subtilisin-like proteinase gene from Bacillus intermedius (aprBi) (AN AY754946) secreting at the stationary growth phase was established. The analysis of aprBi open reading frame revealed three putative translation start sites (TTG, GTG, ATG). Using SignalP online freeware program we have determined the functional activity probability of each of them. To identify the translation start point the modified subtilisin-like protease genes carrying nucleotide replacements in supposed start codons were developed using oligonucleotide-directed mutagenesis. We have investigated the expression of these genetic constructions in protease-deficient strain B. subtilis AJ73. According our results it was concluded that the translation in aprBi gene starts from GTG kodon.


Subject(s)
Bacillus/genetics , Bacterial Proteins/genetics , Codon, Initiator/genetics , Genes, Bacterial/genetics , Serine Endopeptidases/genetics , Software , Bacillus/enzymology , Bacterial Proteins/biosynthesis , Codon, Initiator/metabolism , Peptide Chain Initiation, Translational/genetics , Sequence Analysis, DNA , Serine Endopeptidases/biosynthesis
14.
Mikrobiologiia ; 76(5): 645-51, 2007.
Article in Russian | MEDLINE | ID: mdl-18069325

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.


Subject(s)
Bacillus subtilis/physiology , Bacillus/enzymology , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Serine Endopeptidases/genetics , Spores, Bacterial/growth & development , Bacterial Proteins/metabolism , Genes, Bacterial , Mutation , Phosphorus/metabolism , Plasmids , Protein Kinases/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
15.
Mikrobiologiia ; 76(3): 313-20, 2007.
Article in Russian | MEDLINE | ID: mdl-17633406

ABSTRACT

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.


Subject(s)
Bacillus/metabolism , Subtilisin/biosynthesis , Bacillus/growth & development , Culture Media , Time Factors
16.
Biochemistry (Mosc) ; 72(4): 459-65, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17511612

ABSTRACT

Proteinases secreted during the early and late stationary phases have been isolated from the culture liquid of Bacillus amyloliquefaciens H2 using CM-cellulose ion-exchange chromatography with subsequent FPLC on a Mono S column. Considering the character of hydrolysis of specific chromogenic substrates and the type of inhibition, these enzymes were identified as subtilisin-like proteinases. The molecular weight of both proteinases is 29 kD. The proteolytic activity of the proteinases secreted during the early and late stationary phases towards the synthetic substrate Z-Ala-Ala-Leu-pNA was maximal at pH 8.5 and 9.0, respectively. The maximal activity of both proteinases was observed at 37 degrees C, and the proteins were stable within the pH range of 7.2-9.5. The subtilisin-like proteinases from B. amyloliquefaciens were shown to catalyze synthesis of peptide bonds.


Subject(s)
Bacillus/enzymology , Subtilisins/isolation & purification , Bacillus/growth & development , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Substrate Specificity , Subtilisins/metabolism
17.
Biochemistry (Mosc) ; 72(2): 192-8, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17367297

ABSTRACT

Two subtilisin-like serine proteinases of Bacillus intermedius secreted by the Bacillus subtilis recombinant strain AJ73 (pCS9) on the 28th and 48th h of culture growth (early and late proteinase, respectively) have been isolated by ion-exchange chromatography on CM-cellulose and by FPLC. Molecular weights of both proteinases were determined. The N-terminal sequences of the recombinant protein and mature proteinases of the original strain were compared. Kinetic parameters and substrate specificities of the early and late proteinase were analyzed. Physicochemical properties of the enzymes were studied.


Subject(s)
Bacillus subtilis/enzymology , Bacillus/enzymology , Recombinant Proteins , Serine Endopeptidases , Subtilisin , Amino Acid Sequence , Bacillus/genetics , Bacillus/growth & development , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Chromatography, Ion Exchange , Hydrogen-Ion Concentration , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/isolation & purification , Serine Endopeptidases/metabolism , Substrate Specificity , Subtilisin/chemistry , Subtilisin/genetics , Subtilisin/isolation & purification , Subtilisin/metabolism , Temperature
18.
Mol Biol Rep ; 34(2): 79-87, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17387634

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.


Subject(s)
Bacillus subtilis/genetics , Bacillus/enzymology , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Serine Endopeptidases/genetics , Amino Acid Sequence , Bacillus/genetics , Bacillus subtilis/growth & development , Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Base Sequence , Gene Expression Regulation, Bacterial/drug effects , Glucose/pharmacology , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Sequence Analysis, DNA , Serine Endopeptidases/metabolism , Spores, Bacterial/growth & development , Transcription Factors/genetics
19.
Mikrobiologiia ; 75(5): 642-8, 2006.
Article in Russian | MEDLINE | ID: mdl-17091586

ABSTRACT

The biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius 3-19 by the recombinant strain Bacillus subtilis AJ73(pCS9) was found to be enhanced under salt stress conditions (growth in a medium containing 1 M NaCl and 0.25 M sodium citrate). In a recombinant strain of B. subtilis deficient in the regulatory proteins DegS and DegU, which control the synthesis of degradative enzymes, the expression of the proteinase gene was inhibited. In contrast, in the strain B. subtilis degU32 (Hy), which provides for the over-synthesis of proteins positively regulated by the DegS-DegU system, the biosynthesis of the subtilisin-like proteinase of B. intermedius 3-19 increased by 6-10 times. These data suggest that the DegS-DegU system is involved in the positive regulation of the expression of the subtilisin-like B. intermedius proteinase gene in recombinant B. subtilis strains.


Subject(s)
Bacillus/metabolism , Serine Endopeptidases/biosynthesis , Subtilisins/biosynthesis , Bacillus/genetics , Bacillus/physiology , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Base Sequence , Citrates , Culture Media/chemistry , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Recombination, Genetic , Serine Endopeptidases/genetics , Sodium Chloride , Sodium Citrate , Subtilisins/genetics , Time Factors , Up-Regulation
20.
Mikrobiologiia ; 75(2): 172-8, 2006.
Article in Russian | MEDLINE | ID: mdl-16758864

ABSTRACT

The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).


Subject(s)
Bacillus subtilis/growth & development , Bacillus/enzymology , Recombinant Proteins/biosynthesis , Serine Endopeptidases/biosynthesis , Subtilisin/biosynthesis , Bacillus/genetics , Bacillus subtilis/genetics , Bacillus subtilis/physiology , Culture Media/chemistry , Culture Media/metabolism , Serine Endopeptidases/isolation & purification , Spores, Bacterial/enzymology , Spores, Bacterial/growth & development , Subtilisin/isolation & purification
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