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The population genetic study of hereditary nervous disorders is performed in Mari El (6 regions, total population 224100, including 144100 ethnic Maries and 66100 ethnic Russians). Overall hereditary nervous disorders prevalence is 25,44 per 100000, including 21,86 in Maries and 32,52 in Russians, the load of X-linked and autosomal recessive disorders being heavier than that of autosomal dominant disorders. The hereditary nervous disorders spectrum includes 24 nosologies neuromuscular disorders predominantly. Clinical and genetic characteristics of some cases are presented.

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. © 2013 Pleiades Publishing, Ltd.

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. © 2010 Pleiades Publishing, Ltd.

The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19. kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes. © 2010 Federation of European Biochemical Societies.

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 H 2O2). 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. © 2009 Pleiades Publishing, Ltd.

Subtilisin-like proteinase Bacillus intermedius which is secreted at different stages of bacterial growth (at 28 h and 48 h) were purified from the culture media of recombinant strain Bacillus subtilis JB 20-36(pCS9) by chromatography on CM-cellulose and MonoS columns. MALDI-TOF mass spectroscopy of purified enzymes demonstrated that they were identical in regard to amino acid sequence. The molecular weights of both proteins were 27 kDa. Biochemical analysis revealed differences in Km values for proteinase isolated at different growth stages (1.85 and 0.86 mM for first and second fractions respectively), and in substrate specificity and sensitiveness to Ca2+ ions. Gel-filtration experiments demonstrated that subtilisin-like proteinase B. intermedius was produced as an active monomer (27 kDa) during early stationary phase (28 h of growth) and as a dimer (54 kDa) during the late stationary phase (48 h).

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth. © 2006 Elsevier GmbH. All rights reserved.

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. © Nauka/Interperiodica 2007.

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°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. © Nauka/Interperiodica 2007.

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. © 2007 Springer Science+Business Media, Inc.

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 involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth. © Nauka/Interperiodica 2007.

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). © Pleiades Publishing, Inc., 2006.

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn 2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases. © Pleiades Publishing, Inc., 2006.

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. © 2005 Pleiades Publishing, Inc.

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.

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th hours of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and k cat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.

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.

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.

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 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+ to the medium at concentrations of 1 to 10mM 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. © 2002 Elsevier Science Inc. All rights reserved.

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".

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II-IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed. © 2002 MAIK "Nauka/Interperiodica".

Background: Proteinases are widely used in various fields of medicine, such as the treatment of burns, purulent wounds, or decubitus ulcers. On the basis of new microbial proteinases produced by nonpathogenic organisms, a new generation of medical preparations can be developed. Representatives of the Bacillus genera are nonpathogenic and are suitable for producing various proteases in large quantities. B. intermedius is shown to produce a set of alkaline proteases at the early and late stationary phase of growth. Material/Methods: The activity of alkaline proteinases was determined using synthetic chromogenous substrates Z-Glu-pNA and Z-Ala-Ala-Leu-pNA. To determine β-galactosidase activity, 2-nitro-β-D-galactopyranosid was used. Spores were calculated by phase-contrast microscopy. Results: During the late stages of sporulation B. intermedius 3-19 cells were shown to secrete two proteinases into the medium: glutamyl endopeptidase, with maximum activity at 40 hours of growth, and subtilisin, with maximum activity at 44 hours of growth. Evidence for the secretion of these enzymes into the medium was provided by measuring β-galactosidase activity. Conclusion: Our results show that proteinases from B. intermedius (glutamyl endopeptidase 2 and subtilisin 2) in the late stationary phase of growth are secreted enzymes. This suggests that these enzymes play a role in sporulation.

The effect of nutrients and growth conditions on the accumulation of glutamyl endopeptidase in the culture liquid of Bacillus intermedius 3-19 was studied. Glucose and other readily metabolizable carbon sources were found to suppress the production of the enzyme, whereas inorganic phosphate and ammonium cations enhanced it. Protein substrates, such as casein, gelatin, and hemoglobin, did not affect enzyme production. Some bivalent cations (Ca2+, Mg2+, Co2+) increased the production of glutamyl endopeptidase, but others (Zn2+, Fe2+, Cu2+) acted in the opposite way. The rate of enzyme accumulation in the culture liquid increased as the growth rate of the bacterium decreased, so that the maximum enzyme activity was observed in the stationary growth phase. Based on the results of this investigation, an optimal medium for the maximum production of glutamyl endopeptidase by B. intermedius 3-19 was elaborated. © 2000 MAIK "Nauka/Interperiodica".

Thiol-dependent serine proteinase and glutamylendopeptidase of Bacillus intermedius 3-19 being prevailing enzymes in the total pool of extracellular proteinases (95%) of this microorganism in catalytic active form were detected on the membrane of the cells. Production of these enzymes was maximum on the medium containing inorganic phosphate and gelatin and decreased 2-4-fold on the medium with glucose and lactate. The level of the activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused essential increase in extracellular glutamylendopeptidase activity and promoted the release of membrane-bound enzyme into cultural fluid. Proteolytic activity was detected in cytoplasm also. Proteinases localized in cytoplasm were shown to differ in properties from those secreted.

The activities of proteinases in the culture fluid and cellular fractions of Bacillus intermedius 3-19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. The production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2- to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted. © 2000 MAIK "Nauka/Interperiodica".

The biosynthesis of glutamylendopeptidase from Bacillus intermedius strain 3-19 and localization of the enzyme in the bacterial cells was studied. The synthesis of the enzyme was suppressed by easily metabolizable carbon sources. Inorganic phosphate and NH+ 4 ions stimulated the production of glutamylendopeptidase. Complicated organic substrates such as casein, gelatine, and haemoglobin did not affect the biosynthesis of the enzyme. The divalent metallic ions Ca2+, Mg2+, Co2+ increased the production of glutamylendopeptidase while Zn2+, Cu2+, and Fe2+ reduced the biosynthesis of proteinase. The rate of synthesis of the enzyme increased when the rate of the bacterial growth decreased. The maximum enzyme activity in the culture fluid was determined at the stationary phase of growth. In the cells glutamylendopeptidase was bound to the cytoplasmic membrane, and the maximal enzyme activity was detected in the stationary growth phase. The results facilitated the development of a medium which yielded the maximum glutamylendopeptidase production by B. intermedius strain 3-19.

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.

The results on research of affection "in vitro" of preparate of thiol-dependent serine proteinase from B. intermedius on time of formation and dissolvation of the clots formed from plasma rat blood.

A homogeneous glutamyl endopeptidase splitting peptide bonds of glutamic and, rarely, of aspartic acid residues in peptides and proteins was isolated from Bacillus intermedius 3-19 culture filtrate using chromatography on CM-cellulose and Mono S. The enzyme molecular mass is 29 kD and the p/ is 8.4. The proteinase is inhibited by DFP. The enzyme, like other glutamyl endopeptidases, reveals two pH optima (pH 7.5 and 9.0) for casein and one (pH 8.0) for Z-Glu-pNA hydrolysis. The Km for the hydrolysis of the latter substrate is 6 mM. The enzyme activity is optimal at 55°C. The enzyme is stable in the pH range 6.5-11.0. Its N-terminal sequence shows 56% coinciding residues when compared with that of Bacillus licheniformis glutamyl endopeptidase. Crystal prisms or plates 0.25-0.3 × 0.15 × 0.07-0.1 mm have been grown using the vapor diffusion technique in a hanging drop followed by macroseeding. The crystals belong to the space group B2 with the following unit cell parameters: a = 69.59 Å; b = 61.61 Å; c = 56.11 Å; γ = 117.57°. The X-ray data set to 1.7 Å resolution has been collected on an automatic synchrotron (EMBL Hamburg Station).

A homogeneous glutamyl endopeptidase, splitting peptide bonds of glutamic. rarely - of aspartic acid residues in peptides and proteins, was isolated from Bacillus intermedius 319 culture filtrate using chromatography on CM cellulose and Mono S. The enzyme molecular mass =29kDa. pI 8.4. The pro teinase is inhibited by I)FP. The enzyme, like other glutamyl endopeptidasos, reveals two pl[ optima at pH 7.5 and 9.0 for casein and one - at pH 8.0 for Z-Glu-pNA hydrolysis. K =6 mM was found for hydrolysis of the lat ter substrate. Its activity optimum lies at 55(;. The enzyme is stable at ptf 6.5-11.0. Its N-erminal sequence shows 56 per cent coinciding residues. when compared with that of Bacillus licheniformis glutamyl endopeptidase: VVIGDI) GRTKVA'NTRVAPYXXXXITFGGS-. The crystal prismesor plates of the size 0.23-0.3 x 0.15-0.2 x 0.07-0.1 mm have been grown using the vapour diffusion technique in lhe hanging drop followed by macroseeding. The crystals belong to the spat( group B2 with following unit cell parameters: a=69.59 angstrom; b=61.613 angstrom; c=56.107 angst rom; ,- 117.57. The x-ray data set to 1.7 angstrom resolution was collected on the synchrotron (EMBL Gain burg).

Effects of a thiol-dependent serine proteinase of Bacillus intermedius on peptide substrates and insulin B-chain were studied. The enzyme preferably splits peptide bonds formed by carboxyl groups of hydrophobic amino acids. Ca2+ increases the thermal stability of the proteinase significantly. The kinetic characteristics of hydrolysis of Z-Ala-Ala-Leu-pNA by this enzyme was determined as Km = 1.25 mM and kcat = 0.15 sec-1. The enzyme has high stability to DMFA and isopropanol, and is able to catalyze peptide bond synthesis.

A homogeneous glutamyl endopeptidase splitting peptide bonds of glutamic, rarely of aspartic acid residues in peptides and proteins, was isolated from Bacillus intermedius 3-19 culture filtrate using chromatography on CM cellulose and Mono S. The enzyme molecular mass is equal to 29 kDa, pI 8.4. The protease is inhibited by diisopropylfluorophosphate. The enzyme, like other glutamyl endopeptidases, reveals two pH optima at pH 7.5 and 9.0 for casein and one at pH 8.0 for Z-Glu-pNA hydrolysis. A 6 mM K(m) is found for hydrolysis of the latter substrate. The enzyme activity optimum lies at 55°C, and it is stable at pH 6.5-11.0. Its N-terminal sequence shows 56% coinciding residues when compared with that of Bacillus licheniformis glutamyl endopeptidase.