Isolation and characterization of a subtilisin-like proteinase of Bacillus intermedius secreted by the Bacillus subtilis recombinant strain AJ73 at different growth stages

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

Glutamyl endopeptidase of bacillus intermedius, strain 3-19. purification, properties, cristallization

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)

Crystal growth and preliminary X-ray study of glutamic acid specific serine protease from Bacillus intermedius

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

Optimized medium for the efficient production of bacillus intermedium glutamyl endopeptidase by the recombinant bacillus subtilis

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus 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+

Biosynthesis and localization of glutamylendopeptidase from Bacillus intermedius strain 3-19

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

Effect of nutrients on the accumulation of glutamyl endopeptidase in the culture liquid of bacillus intermedium 3-19

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, while 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

Effect of nutrients on the accumulation of glutamyl endopeptidase in the culture liquid of Bacillus intermedius 3-19

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"

Secreted hydrolases from streptomycin-resistant strains of Bacillus intermedius

Alkaline phosphatases and serine proteinases have been isolated from the culture liquid of streptomycin-resistant strains of Bacillus intermedius using ion-exchange and affinity chromatography and FPLC. Substrate blotting and electrophoresis revealed two phosphatase forms with molecular masses of 40 and 50 kD. The enzyme had maximal activity at pH 9.5 and 50°C and could cleave the phosphate moiety from a range of substrates. It is suggested that both forms of the phosphatase are products of processing that involves a serine proteinase. Two proteinases, with molecular masses of 29 and 33 kD, were purified to homogeneity from the culture liquid of A. intermedius S7. The protein from the major peak was identical in its properties to an earlier described serine proteinase. The minor peak was 5% of the major one. These enzymes had different pH optima. Inhibitor analysis indicated that the minor peak is also a serine proteinase

Secreted hydrolases from streptomycin-resistant strains of Bacillus intermedius

Alkaline phosphatase and serine proteinase have been isolated from streptomycin-resistant strains of Bacillus intermedius using ion-exchange, affinity and FPLC chromatography. Substrate blotting analysis and electrophoresis revealed two phosphatase forms with molecular weight of 40 and 50 kDa. The pH and temperature optima of phosphatase were at pH 9.5 and 50°C. The enzyme showed a broad substrate specificity. It was suggested that the two forms of phosphatase are the products of processing, in which serine proteinase is the participant. Two proteinase peaks with molecular weights of 29 and 33 kDa were isolated from B. intermedius S7, the first peak having only 5% of the activity of the second peak. The major peak was identical to serine proteinase described earlier. The minor peak was distinct from the major one by the pH-optima. Analysis of inhibitors' effects revealed that the minor peak also corresponded to serine proteinase

Proteinases from Bacillus intermedius secreted in the late stages of sporulation

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