182 research outputs found
Characteristics of a novel secreted zinc-dependent endopeptidase of Bacillus intermedius
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. Β© 2010 Pleiades Publishing, Ltd
Purification and characterization of a subtilisin-like proteinases secreted in the stationary growth phase of Bacillus amyloliquefaciens H2
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 37Β°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. Β© Nauka/Interperiodica 2007
ΠΠ»Π°Π·ΠΌΠ΅Π½Π½ΡΠ΅ ΠΏΠΎΠΊΡΡΡΠΈΡ, ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΠ΅ Π²ΡΡΠΎΠΊΠΎΠ±Π°ΡΠ½ΡΠ΅ ΡΠ°Π·Ρ
Results of comparative researches of the initial and processed in a plasma flow oxidic microcomposites, consisting of TiO2, SiO2, Al2O3, ZrO2, and plasma coverings from them β the materials which are characterized by amorphous and crystal structure and strengthened by ultradispersed phases of the stishovit, are presented. It is shown the influence of a type, structure and a way of processing of material (initial powder of different dispersion; the powder obtained in a plasma flow at the different modes and with the normal and accelerated cooling; a plasma layered composite) on the content of silicon oxides, aluminum and titanium, on the type of polymorphic transformations (anatase is found both in powders, and in coverings; the accelerated cooling of spheroids leads to growth of its contents in microcomposites) and also on feature of forming in ceramic materials of a high-bar phase β the stishovit (stishovit it is found only in coverings). It is established that increase in power of the plasma generator leads to increase in extent of amorphicity of plasma-layered composites. In the structure of layered composites three groups of the inclusions, combined by the composition, are revealed: two groups of aluminosilicates and inclusions on the basis of zirconium dioxide. Inclusions of the third group are characterized by two types of structures: homogeneous, consisting of zircon, and plated (with a kernel of dioxide of zirconium and a cover from zircon). The developed layered composites are characterized by high wear resistance, corrosion resistance and antifriction properties. In the conditions of low-temperature plasma at atmospheric pressure the stishovit-containing materials are obtained for the first time.ΠΡΠ΅Π΄ΡΡΠ°Π²Π»Π΅Π½Ρ ΡΠ΅Π·ΡΠ»ΡΡΠ°ΡΡ ΡΡΠ°Π²Π½ΠΈΡΠ΅Π»ΡΠ½ΡΡ
ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠΉ ΠΈΡΡ
ΠΎΠ΄Π½ΡΡ
ΠΈ ΠΎΠ±ΡΠ°Π±ΠΎΡΠ°Π½Π½ΡΡ
Π² ΠΏΠ»Π°Π·ΠΌΠ΅Π½Π½ΠΎΠΌ ΠΏΠΎΡΠΎΠΊΠ΅ ΠΎΠΊΡΠΈΠ΄Π½ΡΡ
ΠΌΠΈΠΊΡΠΎΠΊΠΎΠΌΠΏΠΎΠ·ΠΈΡΠΎΠ², ΡΠΎΡΡΠΎΡΡΠΈΡ
ΠΈΠ· TiO2, SiO2, Al2O3, ZrO2, ΠΈ ΠΏΠ»Π°Π·ΠΌΠ΅Π½Π½ΡΡ
ΠΏΠΎΠΊΡΡΡΠΈΠΉ ΠΈΠ· Π½ΠΈΡ
β ΠΌΠ°ΡΠ΅ΡΠΈΠ°Π»ΠΎΠ², Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΠ·ΡΡΡΠΈΡ
ΡΡ Π°ΠΌΠΎΡΡΠ½ΠΎ-ΠΊΡΠΈΡΡΠ°Π»Π»ΠΈΡΠ΅ΡΠΊΠΎΠΉ ΡΡΡΡΠΊΡΡΡΠΎΠΉ ΠΈ ΡΠΏΡΠΎΡΠ½Π΅Π½Π½ΡΡ
ΡΠ»ΡΡΡΠ°Π΄ΠΈΡΠΏΠ΅ΡΡΠ½ΡΠΌΠΈ ΡΠ°Π·Π°ΠΌΠΈ ΡΡΠΈΡΠΎΠ²ΠΈΡΠ°. ΠΠΎΠΊΠ°Π·Π°Π½ΠΎ Π²Π»ΠΈΡΠ½ΠΈΠ΅ Π²ΠΈΠ΄Π°, ΡΠΎΡΡΠ°Π²Π° ΠΈ ΡΠΏΠΎΡΠΎΠ±Π° ΠΎΠ±ΡΠ°Π±ΠΎΡΠΊΠΈ ΠΌΠ°ΡΠ΅ΡΠΈΠ°Π»Π° (ΠΈΡΡ
ΠΎΠ΄Π½ΡΠΉ ΠΏΠΎΡΠΎΡΠΎΠΊ ΡΠ°Π·Π»ΠΈΡΠ½ΠΎΠΉ Π΄ΠΈΡΠΏΠ΅ΡΡΠ½ΠΎΡΡΠΈ; ΠΏΠΎΡΠΎΡΠΎΠΊ, ΠΏΠΎΠ»ΡΡΠ΅Π½Π½ΡΠΉ Π² ΠΏΠ»Π°Π·ΠΌΠ΅Π½Π½ΠΎΠΌ ΠΏΠΎΡΠΎΠΊΠ΅ ΠΏΡΠΈ ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
ΡΠ΅ΠΆΠΈΠΌΠ°Ρ
ΠΈ Ρ ΠΎΠ±ΡΡΠ½ΡΠΌ ΠΈ ΡΡΠΊΠΎΡΠ΅Π½Π½ΡΠΌ ΠΎΡ
Π»Π°ΠΆΠ΄Π΅Π½ΠΈΠ΅ΠΌ; ΠΏΠ»Π°Π·ΠΌΠ΅Π½Π½ΡΠΉ ΡΠ»ΠΎΠ΅Π²ΠΎΠΉ ΠΊΠΎΠΌΠΏΠΎΠ·ΠΈΡ) Π½Π° ΡΠΎΠ΄Π΅ΡΠΆΠ°Π½ΠΈΠ΅ ΠΎΠΊΡΠΈΠ΄ΠΎΠ² ΠΊΡΠ΅ΠΌΠ½ΠΈΡ, Π°Π»ΡΠΌΠΈΠ½ΠΈΡ ΠΈ ΡΠΈΡΠ°Π½Π°; Π½Π° Π²ΠΈΠ΄ ΠΏΠΎΠ»ΠΈΠΌΠΎΡΡΠ½ΡΡ
ΠΏΡΠ΅Π²ΡΠ°ΡΠ΅Π½ΠΈΠΉ (Π°Π½Π°ΡΠ°Π· ΠΎΠ±Π½Π°ΡΡΠΆΠ΅Π½ ΠΈ Π² ΠΏΠΎΡΠΎΡΠΊΠ°Ρ
, ΠΈ Π² ΠΏΠΎΠΊΡΡΡΠΈΡΡ
; ΡΡΠΊΠΎΡΠ΅Π½Π½ΠΎΠ΅ ΠΎΡ
Π»Π°ΠΆΠ΄Π΅Π½ΠΈΠ΅ ΡΡΠ΅ΡΠΎΠΈΠ΄ΠΎΠ² ΠΏΡΠΈΠ²ΠΎΠ΄ΠΈΡ ΠΊ ΡΠΎΡΡΡ Π΅Π³ΠΎ ΡΠΎΠ΄Π΅ΡΠΆΠ°Π½ΠΈΡ Π² ΠΌΠΈΠΊΡΠΎΠΊΠΎΠΌΠΏΠΎΠ·ΠΈΡΠ°Ρ
), Π° ΡΠ°ΠΊΠΆΠ΅ Π½Π° ΠΎΡΠΎΠ±Π΅Π½Π½ΠΎΡΡΠΈ ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΡ Π² ΠΊΠ΅ΡΠ°ΠΌΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΌΠ°ΡΠ΅ΡΠΈΠ°Π»Π°Ρ
Π²ΡΡΠΎΠΊΠΎΠ±Π°ΡΠ½ΠΎΠΉ ΡΠ°Π·Ρ β ΡΡΠΈΡΠΎΠ²ΠΈΡΠ° (ΡΡΠΈΡΠΎΠ²ΠΈΡ ΠΎΠ±Π½Π°ΡΡΠΆΠ΅Π½ ΡΠΎΠ»ΡΠΊΠΎ Π² ΠΏΠΎΠΊΡΡΡΠΈΡΡ
). Π£ΡΡΠ°Π½ΠΎΠ²Π»Π΅Π½ΠΎ, ΡΡΠΎ ΠΏΠΎΠ²ΡΡΠ΅Π½ΠΈΠ΅ ΠΌΠΎΡΠ½ΠΎΡΡΠΈ ΠΏΠ»Π°Π·ΠΌΠ΅Π½Π½ΠΎΠ³ΠΎ Π³Π΅Π½Π΅ΡΠ°ΡΠΎΡΠ° ΠΏΡΠΈΠ²ΠΎΠ΄ΠΈΡ ΠΊ ΡΠ²Π΅Π»ΠΈΡΠ΅Π½ΠΈΡ ΡΡΠ΅ΠΏΠ΅Π½ΠΈ Π°ΠΌΠΎΡΡΠΈΠ·Π°ΡΠΈΠΈ ΠΏΠ»Π°Π·ΠΌΠ΅Π½Π½ΡΡ
ΡΠ»ΠΎΠ΅Π²ΡΡ
ΠΊΠΎΠΌΠΏΠΎΠ·ΠΈΡΠΎΠ². Π ΡΡΡΡΠΊΡΡΡΠ΅ ΡΠ»ΠΎΠ΅Π²ΡΡ
ΠΊΠΎΠΌΠΏΠΎΠ·ΠΈΡΠΎΠ² ΠΎΠ±Π½Π°ΡΡΠΆΠ΅Π½ΠΎ ΡΡΠΈ Π³ΡΡΠΏΠΏΡ Π²ΠΊΠ»ΡΡΠ΅Π½ΠΈΠΉ, ΠΎΠ±ΡΠ΅Π΄ΠΈΠ½Π΅Π½Π½ΡΡ
ΠΏΠΎ ΡΠΎΡΡΠ°Π²Ρ: Π΄Π²Π΅ Π³ΡΡΠΏΠΏΡ Π°Π»ΡΠΌΠΎΡΠΈΠ»ΠΈΠΊΠ°ΡΠΎΠ² ΠΈ Π²ΠΊΠ»ΡΡΠ΅Π½ΠΈΠΉ Π½Π° ΠΎΡΠ½ΠΎΠ²Π΅ Π΄ΠΈΠΎΠΊΡΠΈΠ΄Π° ΡΠΈΡΠΊΠΎΠ½ΠΈΡ. ΠΠΊΠ»ΡΡΠ΅Π½ΠΈΡ ΡΡΠ΅ΡΡΠ΅ΠΉ Π³ΡΡΠΏΠΏΡ Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΠ·ΡΡΡΡΡ Π΄Π²ΡΠΌΡ Π²ΠΈΠ΄Π°ΠΌΠΈ ΡΡΡΡΠΊΡΡΡ: ΠΎΠ΄Π½ΠΎΡΠΎΠ΄Π½ΠΎΠΉ, ΡΠΎΡΡΠΎΡΡΠ΅ΠΉ ΠΈΠ· ΡΠΈΡΠΊΠΎΠ½Π°, ΠΈ ΠΏΠ»Π°ΠΊΠΈΡΠΎΠ²Π°Π½Π½ΠΎΠΉ (Ρ ΡΠ΄ΡΠΎΠΌ Π΄ΠΈΠΎΠΊΡΠΈΠ΄Π° ΡΠΈΡΠΊΠΎΠ½ΠΈΡ ΠΈ ΠΎΠ±ΠΎΠ»ΠΎΡΠΊΠΎΠΉ ΠΈΠ· ΡΠΈΡΠΊΠΎΠ½Π°). Π Π°Π·ΡΠ°Π±ΠΎΡΠ°Π½Π½ΡΠ΅ ΡΠ»ΠΎΠ΅Π²ΡΠ΅ ΠΊΠΎΠΌΠΏΠΎΠ·ΠΈΡΡ ΠΎΡΠ»ΠΈΡΠ°ΡΡΡΡ Π²ΡΡΠΎΠΊΠΈΠΌΠΈ ΠΈΠ·Π½ΠΎΡΠΎΡΡΠΎΠΉΠΊΠΎΡΡΡΡ, ΠΊΠΎΡΡΠΎΠ·ΠΈΠΎΠ½Π½ΠΎΠΉ ΡΡΠΎΠΉΠΊΠΎΡΡΡΡ ΠΈ Π°Π½ΡΠΈΡΡΠΈΠΊΡΠΈΠΎΠ½Π½ΡΠΌΠΈ ΡΠ²ΠΎΠΉΡΡΠ²Π°ΠΌΠΈ. Π ΡΡΠ»ΠΎΠ²ΠΈΡΡ
Π½ΠΈΠ·ΠΊΠΎΡΠ΅ΠΌΠΏΠ΅ΡΠ°ΡΡΡΠ½ΠΎΠΉ ΠΏΠ»Π°Π·ΠΌΡ ΠΏΡΠΈ Π°ΡΠΌΠΎΡΡΠ΅ΡΠ½ΠΎΠΌ Π΄Π°Π²Π»Π΅Π½ΠΈΠΈ ΡΡΠΈΡΠΎΠ²ΠΈΡ-ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΠ΅ ΠΌΠ°ΡΠ΅ΡΠΈΠ°Π»Ρ ΠΏΠΎΠ»ΡΡΠ΅Π½Ρ Π²ΠΏΠ΅ΡΠ²ΡΠ΅
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. Β© 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)
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
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 Γ
, b = 55.84 Γ
, c = 60.40 Γ
, Ξ² = 117.6Β° X-ray diffraction data to 1.68 Γ
resolution were collected using synchrotron radiation (EMBL, Hamburg) and an imaging plate scanner. Β© 1999 Elsevier Science B.V. All rights reserved
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
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 50Β°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
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