Bacillus Clausii

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Altan Erarslan - One of the best experts on this subject based on the ideXlab platform.

  • Water miscible mono alcohols' effect on the proteolytic performance of Bacillus Clausii serine alkaline protease.
    Applied biochemistry and biotechnology, 2013
    Co-Authors: Yonca Avcı Duman, Dilek Kazan, Aziz Akin Denizci, Altan Erarslan
    Abstract:

    In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the V m, k cat and k cat/K m values of Bacillus Clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the K m value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (K I) were in the range of 1.32–3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG ≠ and ΔG ≠ E − T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG ≠ ES value of the enzyme remained almost the same. The constant K m and ΔG ≠ ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The k cat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG ≠ and ΔG ≠ E − T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

  • Studies on Alkaline Serine Protease Produced by Bacillus Clausii GMBE 22
    Preparative biochemistry & biotechnology, 2009
    Co-Authors: Dilek Kazan, Aziz Akin Denizci, Hulya Bal, Nurcin Celik Ozturk, Hasan Umit Ozturk, Aydan Salman Dilgimen, Dilek Coskuner Ozturk, Altan Erarslan
    Abstract:

    An alkali tolerant Bacillus strain having extracellular serine alkaline protease activity was newly isolated from compost and identified as Bacillus Clausii GMBE 22. An alkaline protease (AP22) was 4.66-fold purified in 51.5% yield from Bacillus Clausii GMBE 22 by ethanol precipitation and DEAE-cellulose anion exchange chromatography. The purified enzyme was identified as serine protease by LC-ESI-MS analysis. Its complete inhibition by phenylmethanesulfonylfluoride (PMSF) also justified that it is a serine alkaline protease. The molecular weight of the enzyme is 25.4 kDa. Optimal temperature and pH values are 60 degrees C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The K(m) and k(cat) values for hydrolysis of this substrate are 0.347 mM and 1141 min(-1) respectively. The enzyme was affected by surface active agents to varying extents. The enzyme is stable for 2 h at 30 degrees C and pH 10.5. AP22 is also stable for 5 days over the pH range 9.0-11.0 at room temperature. AP22 has good pH stability compared with the alkaline proteases belonging to other strains of Bacillus Clausii reported in the literature.

  • Water miscible mono alcohols effect on the structural conformation of Bacillus Clausii GMBAE 42 serine alkaline protease
    Journal of Molecular Catalysis B-enzymatic, 2009
    Co-Authors: Dilek Coskuner Ozturk, Dilek Kazan, Aziz Akin Denizci, Dario Grimoldi, Francesco Secundo, Altan Erarslan
    Abstract:

    Abstract Proteases are largely employed in biocatalysis. In order to increase the number of their applications it is useful to shed light on the reasons that cause a non-optimal activity of these enzymes when used in inactivating experimental conditions (e.g., in the presence of co-solvent to favor substrate dissolution). To this end the effect of different mono alcohols on the activity and the conformation of alkaline protease from Bacillus Clausii GMBAE 42 was investigated. We found that the enzyme in the presence of 20–25% of methanol, ethanol, 1-propanol or 2-propanol halves its activity. At the concentration of 10%, all the alcohols caused a slightly more intense far-UV (CD) circular dichroism signal of the protease at around 208 and 220 nm with respect to the protein in only buffer, which suggests an increase of helicity in the secondary structure of the protein. Monitoring the shift of the fluorescence emission of the protease with respect to that of the standard N -acetyl- l -tryptophan-ethyl ester, we suggest that with all the alcohols tested the decrease of activity might be due to the loss of tertiary structure (even though at a lower extents in methanol and ethanol compared to 1-propanol and 2-propanol).

  • Purification and characterization of a serine alkaline protease from Bacillus Clausii GMBAE 42
    Journal of Industrial Microbiology and Biotechnology, 2005
    Co-Authors: Dilek Kazan, Aziz Akin Denizci, Mine N. Kerimak Öner, Altan Erarslan
    Abstract:

    An extracellular serine alkaline protease of Bacillus Clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH_4)_2SO_4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca^2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol^−1 (44.30 kJ mol^−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n -ethylmaleimidate, n -bromosuccinimide, diethylpyrocarbonate or n -ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N -Suc-Ala-Ala-Pro-Phe- p NA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N -Suc-Ala-Ala-Pro-Phe- p NA and 4.21×10^3 min^−1, respectively.

Tsutomu Sato - One of the best experts on this subject based on the ideXlab platform.

C.-s. Chang - One of the best experts on this subject based on the ideXlab platform.

  • Oxidant and SDS-stable alkaline protease from a halo-tolerant Bacillus Clausii I-52: enhanced production and simple purification.
    Journal of applied microbiology, 2005
    Co-Authors: H.-s. Joo, C.-s. Chang
    Abstract:

    H.-S. JOO AND C.-S. CHANG. 2004. Aims: An investigation was carried out on the enhancement of protease production and simple purification of an oxidant and SDS-stable alkaline protease produced by Bacillus Clausii I-52 of industrial significance. Methods and Results: The supplementation with 0AE4% (w/v) NaCl and 0AE05% (w/v) FeSO4AE7H2O in a culture medium caused an increase in the protease production. The enzyme was purified to homogeneity with overall recovery of 79% and 10-fold purification from culture supernatant using Diaion HPA75, phenyl-Sepharose and DEAE-Sepharose column chromatographies. The protease was a halo-tolerant enzyme with apparent molecular mass of 28 kDa, and the Km and kcat values for N-Succinyl-Ala-Ala-Pro-Phe-pNA at 45� C and pH 11AE0 were determined to be 83AE9 lmol l )1 and 238AE 6s )1 respectively. Conclusions: Bacillus Clausii I-52 was identified as a halo-tolerant bacterium, and the extracellular alkaline protease produced by B. Clausii I-52 also showed extreme halo-tolerance. The enzyme stability towards SDS and H2O2 could be increased by adding NaCl or propylene glycol to the enzyme solution. Significance and Impact of the Study: The alkaline protease secreted by B. Clausii I-52 is significant from an industrial perspective because of its stability against surfactants and oxidants as well as its tolerance towards high salinity. These enzymatic properties suggest its suitable application for industrial purposes.

  • Oxidant and SDS-stable alkaline protease from Bacillus Clausii I-52: production and some properties.
    Journal of applied microbiology, 2003
    Co-Authors: H.‐s. Joo, C. Ganesh Kumar, Seung R. Paik, G.‐c. Park, C.-s. Chang
    Abstract:

    Aims: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus Clausii of industrial significance. Methods and Results: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l−1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4·7H2O, 0·1; Na2CO3, 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60°C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. Clausii belongs to the family of serine proteases. Conclusions:Bacillus Clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2. Significance and Impact of the Study: The alkaline protease from B. Clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations.

Tsutomu Hoshino - One of the best experts on this subject based on the ideXlab platform.

Dilek Kazan - One of the best experts on this subject based on the ideXlab platform.

  • Water miscible mono alcohols' effect on the proteolytic performance of Bacillus Clausii serine alkaline protease.
    Applied biochemistry and biotechnology, 2013
    Co-Authors: Yonca Avcı Duman, Dilek Kazan, Aziz Akin Denizci, Altan Erarslan
    Abstract:

    In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the V m, k cat and k cat/K m values of Bacillus Clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the K m value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (K I) were in the range of 1.32–3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG ≠ and ΔG ≠ E − T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG ≠ ES value of the enzyme remained almost the same. The constant K m and ΔG ≠ ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The k cat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG ≠ and ΔG ≠ E − T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

  • Studies on Alkaline Serine Protease Produced by Bacillus Clausii GMBE 22
    Preparative biochemistry & biotechnology, 2009
    Co-Authors: Dilek Kazan, Aziz Akin Denizci, Hulya Bal, Nurcin Celik Ozturk, Hasan Umit Ozturk, Aydan Salman Dilgimen, Dilek Coskuner Ozturk, Altan Erarslan
    Abstract:

    An alkali tolerant Bacillus strain having extracellular serine alkaline protease activity was newly isolated from compost and identified as Bacillus Clausii GMBE 22. An alkaline protease (AP22) was 4.66-fold purified in 51.5% yield from Bacillus Clausii GMBE 22 by ethanol precipitation and DEAE-cellulose anion exchange chromatography. The purified enzyme was identified as serine protease by LC-ESI-MS analysis. Its complete inhibition by phenylmethanesulfonylfluoride (PMSF) also justified that it is a serine alkaline protease. The molecular weight of the enzyme is 25.4 kDa. Optimal temperature and pH values are 60 degrees C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The K(m) and k(cat) values for hydrolysis of this substrate are 0.347 mM and 1141 min(-1) respectively. The enzyme was affected by surface active agents to varying extents. The enzyme is stable for 2 h at 30 degrees C and pH 10.5. AP22 is also stable for 5 days over the pH range 9.0-11.0 at room temperature. AP22 has good pH stability compared with the alkaline proteases belonging to other strains of Bacillus Clausii reported in the literature.

  • Water miscible mono alcohols effect on the structural conformation of Bacillus Clausii GMBAE 42 serine alkaline protease
    Journal of Molecular Catalysis B-enzymatic, 2009
    Co-Authors: Dilek Coskuner Ozturk, Dilek Kazan, Aziz Akin Denizci, Dario Grimoldi, Francesco Secundo, Altan Erarslan
    Abstract:

    Abstract Proteases are largely employed in biocatalysis. In order to increase the number of their applications it is useful to shed light on the reasons that cause a non-optimal activity of these enzymes when used in inactivating experimental conditions (e.g., in the presence of co-solvent to favor substrate dissolution). To this end the effect of different mono alcohols on the activity and the conformation of alkaline protease from Bacillus Clausii GMBAE 42 was investigated. We found that the enzyme in the presence of 20–25% of methanol, ethanol, 1-propanol or 2-propanol halves its activity. At the concentration of 10%, all the alcohols caused a slightly more intense far-UV (CD) circular dichroism signal of the protease at around 208 and 220 nm with respect to the protein in only buffer, which suggests an increase of helicity in the secondary structure of the protein. Monitoring the shift of the fluorescence emission of the protease with respect to that of the standard N -acetyl- l -tryptophan-ethyl ester, we suggest that with all the alcohols tested the decrease of activity might be due to the loss of tertiary structure (even though at a lower extents in methanol and ethanol compared to 1-propanol and 2-propanol).

  • Purification and characterization of a serine alkaline protease from Bacillus Clausii GMBAE 42
    Journal of Industrial Microbiology and Biotechnology, 2005
    Co-Authors: Dilek Kazan, Aziz Akin Denizci, Mine N. Kerimak Öner, Altan Erarslan
    Abstract:

    An extracellular serine alkaline protease of Bacillus Clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH_4)_2SO_4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca^2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol^−1 (44.30 kJ mol^−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n -ethylmaleimidate, n -bromosuccinimide, diethylpyrocarbonate or n -ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N -Suc-Ala-Ala-Pro-Phe- p NA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N -Suc-Ala-Ala-Pro-Phe- p NA and 4.21×10^3 min^−1, respectively.

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