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Comparative Studies of the Characteristics of Two Alkaline Proteases from Haloalkaliphilic bacterium D-15-9 and Oceanobacillus onchorynchii Mi-10-54

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Abstract

The study describes purification and characterization of two alkaline proteases in comparative manner from haloalkaliphilic bacteria obtained from two different geographical locations. The enzymes from Haloalkaliphilic bacterium D-15-9 (D-15-9) and Oceanobacillus onchorynchii Mi-10-54 (Mi-10-54) purified by ammonium sulfate fractionation and hydrophobic interaction chromatography were characterized for pH, temperature, metal ions and NaCl stability. The apparent molecular weight of the enzymes was 40 and 28 kDa for D-15-9 and Mi-10-54, respectively. Both proteases optimally catalyzed the reactions at 50°C, pH 10.5–10.0 and 0.25–0.5 M NaCl. The Mi-10-54 protease was more thermally stable in comparison to D-15-9 enzyme. While NaCl did not significantly affect the temperature optima of the D-15-9 protease at lower NaCl concentrations, it shifted it from 50 to 80°C in the presence of 3 M NaCl. However, a similar pattern was not evident for the Mi-10-54 protease. On a similar note, there was a shift in the temperature optima from 50 to 60°C in the presence of 10 mM Ca2+ for Mi-10-54 protease, while D-15-9 protease did not display the similar effect. Instead, the D-15-9 protease denatured easily at higher temperatures and Ca2+. Both the enzymes were stable in urea, metal ions, oxidizing and reducing agents and inhibitors. The sensitivity to PMSF suggested that both enzymes were serine proteases. Moderate stability against hydrogen peroxide was also evident for both proteases.

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ACKNOWLEDGMENTS

VHR is grateful for the JRF and SRF Fellowship under the DBT Multi-Institutional Project. The majority of the research work included in this report was carried as part of the DBT Multi-Institutional Project involving Prof. S. K. Khare (IIT Delhi) and Dr. Sanjay Kapoor (University of Delhi South Campus, New Delhi). SPS acknowledges DST-SERB International Travel Fellowships to present his work in Hamburg (Germany), Cape Town (South Africa) and Kyoto (Japan). SPS also acknowledge award of UGC BSR Faculty Fellowship. VHR acknowledges SERB- Young Scientist Award and DST-SERB International Travel Fellowship. Facilities and infrastructure created under UGC-CAS Program, MoES Project and DST-FIST are duly acknowledged.

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Authors and Affiliations

  1. UGC Centre for Advanced Studies (CAS), Department of Biosciences, Saurashtra University, Rajkot, India

    V. H. Raval, D. S. Rathore & S. P. Singh

  2. Department of Microbiology & Biotechnology, Gujarat University, Navrangpura, Ahmedabad, Gujarat, India

    V. H. Raval

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  1. V. H. Raval
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V.H. Raval has carried out the experiment. D.S. Rathore has framed the manuscript and S.P. Singh has designed the experiment, critical suggestions and examination of manuscript.

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Correspondence to S. P. Singh.

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Raval, V.H., Rathore, D.S. & Singh, S.P. Comparative Studies of the Characteristics of Two Alkaline Proteases from Haloalkaliphilic bacterium D-15-9 and Oceanobacillus onchorynchii Mi-10-54. Appl Biochem Microbiol 58, 551–563 (2022). https://doi.org/10.1134/S0003683822050131

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