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Stability and Detergent Compatibility of a Predominantly β-Sheet Serine Protease from Halotolerant B. aquimaris VITP4 Strain

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Abstract

The present study deals with the characterization of halotolerant protease produced by Bacillus aquimaris VITP4 strain isolated from Kumta coast, Karnataka, India. The studies were performed at 40 °C and pH 8 in Tris buffer. Metal ions such as Mn2+ and Ca2+ increased the proteolytic activity of the enzyme by 34 and 30 %, respectively, at 10 mM concentration. Cu2+ at 1 mM concentration was found to enhance the enzyme activity by 16 %, whereas inhibition was observed at higher concentration (>5 mM). Slight inhibition was observed even with lower (>1 mM) concentrations of Zn2+, Hg2+, Fe3+, Ni2+, and Co2+.The activity of protease was completely inhibited by phenylmethylsulfonyl fluoride, indicating that the VITP4 protease is a serine protease. The presence of ethylenediaminetetraacetic acid and 1,10-phenanthroline (>5 mM) moderately inhibited the activity, suggesting that the enzyme is activated by metal ions. The protease was purified to homogeneity with a purification fold of 15.7 with ammonium sulfate precipitation and 46.65 with gel filtration chromatography using Sephadex G-100, resulting in a specific activity of 424 ± 2.6 U mg−1. The VITP4 protease consists of a single polypeptide chain with a molecular mass of 34.7 kDa as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization–time of flight. Among the different substrates used (casein, egg albumin, gelatin, and bovine serum albumin), the activity was higher with casein with V max, K m, and k cat values of 0.817 mg ml min−1, 0.472 mg ml−1, and 2.31 s−1, respectively. Circular dichroism studies revealed that the VITP4 protease has a predominantly β-sheet structure (51.6 %) with a temperature for half denaturation of 85.8 °C in the presence of 1 mM CaCl2. Additionally, the VITP4 protease was found to retain more than 70 % activity in the presence of 10 mM concentration of different detergents (CTAB, urea, and sodium dodecyl sulfate) and surfactants (Triton X-100, Tween-20, and Tween-80), and the results of wash performance test with various commercial detergents confirmed that it can be used in detergent formulations.

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Acknowledgments

C. Jabeena Thaz is a recipient of a UGC-MAN Fellowship (201011-MANF-MUS-AND-1551). The research facility provided by the VIT University (Vellore, India) is gratefully acknowledged.

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  1. VIT University, Vellore, Tamil Nadu, India

    Chittoor Jabeena Thaz & Gurunathan Jayaraman

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  1. Chittoor Jabeena Thaz
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Correspondence to Gurunathan Jayaraman.

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Thaz, C.J., Jayaraman, G. Stability and Detergent Compatibility of a Predominantly β-Sheet Serine Protease from Halotolerant B. aquimaris VITP4 Strain. Appl Biochem Biotechnol 172, 687–700 (2014). https://doi.org/10.1007/s12010-013-0524-4

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