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Molecular and biochemical characterization of a thermostable keratinase from Bacillus altitudinis RBDV1

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Abstract

A thermostable keratinase designated as KBALT was purified from Bacillus altitudinis RBDV1 from a poultry farm in Gujarat, India. The molecular weight of the native KBALT (nKBALT) purified using ammonium sulfate and ion exchange and gel permeation chromatography with a 40% yield and 80-fold purification was estimated to be ~ 43 kDa. The gene for KBALT was successfully cloned, sequenced and expressed in Escherichia coli. Recombinant KBALT (rKBALT) when purified using a single step Ni–NTA His affinity chromatography achieved a yield of 38.20% and a 76.4-fold purification. Comparison of the deduced amino acid sequence of rKBALT with known proteases of Bacillus species and inhibitory effect of PMSF suggest that rKBALT was a subtilisin-like serine protease. Both native and rKBALT exhibited higher activity at 85 °C and pH 8.0 in the presence of Mg2+, Mn2+, Zn2+, Ba2+ and Fe3+ metal ions. Interestingly, 70% of their activity was retained at temperatures ranging from 35 to > 95 °C. The keratinolytic activity of both nKBALT and rKBALT was enhanced in the presence of reducing agents. They exhibited broad substrate specificity towards various protein substrates. KBALT was determined for its kinetic properties by calculating its Km (0.61 mg/ml) and Vmax (1673 U/mg/min) values. These results suggest KBALT as a robust and promising contender for enzymatic processing of keratinous wastes in waste processing plants.

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Acknowledgements

Authors are thankful to P.G. Department of Biosciences, Sardar Patel University for providing facilities to conduct research.

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Authors and Affiliations

  1. P. G. Department Of Biosciences, Satellite Campus, Sardar Patel Maidaan, Bakrol-Vadtal Road, Sardar Patel University, P.O. Box No. 39, Vallabh Vidyanagar, Gujarat, 388120, India

    Vishakha A. Pawar, Anil S. Prajapati & R. B. Subramanian

  2. Department of Biochemistry, P. D. Patel Institute of Applied Sciences, Charotar University of Science and Technology, Changa, Anand, Gujarat, India

    Rekha C. Akhani & Darshan H. Patel

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  1. Vishakha A. Pawar
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  2. Anil S. Prajapati
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  3. Rekha C. Akhani
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  4. Darshan H. Patel
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  5. R. B. Subramanian
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Correspondence to R. B. Subramanian.

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Pawar, V.A., Prajapati, A.S., Akhani, R.C. et al. Molecular and biochemical characterization of a thermostable keratinase from Bacillus altitudinis RBDV1. 3 Biotech 8, 107 (2018). https://doi.org/10.1007/s13205-018-1130-5

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