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Production, Purification, and Biochemical Characterization of Thermostable Metallo-Protease from Novel Bacillus alkalitelluris TWI3 Isolated from Tannery Waste

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Abstract

Protease enzymes in tannery industries have enormous applications. Seeking a potential candidate for efficient protease production has emerged in recent years. In our study, we sought to isolate proteolytic bacteria from tannery waste dumping site in Tamilnadu, India. Novel proteolytic Bacillus alkalitelluris TWI3 was isolated and tested for protease production. Maximum protease production was achieved using lactose and skim milk as a carbon and nitrogen source, respectively, and optimum growth temperature was found to be 40 °C at pH 8. Protease enzyme was purified using ammonium sulfate precipitation method and anion exchange chromatography. Diethylaminoethanol (DEAE) column chromatography and Sephadex G-100 chromatography yielded an overall 4.92-fold and 7.19-fold purification, respectively. The 42.6-kDa TWI3 protease was characterized as alkaline metallo-protease and stable up to 60 °C and pH 10. Ca2+, Mn2+, and Mg2+ ions activated the protease, while Hg2+, Cu2+, Zn2+, and Fe2+ greatly inhibited it. Ethylenediaminetetraacetic acid (EDTA) inhibited TWI3 protease and was activated by Ca2+, which confirmed that TWI3 protease is a metallo-protease. Moreover, this protease is capable of dehairing goat skin and also removed several cloth stains, which makes it more suitable for various biotechnological applications.

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Acknowledgments

The authors are thankful to the Department of Biology, Gandhigram Rural Institute-Deemed University, Gandhigram, Tamilnadu, India, for providing laboratory facility to carry out the entire research. We also thankful to Mr. Narendrakumar for English correction.

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Authors and Affiliations

  1. Department of Biology, Gandhigram Rural Institute-Deemed University, Gandhigram, 624302, Tamilnadu, India

    Marimuthu Anandharaj, Balayogan Sivasankari, Nagarajan Siddharthan, Rizwana Parveen Rani & Subramaniyan Sivakumar

  2. Biodiversity Research Center, Academia Sinica, Taipei, 11529, Taiwan

    Marimuthu Anandharaj

  3. Department of Biotechnology, Indian Institute of Technology Madras, Chennai, 600036, Tamilnadu, India

    Subramaniyan Sivakumar

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  1. Marimuthu Anandharaj
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  2. Balayogan Sivasankari
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Correspondence to Balayogan Sivasankari.

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Anandharaj, M., Sivasankari, B., Siddharthan, N. et al. Production, Purification, and Biochemical Characterization of Thermostable Metallo-Protease from Novel Bacillus alkalitelluris TWI3 Isolated from Tannery Waste. Appl Biochem Biotechnol 178, 1666–1686 (2016). https://doi.org/10.1007/s12010-015-1974-7

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  • DOI: https://doi.org/10.1007/s12010-015-1974-7

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