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Cloning, expression, and characterization of thermostable α-amylase of a thermophilic bacterium isolated from hot-spring of Sikkim and sustainable immobilization on coconut coir

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Abstract

Cloning and expression of the α-amylase gene (AmyK2) of thermophilic Bacillus subtilis k2cm originated from Yume Samdong hot spring, North Sikkim, India was done in Escherichia coli BL-21(DE3). The 55.0 kDa purified recombinant enzyme exhibited optimum activity at 70 °C and pH 7.0 with significant stability in temperature and pH ranges from 30 to 90 °C and 6.0–8.0, respectively. The α-amylase is Ca+ 2 independent, and can act with reasonably high efficiency in absence of any metal. Moreover, its activity increased in the presence of Fe+ 2 ion and was inhibited by Hg+ 2 ion and EDTA. The recombinant enzyme showed a half-life of 53 min at 70 °C and its Vmax and Km values were 22.22 U/mg and 5.06 mg/ml, respectively. Immobilization of the purified enzyme on low-cost coconut coir with high immobilization yield (98.27% specific activity), increased half-life (71 min), and higher thermostability with successive use up to 8 cycles with high efficacy validates the techno-economic merit of use of the immobilized biocatalyst. As a whole, cloning the α-amylase gene of thermostable bacteria into the mesophilic organism and subsequent immobilization of the enzyme will unravel its secrets within the confines of the laboratory which could expedite its commercial exploitation in future.

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Highlights of the Study

  • • The recombinant α-amylase AmyK2 is highly thermotolerant, stable in a wide range of pH and Ca+ 2 independent enzyme.

  • • The enzyme having low Km for starch; therefore, it exhibits high affinity for any starchy substrate.

  • • The enzyme was successfully immobilized at its native state with coconut coir and effective after successive reuse.

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Acknowledgements

All authors are grateful to the Department of Biotechnology, Government of India, for financial assistance.

Funding

This study has received funding from Department of Biotechnology, Government of India.

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Author notes

  1. Suman Kumar Halder

    Present address: Department of Microbiology, Vivekananda Mahavidyalaya, Burdwan, West Bengal, 713103, India

  2. Krishnendu Mondal and Prayatna Sharma contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Vidyasagar University, Midnapore, West Bengal, 721102, India

    Krishnendu Mondal, Suman Kumar Halder & Keshab Chandra Mondal

  2. Department of Microbiology, Sikkim University, Gangtok, Sikkim, 737102, India

    Prayatna Sharma, Santosh Kumar, Sonia Tamang & Nagendra Thakur

  3. Fermentation and Microbial Biotechnology Division, CSIR-IIIM, Jammu and Kashmir, Jammu, 180001, India

    Ishfaq Nabi Najar

  4. Center for Life Sciences, Vidyasagar University, Midnapore, West Bengal, 721102, India

    Shubhadeep Mondal

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Contributions

KCM and NT designed the study. KM and PS conducted the research. KM, INN, SM, SK, ST and SKH analyzed the data. KM, PS and INN prepared the primary manuscript. KCM, SKH and NT prepared the final manuscript and revised the manuscript. KCM and NT had the primary responsibility of the final content.

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Correspondence to Nagendra Thakur or Keshab Chandra Mondal.

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Mondal, K., Sharma, P., Najar, I.N. et al. Cloning, expression, and characterization of thermostable α-amylase of a thermophilic bacterium isolated from hot-spring of Sikkim and sustainable immobilization on coconut coir. Syst Microbiol and Biomanuf (2024). https://doi.org/10.1007/s43393-024-00271-x

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