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In-Vitro Refolding and Characterization of Recombinant Laccase (CotA) From Bacillus pumilus MK001 and Its Potential for Phenolics Degradation

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Abstract

Among lignocellulolytic enzymes, laccases are the most versatile, broadly specific, and largely studied enzyme with a wide range of biotechnological potential. Putative laccase (CotA) from Bacillus pumilus MK001 was cloned and expressed in E. coli. In addition to soluble bioactive fraction, inactive inclusion body fraction was also harvested and refolded under optimized conditions resulting in 64 % of refolding efficiency. The enzyme was found to be thermostable exhibiting a half-life of 60 min at 80 °C. UV thermal CD spectra also supported the observation as about 9 % increase in β-sheets was recorded after thermal induction. The 3D CotA structure was constructed through homology modeling and the best selected model was verified through PROCHECK, ERRAT, Verify 3D, and PROSA servers. Final 3D model showed potential binding affinities with ferulic acid, caffeic acid, and vanillin. Results of the docking studies were further validated by HPLC analysis which signified the efficient bioconversion ability of CotA.

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Acknowledgments

The financial assistance from the University of Delhi South Campus and DU/DST PURSE Grant to RCK is highly acknowledged. Also the fellowship grants from Indian Council of Medical Research (ICMR), New Delhi to SK and from Council of Scientific and industrial research (CSIR) to KKJ is gratefully acknowledged.

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Authors and Affiliations

  1. Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Sandeep Kumar, Kavish Kr. Jain & Ramesh Chander Kuhad

  2. Department of Biophysics, University of Delhi South Campus, New Delhi, 110021, India

    Shikha Rani & Manisha Goel

  3. Uttarakhand State Council for Science and Technology [UCOST], Vigyan Dham, Post Office - Jhajra, Dehradun, Uttarakhand, 248007, India

    Kailash N. Bhardwaj

  4. Central University of Haryana, Jant-Pali Village, Mahendergarh District, Pali, Haryana, 123029, India

    Ramesh Chander Kuhad

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  1. Sandeep Kumar
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Correspondence to Ramesh Chander Kuhad.

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Kumar, S., Jain, K.K., Rani, S. et al. In-Vitro Refolding and Characterization of Recombinant Laccase (CotA) From Bacillus pumilus MK001 and Its Potential for Phenolics Degradation. Mol Biotechnol 58, 789–800 (2016). https://doi.org/10.1007/s12033-016-9978-2

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