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Characterization of recombinant endo-1,4-β-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach

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Abstract

Increased demand of enzymes for industrial use has led the scientists towards protein engineering techniques. In different protein engineering strategies, rational approach has emerged as the most efficient method utilizing bioinformatics tools to produce enzymes with desired reaction kinetics; physiochemical (temperature, pH, half life, etc) and biological (selectivity, specificity, etc.) characteristics. Xylanase is one of the widely used enzymes in paper and food industry to degrade xylan component present in plant pulp. In this study endo 1,4-β-xylanase (Xyl-11A) from Bacillus halodurans C-125 was cloned in pET-22b (+) vector and expressed in Escherichia coli BL21 (DE3) expression strain. The enzyme had Michaelis constant Km of 1.32 mg ml−1 birchwoodxylan (soluble form) and maximum reaction velocity (Vmax) 73.53 mmol min−1 mg−1 with an optimum temperature of 75 °C and pH 9.0. The thermostability analysis showed that enzyme retained more than 80% of its residual activity when incubated at 75 °C for 2 h. In addition, to increase Xyl-11A thermostability, an in-silico analysis was performedto identify the hot spot amino acid residues. Consensus-based amino acid substitution was applied to evaluate multiple sequence alignment of homologs and identified 20 amino acids positions by following Jensen-Shnnon Divergence method. 3D models of 20 selected mutants were analyzed for conformational transition in protein structures by using NMSim server. Two selected mutants T6K and I17M of Xyl-11A retained 40, 60% residual activity respectively, at 85 °C for 120 min as compared to wild type enzyme which retained 37% initial activity under same conditions, confirming the enhanced thermostability of mutants. The present study showed a good approach for the identification of promising amino acid residues responsible for enhancing the thermostability of enzymes of industrial importance.

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Acknowledgements

We are thankful to Higher Education commission (HEC) Pakistan for providing the funding for this research project [Grant No. PM-IPFP/HRD/HEC/2011/0018] and scholarship funding for HEC Indigenous Ph D Fellowship 5000 Phase II” Award No. 315-2158-2BS3-029.

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Authors and Affiliations

  1. Institute of Biochemistry and Biotechnology, University of the Punjab, P. O Box No, 54590, Lahore, Pakistan

    Malik Siddique Mahmood & Mahjabeen Saleem

  2. Department of Biochemistry and Molecular Biology, University of Science and Technology, Hefei, China

    Faiz Rasul

  3. Department of Biochemistry and Biotechnology, University of Gujrat, P. O Box No. 50700, Gujrat, Pakistan

    Amber Afroz, Muhammad Faheem Malik, Naeem Mehmood Ashraf, Umar Rashid & Nadia Zeeshan

  4. Department of Biosciences, University of Gujrat, Gujrat, Pakistan

    Shumaila Naz

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  1. Malik Siddique Mahmood
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Correspondence to Nadia Zeeshan.

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Mahmood, M.S., Rasul, F., Saleem, M. et al. Characterization of recombinant endo-1,4-β-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach. Mol Biol Rep 46, 3651–3662 (2019). https://doi.org/10.1007/s11033-019-04751-5

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