Abstract
The biotechnological application of bacterial xylanases requires a high thermostability, a catalytically active state for a broad pH range. The Bacillus amyloliquefaciens (MTCC 1270) xynA gene was amplified and cloned into the pCold vector and was expressed in Escherichia coli to evaluate the expressed proteins’ thermostability. The pCold, compared to other similar vectors, has unique properties—including pH and temperature tolerance due to the presence of the cspA promoter. The recombinant xynA-pCold (rxynApC) showed the expression of xynA gene with a molecular weight of ~ 27 kDa, confirmed on SDS-PAGE. The rxynApC exhibits optimal activity at 70 °C and pH 8.0. The residual activity of the recombinant enzyme was 90% at pH 8.0. The thermal decomposition temperature (Td) value for the rxynApC enzyme was 93.33 °C obtained from the thermogravimetric analysis, indicating the potent stability of the cloned enzyme. The specific activity of native xylanase and rxynApC under optimal conditions was 32.35 and 105.5 U/mg, respectively. The structural model of the xynA gene was predicted using the in silico tool along with the active site (containing four important Tyr-166, Gly-7, Try-69 and Arg-112 amino acids). The predicted biophysical parameters of the in silico model were similar to the experimental results. The unique feature of the cspA promoter is that it gave a high expression of rxynApC enzyme having alkali and thermostable properties with high yield in surrogate host E. coli. Thus, the recombinant xynA gene can potentially be applied to different industrial needs by looking at its thermostability and enhanced enzyme activity.
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Acknowledgements
We thank the Department of Science and Technology for providing INSPIRE Fellowship (No. DST/INSPIRE Fellowship/[IF170394].
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This work was supported by the Department of Science and Technology, Delhi, India (No. DST/INSPIRE Fellowship/[IF170394], Dharti Keyur Patel.
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Material preparation, data collection, and analysis were performed. The first draft of the manuscript was written by Dharti Patel. The idea and design of work with critical revision the work was done by Dr. Gayarti Dave.
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Patel, D.K., Dave, G. pCold-assisted expression of a thermostable xylanase from Bacillus amyloliquefaciens: cloning, expression and characterization. 3 Biotech 12, 245 (2022). https://doi.org/10.1007/s13205-022-03315-y
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DOI: https://doi.org/10.1007/s13205-022-03315-y