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bioRxiv · 10.1101/283226

In vivo cloning of β 1-4 endoglucanase gene of Serratia liquefaciens using Muduction and its in silico analysis

Gowri Sankar, S.·Asnet Mary, J.·John Vennison, S.·Alwin Prem Anand, A.
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Abstract

Cellulose is the major structural component in the plant cell wall. The bio-degradation of cellulose molecules is facilitated by cellulase. In the present study, in vivo cloning of cellulase ({beta}-1, 4 endoglucanase) gene from a cellulolytic bacterium Serratia liquefaciens into E. coli DH5 has been performed using mini-Mu phage transduction. The enzyme activity of cloned endoglucanase was 81.2U/mg at optimum temperature (40{degrees}C) and 80.2U/mg at optimum pH7, while the wildtype has 65.9U/mg and 64.9U/mg respectively. The conserved domain analysis shows that S. liquefaciens endoglucanase belongs to GH8 family. The nucleotide sequence analysis of wildtype and cloned endoglucanase shows that mutations were found at residues 51(Lys - Asn), 203(Trp-Cys), 246(Thr-Iso), 260(Gly-Ala) and 288(Phe-Leu). The structural analysis shows the active site of wildtype endoglucanase is a narrow groove which lies parallel to the central axis, whereas cloned endoglucanase is broad and tilted to [~]70{degrees} from the central axis. The increased enzyme activity in the cloned endoglucanase is due to the structural modification conferred by changes in amino acid resulting in widening of the cleft in the active site.

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Gowri Sankar, S., Asnet Mary, J., John Vennison, S., Alwin Prem Anand, A.. 2018-03-15. In vivo cloning of β 1-4 endoglucanase gene of Serratia liquefaciens using Muduction and its in silico analysis. https://doi.org/10.1101/283226

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