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Cloning, expression and characterization of a metagenome derived thermoactive/thermostable pectinase

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Abstract

The gene encoding a thermostable pectinase was isolated from a soil metagenome sample. The gene sequence corresponded to an open reading frame of 1,311 bp encoding a translation product of 47.9 kDa. It showed maximum (93 %) identity to a Bacillus licheniformis glycoside hydrolase. Deduced amino acid analysis showed an absence of highly conserved cysteine residues in the N-terminal region at positions 24 and 42, and in the C-terminal region at positions 389, 394, 413 and 424. pQpecJKR01 (pQE30 expression vector containing the pectinase gene) was expressed in Escherichia coli strain M15 as a recombinant fusion protein containing an N-terminal 6× His tag. Biochemical properties of this pectinase were novel. The enzyme had temperature and pH optima of 70 °C and 7.0, respectively, but was active over a broad temperature and pH range. The enzyme was stable at 60 °C with a half-life of 5 h and the enzyme activity was inhibited by 0.1 % diethyl pyrocarbonate and 5 mM dicyclohexyl carbodiimide. The enzyme could be of great use in industrial processes due to its activity over a broad pH range and at high temperature.

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Acknowledgments

Fellowship granted to RS by University Grants Commission (UGC), India is acknowledged.

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Authors and Affiliations

  1. Department of Biotechnology, Panjab University, Sector-14, Chandigarh, 160014, India

    Rajvinder Singh, Kashmir Singh & Jagdeep Kaur

  2. Department of Biotechnology, GGSD College, Chandigarh, India

    Samriti Dhawan

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  1. Rajvinder Singh
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Correspondence to Jagdeep Kaur.

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Singh, R., Dhawan, S., Singh, K. et al. Cloning, expression and characterization of a metagenome derived thermoactive/thermostable pectinase. Mol Biol Rep 39, 8353–8361 (2012). https://doi.org/10.1007/s11033-012-1685-x

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