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Molecular Biology Reports

, Volume 39, Issue 8, pp 8353–8361 | Cite as

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

  • Rajvinder Singh
  • Samriti Dhawan
  • Kashmir Singh
  • Jagdeep Kaur
Article

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.

Keywords

Pectinase GH family 28 Metagenomics Thermostability Recombinant expression 

Notes

Acknowledgments

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

Supplementary material

11033_2012_1685_MOESM1_ESM.doc (196 kb)
Supplementary material 1 (DOC 196 kb)

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Rajvinder Singh
    • 1
  • Samriti Dhawan
    • 2
  • Kashmir Singh
    • 1
  • Jagdeep Kaur
    • 1
  1. 1.Department of BiotechnologyPanjab UniversityChandigarhIndia
  2. 2.Department of BiotechnologyGGSD CollegeChandigarhIndia

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