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Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum

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Abstract

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca2+ or Mg2+ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca2+ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.

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Abbreviations

PME:

Pectin methylesterase

DE:

Degree of esterification

HMP:

High methoxyl pectin

LMP:

Low methoxyl pectin

CP:

Citrus pectin

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Acknowledgements

The research was supported by funding from Indian Institute of Technology Guwahati, Guwahati from Ministry of Human Resource Development, India. The authors are grateful to Central instrument facility (CIF), IIT Guwahati, for providing the facility of MALDI-TOF MS analysis.

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  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

    Vikky Rajulapati & Arun Goyal

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  1. Vikky Rajulapati
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Correspondence to Arun Goyal.

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Rajulapati, V., Goyal, A. Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum . Mol Biotechnol 59, 128–140 (2017). https://doi.org/10.1007/s12033-017-9997-7

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