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

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 35.2kDa on SDS-PAGE gels. The molecular mass, 35.5kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5mg/mL). CtPME showed maximum activity (18.1U/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 5mM 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 5mM Ca2+ ions, and the addition of 5mM EDTA shifted back the melting peak to 80°C. CtPME can be potentially used in food and textile industry applications.