A thermotolerant and pH stable rhamnogalacturonan acetylesterase (CtPae12B), a family 12 carbohydrate esterase from Clostridium thermocellum with broad substrate specificity

Abstract

The gene encoding rhamnogalacturonan acetylesterase, CtPae12B from Clostridium thermocellum was cloned, expressed, purified and biochemically characterized. Purified CtPae12B was soluble and exhibited homogenous single band. Phylogenetically it was most closely related to an RGAE, YesT from B. subtilis. CtPae12B production was maximum with LB medium. CtPae12B showed optimal temperature, 65 °C and thermostability with half-life, 5.1 h at 80 °C. CtPae12B was alkaliphilic with optimal pH, 8.0, while it displayed stability at both acidic and alkaline pH ranges. Inhibition of CtPae12B activity by PMSF showed the importance of nucleophilic serine in the catalytic triad. The metal ions, chemical or chelating agents used, did not enhance CtPae12B activity, which was also corroborated by protein melting study. The enzymatic activity of CtPae12B remained unaffected by 5 M urea. CtPae12B showed broad substrate specificity as it displayed activity against a range of synthetic substrates showing highest Vmax, 770 U/mg and Km, 1.2 mM with β-D-gluco pentaacetate. CtPae12B could deacetylate both pectic and xylan substrates showing highest Vmax, 770 U/mg and Km, 13.4 mg/mL with potato rhamnogalacturonan and Vmax, 105 U/mg and Km, 7.1 mg/mL with acetylated birchwood xylan. The thermostability, pH stability and broad substrate specificity of CtPae12B makes it a versatile enzyme for industrial applications.