Ahmed_2022_Int.J.Biol.Macromol__

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, 65s degreesC and thermostability with half-life, 5.1sh at 80s degreesC. 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 5sM urea. CtPae12B showed broad substrate specificity as it displayed activity against a range of synthetic substrates showing highest V(max), 770sU/mg and K(m), 1.2smM with beta-D-gluco pentaacetate. CtPae12B could deacetylate both pectic and xylan substrates showing highest V(max), 770sU/mg and K(m), 13.4smg/mL with potato rhamnogalacturonan and V(max), 105sU/mg and K(m), 7.1smg/mL with acetylated birchwood xylan. The thermostability, pH stability and broad substrate specificity of CtPae12B makes it a versatile enzyme for industrial applications.