Thermostable Recombinant β‑(1→4)-Mannanase from C. thermocellum: Biochemical Characterization and Manno-Oligosaccharides Production

Functional attributes of a thermostable β-(1→4)-mannanase were investigated from Clostridium thermocellum ATCC 27405. Its sequence comparison the exhibited highest similarity with Man26B of C. thermocellum F1. The full length CtManf and truncated CtManT were cloned in the pET28a(+) vector and expressed in E. coli BL21(DE3) cells, exhibiting 53 kDa and 38 kDa proteins, respectively. On the basis of the substrate specificity and hydrolyzed product profile, CtManf and CtManT were classified as β-(1→4)-mannanase. A 1.5 fold higher activity of both enzymes was observed by Ca2+ and Mg2+ salts. Plausible mannanase activity of CtManf was revealed by the classical hydrolysis pattern of carob galactomannan and the release of manno-oligosaccharides. Notably highest protein concentrations of CtManf and CtManT were achieved in tryptone yeast extract (TY) medium, as compared with other defined media. Both CtManf and CtManT displayed stability at 60 and 50 °C, respectively, and Ca2+ ions imparted higher thermostability, resisting their melting up to 100 °C.