Cloning, over-expression and characterization of an alkali-tolerant endo-β-1,4-mannanase from Penicillium freii F63

A glycosyl hydrolase family 5 endo-β-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 g l–1 in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60°C and exhibited good stability over a broad pH range from acidic to alkaline (> 85.0% activity at pH 4.0–9.0, > 70.0% activity at pH 10.0 and 43.7% even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co2 +, Cu2 +, Mn2 + and β-mercaptoethanol and was strongly inhibited by Hg2 + and SDS. The specific activity, Km and Vmax values were 47.5 U mg− 1, 7.8 mg ml− 1 and 70.4 μmol min− 1 mg− 1, respectively, for locust bean gum, and 40.3 U mg− 1, 2.3 mg ml− 1 and 61.7 μmol min− 1 mg− 1, respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries.