Highly efficient expression and characterization of a beta-mannanase from Bacillus subtilis in Pichia pastoris

摘要

A -mannanase gene (Man5) from Bacillus subtilisBS5 was cloned by PCR and integrated into the genome of Pichia pastorisGS115 via pPIC9 vector. The recombinant Man5 with a molecular mass of 43kDa was successfully expressed and secreted into the culture medium. After methanol induction in a shake flask for 96H, the recombinant Man5 protein reached 375 mu g/mL in concentration, with an enzyme activity of 892U/mL. The recombinant Man5 was purified 3.35-fold with 60% yield by using HiTrap DEAE FF and HiTrap Phenyl FF columns. The specific activity of the purified enzyme was 7,978U/mg. The optimum temperature and pH of the recombinant Man5 were 50 degrees C and 6.0, respectively. Studies of substrate specificity showed that the optimum substrate for the Man5 was konjac flour, suggesting that it has great potential as an effective additive in the food industry.