Production Optimization of an Active β-Galactosidase of Bifidobacterium animalis in Heterologous Expression Systems.

β-Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The β-galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a β-galactosidase gene (bg42-106) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0-8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of β-galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.