Overexpression Of β-Galactosidase From Bacillus Circulans In Pichia Pastoris

Lactase can be used for treatment of "lactose intolerance" and production of low-lactose milk, whey and other dairy products. Main problems of lactase production are low yield, complicate process of deriving from yeasts and narrow applied range.The lacO gene encoding a Galactosidase from Bacillus circulansand was modified to the lacM gene without changing its amino acid sequence. According to the bias in codon choice of yeast and the factors which affect the gene transcription, translation and the stability of mRNA in eukaryote, the lacM gene which be fit for expression in Pichia pastoris was designed and synthesized. The lacM gene was cloned into vector of pPIC9 and spliced with the signal peptide encoding sequence of Pichia pastoris alpha-factor in reading frame correctly, and constituted the recombinant vector of pPlC9-LacM. The lacM was driven by the alcohol oxidase gene promoter. A high-expression strain was constructed by transforming pPIC9-LacM into Pichia pastoris GS115.The assay result revealed that the recombinant lactose gene was expressed highly and secrete effectually in Pichia pastoris, and the expressed product had the normal bioactivity. The enzyme activity in the medium was reached 2237.9U/mL after inducing by methanol for 144h, which is 3~5 folds of that in recombinant Pichia pastoris with unmodified lacO gene. Specific activity of LACM was about 1638U/mg. SDS-PAGE revealed that the molecular weight of the recombinant expressed lactase was about 66kD. The LACM shows optimum pH value of 4.6 to 6.4, optimum temperature of 55 癈 and good pH stability from 3.0 to 8.0. Most metal ions and chemicals have no effect on the activity of LACM. Zn2+, K+ and Fe2+ can slightly activate the enzyme, and Cu2+> SDS were potent inhibitor for LACM.