Gene Cloning, Expression And Applications Of β-Galactosidase

P-galactosidase usually named lactase, which can hydrolyze lactose into galactose and glucose, also catalyse the formation of galacto-oligosaccharides. Lactase not only could be used for relieve lactose intolerance, but also used in improving dairy products flavor. This study using genetic engineering technology to research lactase from Lactobacillus crispatus B164. This thesis mainly focuses on lactase gene cloning, prokaryotic expression, enzyme properties and applications in production of low-lactose milk.The results as follows:1. Phylogenetic analysis of the 16S rDNA DNA sequence from the strain B164 resulted in that it can be classed in the genus Lactobacillus crispatus. A TAIL-PCR approach used to clone lactase gene bg42-164. Sequences analysis reveals that the lactase gene with the length of encoding a polypeptide of 676 amino acid residues, and no signal peptide. Sequences alignment indicated that bg42-164 belong to the glycosyl hydrolase 42 family.2. The lactase gene bg42-164 ligased with pET-30a(+) and expressed in Escherichia coli BL21 (DE3). The recombinant lactase was purified and characterized. The lactase BG42-164 had a molecular weight of 66 kD. The optimum reaction condition was 50℃and pH 6.0. After treatment of 47℃for 30min, the residue lactase activity was still more than 90%. BG42-164 was significantly activated by metal ions of Mn2+, Zn2+ and Mg2+, but inhibited by the metal ions of K+ and Cu2+, especially SDS. Specific activity of BG42-164 was about 159.77U/mg protein. At 50℃and pH6.0, the Km was 3.559 m mol-L-1 and the Vmax was 19.231n mol-min-1. The lactase gene bg42-164 also ligased with pPIC9 and transformed in Pichia pastoris GS115, unfortunately there is no protein secret out the cell.3. Study on the hydrolysis of lactose in milk by different unit of lactase BG42-164, different treatment peroid at 50℃,37℃and 18℃. HPLC is used to determined the residue lactose of the milk. The experiment result made clear that when lactase BG42-164 is add with 5000 U·mL-10.1mL in 2mL milk,100% of the lactose has been hydrolyzed at 50℃within 2 hours; 99% of the lactose has been hydrolyzed at 37℃within 6 hours; 59% of the lactose has been hydrolyzed at 18℃within 9 hours. Accordingly, the parameter of the low-lactose milk production use BG42-164 were 50℃,2 h, with lactase 5000 U·mL-10.1mL in 2mL milk.