Gene Cloning Of Lactase

The enzyme β-beta-D-galactosidase(beta-D-galactoside galactohydrolase, EC3.2.1.23, also referred to as lactase)hydrolysies the disaccharide lactose to glucode and galactose,and also can transfer galactose. Lactase is mainly used in producing oligo-galactose and improving dairy products, as well as in the research related to protein synthesis and heredity factor.So lactase is valuable in theory and practice.This paper mainly dealt with isolation,purification L.delbrueckii bulgaricus from Dairy Starter Culture;and characterization of β-galactosidase produced by L.delbrueckii bulgaricus. The lactase gene from it was researched aslo in the thesis. In addition, we analysed the L.lac genomia DNA and amino acid sequences.Aaccording to facultative anoxybionsis property of L.delbrueckii bulgaricu which diferent from Streptococcus thermophilus, L.delbrueckii bulgaricu were isolated and purified from Dairy Starter Culture by anoxybionsis andsteak plating.The lactase solution was prepared which produced by L.delbrueckii bulgaricus. It was determinated of the optimum pH, stability, optimum reaction tempreture, thermal stability with ONPG as a substrate. Its optimum pH was 6. 6 to 7. 0,and the optimum tempreture was 42℃ to 47℃. It had better basicity-tolerance and worse acidity-tolerance. So the lactase was suitable to whey at pH7. 0, but couldn't react at high tempreture.Specific primers were designed according to the sequence of the beta-galactosidase gene and Primer Premier 5 from L.delbrueckii bulgaricus. L.lac genomic DNA was amplified by PCR and subsequently cloned into the plasmidpGEM T-Easy.Sequences analysis revealed that the genomic DNA with length of 3130bp encoding a polypeptide of 1007 amino acid residues. Hydrophobicity Profile and Hydrophilicity Profile were found between 800-840 and 700-730 site. 8 potential N-glycosylation sites and 8 Protein kinase C phosphorylation site and 18 Casein kinase Ⅱ phosphorylation site and 2 Tyrosine kinase phosphorylation site and 19 N-myristoylation site and 1 Glycosyl hydrolases family 2 signature 1 and 1 Glycosyl hydrolases family 2 acid/base catalyst are assumed. It was showed a very low homology of Comparing the gene DNA and the deduced amino acid sequences with the lactase sequences from various organism sources.