Construction Of Recombinant Lactococcus Lactis With Expressing And Secreting High Activity β-galactosidase And A Primary Study On The Property Of Producing The Enzyme

ObjectiveUsed high β-galactosidase activity Lactobacillus bulgaricus strains β -galactosidase gene as original gene, Lactococcus lactis usp45 gene synthesized by PCR as secretion signal gene, and expression plasmid vector pMG36e to construct recombinant plasmids pMG36e-lacZ and pMG36e- usp -lacZ . Obtained Lactococcus lactis recombinant strains with expression and secretion high activity β-galactosidase after electrotransformed Lactococcus lactis MG1363 with recombinant plasmids, and studied their property of producing enzyme. These studies could establish basis for the construction of food-grade recombinant bacteria secretion of high activity β -galactosidase and using them for therapy and studies on lactase deficience.MethodsThis study contained 5 parts.1. The identification of β-galactosidase gene origin bacteria and the studies on their property of producing enzyme.Carbohydrate ferment tests were used to identify the two strains of Lactobacillus bulgaricus. 16SrRNA sequence analysis methods were used to identify Lactobacillus bulgaricus wch9901 which separated from yoghourt. To Study the property of producing enzyme of these two strains of Lactobacillus bulgaricus, we observed the effects of incubation time, carbon source in culture medium, shaker speed and oxygen concentration to their P-galactosidase production.2. The screening of human gastrointestinal Lactobacillus strains with distinct acid and bile salt resistance.To screen the bacteria with distinct acid and bile salt resistance, we determined the ability of resisting acid and bile salt of 104 strains of human gastrointestinal Lactobacillus separated from human stomach samples and fecal samples.