Research On The Recombinant Efficient Expression Of Bsh Gene In Lactobacillus Plantarum

Bile salt hydrolase(BSH)is an enzyme produced by several bacterial species in the human or animal gastrointestinal tract that catalyzes the glycine-or taurine-linked bile salt deconjugation reaction.There are a lot of refrences focused on bsh gene cloning and expression,however,the results of the expression are different in Escherichia coli,Lactococcus lactis and Yeast,This research established a method for the recombinant expression of bsh gene in Lactobacillus plantarum.bsh gene were amplified by polymerase chain reaction(PCR)using total genome of health ICF mice feces as template,and the products were inserted into pSIP expression plasmid which could be induced to express gene in L.plantarum.The strategy to achieve bsh gene deletion L.plantarum is based on Cre-lox recombination system.And the mutant used as a host bacteria.The results not only eliminated the interference of the bsh gene product in host bacteria,but also improved the accuracy of detection of gene expression,because the antibiotic resistance selection marker on the chromosome of the host was removed.On this basis,several bsh gene recombinant strains were obtained,such as WB461?WB464.The BSH assay was earried out by determining the amount of liberated amino acids from the conjugated bile salts according to the ninhydrin method.One unit of BSH activity was defined as the amount of enzyme that releases 1 ?M of amino acid from the substrate per minute.The method has strong operability and high repeatability.This study detected BSH activity of four recombinant strains WB461?WB464 with different bsh genes and the results showed that bsh genes of the recombinant strains were expressed.WB462 recombinant strain with bsh gene drived from L reuteri has the highest activity(1.725 U/mL).In summary,the research realized the recombinant expression of bsh gene in L.plantarum without bsh gene successfully and provided a method for the recombinant expression of other bsh genes.This will lay a good foundation for further research of BSH's features.It will also provide a theoretical basis for the development and utilization of lactic acid bacteria in food industry.