Screening Of Conjugated Linoleic Acid-producing Lactic Acid Bacteria And Clone And Expression The Linoleate Isomerase Gene

Conjugated linoleic acid (CLA) containing cis-or trans-conjugated double bond is a kind of octadecadienoic acid and is a general term for a set of locations and conformational isomers of the essential amino acids linoleic acid (LA). Since CLA is a kind of functional fatty acid which has many physiological functions, such as anti-cancer, anti-atherosclerosis, immunity regulation, growth and development promotion, weight loss and so on, much attention has been paid on it in recent years. In this study, we used the CLA product strain lactic acid bacteria S2which was screened from the northeast traditional fermented sauerkraut juice as the original strain and cloned the linoleic acid isomerase gene using genetic engineering techniques. Then the gene was connected with the pMD18-T vector and constructed a recombinant plasmid by inserting it into expression plasmid pET-28a. The recombinant E.coli was constructed by transforming the recombinant plasmid into E.coli BL21(DE3) and induced by IPTG. The recombinant linoleic acid isomerase was purified by nichel column and then determined the enzyme activity. Research and the results are as follows:1.This study is using UV spectrophotometry to determine the production of Conjugated linoleic acid produced by two strains of CLA Production lactobacillus (A53.2and S2) screened from Pickle juice, and the production was14.91μg/mL and10.082μg/mL, respectively. We identified the two stains as Lactobacillus rhamnosus A53.2and Lactobacillus plantarum S2through the combination of the methods of observing the morphology of strain, discussing the physiological and biochemical properties,16SrDNA Molecular Identification Method and the method of constructing the phylogenetic tree, ect.2.Iinoleic acid isomerase gene was amplified by PCR through the design of specific primers and genomic DNA of Lactobacillus plantarum S2as a template. And it was cloned to the carrier pMD18-T and sequenced. The results showed that in comparison with the nucleotide sequence of linoleic acid isomerase whose gene bank accession is FR732048.1, the nucleotide sequence homology of linoleic acid isomerase was99%, while its putative amino acid sequence homology was98%. Theoretical relative molecular mass of the gene is about65kDa, because it can code573amino acids.3.Constructing recombinant expression plasmid pET-LAI and transforming recombinant plasmid into the expression host strain E.coli BL21(DE3), to induce gene expression of recombinant bacteria linoleic acid isomerase with inducer IPTG. Through preliminary optimization induction temperature and inducer concentration, identified the optimal conditions of the induced expression:induction temperature is25℃, final concentration of IPTG is0.6mmol/L, and induction time is13h.4.Utilized Nickel column to purify recombinant linoleic acid isomerase,and carried out SDS-PAGE analysis after eluting by different concentrations of imidazole,the results showed that the elution was best when the concentration of imidazole was200mmol/L, emergence of a single band, and protein relative molecular mass was consistent with the predicted results. Measured enzyme activity of purified recombinant linoleate isomerase which was63.51U, and confirmed that recombinant linoleic acid isomerase has activity.