Cloning Of Cyclopropane Fatty Acid Synthase Gene From Lactococcus Lactis And Expression In Escherichia Coli

Objective: In order to set up a method of cyclopropane fatty acid in Lactococcus lactis analysis by gas chromatography, and to analysis the cyclopropane fatty acid of Lactococcus lactis subsp. cremoris MG1363。After cfa gene was cloned, it was cloned into the expression vector pGEX-6P-1 to constructe of the prokaryotic expression vector, which was prepared for study of the function of cyclopropane fatty acid.Methods: Several methods of methyl ester from different references were used, and their effects were compare., and then the cyclopropane fatty acid of Lactococcus lactis subsp. cremoris MG1363 was analysed by GC. The cfa gene was amplified by PCR using the Lactococcus lactis subsp. cremoris MG1363 genomic DNA, and the amplified fraction was ligated into the vector pMD19-T for DNA sequencing. The identified cfa gene was cloned into the expression vector pGEX-6P-1 and the recombinant of pGEX-6P-cfa was obtained. The recombinant expression vector was induced and expressed in E. Coli DH5α, and then it wan analysised by SDS-PAGE and GC.Results:(1)Several methods of methyl ester from different references were used, and their effects were compared by comparing the peak area of their gas chromatogram. After comparing several methods of methyl ester, we found that the method of methyl ester catalyzed boron trifluoride is better than others.(2)The fatty acid of Lactococcus lactis subsp. cremoris MG1363 was analysed by GC. The main kind of fatty acid of Lactococcus lactis subsp. cremoris MG1363 was determined. Lactococcus lactis subsp. cremoris MG1363 has a kind of cyclopropane fatty acid as well as other kinds of fatty acid.(3)The cfa gene was amplified by PCR using the Lactococcus lactis subsp. cremoris MG1363 genomic DNA, and the amplified fraction was ligated into the vector pMD19-T for DNA sequencing. The result of amplification for cfa by PCR and sequencing show that the cfa gene of Lactococcus lactis subsp. cremoris MG1363 had benn cloned successfully.(4)The identified cfa gene was cloned into the expression vector pGEX-6P-1 and the recombinant of pGEX-6P-cfa was obtained.(5)The recombinant expression vector was induced and expressed in E. Coli DH5α. The result of SDS-PAGE showed that the relative molecularweight of the expression p roductwas about 71KDa. The resultant analysed by gas chromatography (GC) is the recombinants also have cyclopropane Fatty Acid(19C). Conclusion: The cfa gene from Lactococcus lactis subsp. cremoris MG1363 had been expressed successfully in E. Coli, and the molecular weight of the soluble protein was 71 kDa, the phenomenon that the recombinants also had cyclopropane Fatty Acid (19C) show that the enzyme is active.