Study On Enzymatic Characterization And Necessary Groups Of Linoleate Acid Isomerase From Lactobacillus Plantarum P-8

CLA(conjugated linoleic acids)is a fatty acids with a variety of physiological activities,such as anti-cancer,lowering cholesterol,lowering blood fat,enhancing immunity.It has geometric and positional linoleic acid isomer which have cis or trans conjugated double bond.Linoleic acid isomerase(LAI)is a very important isomerase which could convert linoleic acid into conjugated linoleic acid.The Lactobacillus Plantarum P-8 also have LAI.But we don't know the structure,function and catalytic mechanism about the LAI.In this experiment,in order to explore enzymatic characterization and some necessary amino acid groups LAI from the Lactoba.cillus Plantarum P-8,we conformed recombinant strain:BL21-pET28a-LAI.We optimized the expression condition and analysed the enzymatic characterization.The results show that the optimum enzyme expression condition is 0.1 mM IPTG,130 rpm,and 14 hours,the optimum substrate concentration of purified LAI is 0.3 mg/mL,the optimum temperature is 37?,the optimum enzyme dosage is 100 ?L,the optimum pH is 7.0.Km=211.368 g/L,Vmax=75.188 mg/L X h.We analyed the structure of LAI by Specia lized BLAST and vecter NTI,found the possible necessary groups.We used DNAMAN to design six pairs of primers for six site-directed mutagenesis.Using the Quick Change method.Successfully construct the mutant plasmid:pET28a-T563C-LAI,pET28a-G320T-LA,pET28a-A515C-LAI,pET28a-G203C-LAI,pET28a-A389T-LAI and pET28a-G458T-LAI that express mutant F188S-LAI,R107L-LAI,H172P-LAI,G68A-LAI,N130I-LAIandS153I-LAI and identify.Then we induced the recombinant enzyme expression with IPTG,broke the cells,centrifuged the liquid and remained the supernatant.Then we purificated the protein:LAI?F188S-LAI?R107L-LAI?H172P-LAI?G68A-LAI?N130I-LAI and S153I-LAI.We use gas chromatography to text the enzyme activity.Then we could know which site is the necessary groups.The result show that the activity of F188S-LAI?R107L-LAI?H172P-LAI and G68A-LAI was decreased significantly,but activity of N130I-LAI and S153I-LAI was increased significantly.The change of the six sites caused a great change of enzyme activity.So in LAI the 188 F(proline)?107 R(Arginine)?172 H(histidine)?130 N(asparagine)and 153 S(serine),are necessary groups.