Heterologous Expression Of Linoleic Acid Isomerase In Prokaryotes

trans10,cis12-Conjugated linoleic acid?t10,c12-CLA?has a lot of biological functions such as anti-tumor,anti-atherosclerosis,reducing intracellular lipid accumulation,and regulating immunity.As a novel food functional factor,t10,c12-CLA shows a great market potential in food and pharmaceutical industries.Linoleic acid isomerase?PAI?,which is derived from Propionibacterium acnes,can efficiently and specifically catalyze the isomerization of linoleic acid?LA?to t10,c12-CLA,therefore,PAI has been expressed in various hosts.However,the heterologous expression of PAI has low expression and enzyme activity,especially in Escherichia coli,PAI forms a large number of non-physiological inclusion bodies,which seriously affects the application of it.Therefore,in order to solve the problems in the heterogenous expression of PAI,8 soluble expression strains in E.coli were constructed by genetic engineering methods,and the effects of promoters and fusion tags on the soluble expression of PAI were studied.Finally,a recombinant E.coli strain with high yield of soluble PAI was obtained.The fusion protein MBP-PAI was then isolated and purified,and its enzymatic properties were studied.This study provides a new solution to solve the problem which PAI has a low solublility in heterologous expression of E.coli.At the same time,this study is the first attempt to secrete PAI by Bacillus subtilis.The results are as follows:?1?Recombinant strains containing promoters T7,CspA,trc and fusion tags?His?₆,Fh8,MBP were successfully constructed in E.coli:E.coli BL21?DE3??pET24a-Fpai?,E.coli BL21?DE3??pET24a-Mpai?,E.coli BL21?pCold-Hpai?,E.coli BL21?p Cold-Fpai?,E.coli BL21?pCold-Mpai?,E.coli JM109?pTrc-Hpai?,E.coli JM109?pTrc-Fpai?,E.coli JM109?pTrc-Mpai?.?2?The effects of fusion tags and promoters on total expression,soluble expression,and relative enzymatic activity of PAI were studied.In terms of total expression,compared with?His?₆,the MBP-tag promoted the total expression of PAI,whereas Fh8-tag severely affected it due to the large difference in gene codon between Fh8-tag and E.coli.Therefore,it was impossible to do further analysis of Fh8-tag.Promoters also affected the total expression of PAI,namely,the stronger the promoter,the higher the total expression of PAI,which was T7>CspA>trc.In terms of the soluble expression level of PAI,it was influenced by fusion tags.Among the three fusion tags,MBP had the best solubilizing effect.In terms of relative enzyme activity,the effects of the tags and promoters were consistent with their effects on soluble expression levels.Therefore,MBP-tag and T7 promoter were the best combination,which made E.coli BL21?DE3??pET24a-Mpai?the best recombinant strain expressing the highest soluble PAI in this study.?3?The E.coli BL21?DE3??pET24a-Mpai?strain with the highest soluble expression level of PAI was used to optimize the inducing conditions.The optimized conditions were:induction temperature 20°C,IPTG concentration 0.1 mmol/L and induction time 18 h.By using affinity chromatography,the electrophoretically pure MBP-PAI was successfully isolated from recombinant strain.After purified,the specific enzymatic activity of MBP-PAI was 1083.61 nmol/min/mg,the protein yield was about 54.52%,and the purification fold was about 41.2 times.?4?The enzymatic properties of the purified MBP-PAI were studied.The optimum temperature was 37°C,the optimum pH was 7.5,and the stability of MBP-PAI was different at different temperatures.The metal ion K⁺could increase the enzyme activity by 3%,Na⁺had little effect on the enzyme activity,and other metal ions inhibited the catalytic activity of MBP-PAI.Among the four surfactants used in this study,only Tween-20 was able to increase MBP-PAI at low concentrations.The activity of MBP-PAI was increased by 5.86%when Tween-20 was added at a concentration of 0.1 mmol/L,and the activity of MBP-PAI was increased by 53.57%when Tween-20 was added at a concentration of 1 mmol/L.Substrate had no inhibition effect on MBP-PAI.The K_m of MBP-PAI was 253.89?mol/L,and the V_maxax was 2252.76 nmol/min/mg.?5?The secretory expression strain B.subtilis WB800?pMA5-phoD-pai?,B.subtilis WB800?pMA5-YwbN-pai?,B.subtilis WB800?pMA5-Eno-pai?and B.subtilis WB800?pMA5-gapA-pai?were constructed.However,the above four recombinant strains failed to express PAI using a variety of methods which due to the fact that the pai gene contains more rare codons.It may also be related to the fact that the promoter this study used was a non-inducible promoter.