Preparation And Activity Analysis Of Microcapsulated Recombinant Linoleic Acid Isomerase With Polyelectrolyte

Conjugated linoleic acid is a kind of natural fatty acid which has many physiological functions. Nowadays, it has been used in food, health products, feed industry and others fields, and it has a more broad application prospects. Linoleic acid isomerase as a key enzyme for biological transformation of conjugated linoleic acid, it has extremely important research significance.At present, the production of microbial fermentation can get single isomer CLA which have high activity, but there are some problems in the production process, such as substrate inhibit the growth of bacteria, microbial cultures easy to pollution and so on. Enzymatic production of CLA can be solved the problems in microbial fermentation, and the production condition is mild, the production equipment is simple, no pollution to the environment. So, to obtain the LAI which has high stability, high purity and high activity is path to achieve CLA production.In this study, putting the recombinant strain JM109-LAI-pQE30which is constructed by genetic engineering techniques as the research object. From JM109-LAI-pQE30induced expression of the recombinant LAI and purified the recombinant LAI by the chromatograph. In order to improve the stability of the recombinant LAI, Then uses polyelectrolyte layer self-assembly technique to microcapsulated LAI. The conclusion is as follow:(1) Optimal conditions of recombinant LAI induced expression:the timing of induction culture was recombinant strain growth7h after, induction time was10h, inducer concentration was0.15mM, and induction culture temperature was30℃. Under the optimal conditions of recombinant LAI induced expression, the enzyme specific activity of LAI was(3.85±0.075)×103U/mL.(2) Through the chromatograph for purification, the specific activity of the purified LAI was2.16×103U/mg, enzyme activity recovery was87%and purification fold was23.3times.(3)Using polyelectrolyte self-assembly technique microcapsulated recombinant LAI and the result is as follow:(a) Templates and microcapsulated LAI in optical microscopy and laser scanning confocal microscope are spherical,(b) The best ratio is1:1:2for the CaCl2:NaCO3:LAI of template,(c) The highest specific enzyme activity was (2.12±0.085)×103U/mg when coated LAI of PAH/PSS bilayers was four. Then using four PAH/PSS bilayers microcapsulated LAI, optimum reaction temperature of microcapsulated LAI was50℃, optimum reaction pH of microcapsulated LAI was5, the microcapsulated LAI can be stable when the range of temperature was20℃to60℃and pH was2to7, the half-life of microcapsulated LAI was95.4h.