Study On Screening Of Mutant For Producing Linoleic Acid Isomerase And Catalytic Performance

Conjugated linoleic acid(CLA)has many important physiological functions,such as anti-tumor,enhancing immunity and reducing weight.The methods of producting CLA mainly include chemical synthesis and biosynthesis.The biosynthesis has strong specificity and high content of active isomers in products compared with chemical synthesis.It is important to obtain strains that produce high yield of linoleic acid isomerase for production of CLA.In this paper,L.acidophilus was subjected to ARTP mutagenesis to screen strains for producing high yield of linoleic acid isomerase and to optimize the conditions for producing enzyme.The model of kinetic for producing enzyme on mutants was built.The effects of reaction conditions and catalytic systems on the catalytic performance of linoleic acid isomerase were discussed,and the reaction conditions were optimized.A mutant named B-5 for producing linoleic acid isomerase was obtained by ARTP mutagenesis,and the enzyme activity reached 964.4 U/m L,which was 2.26-fold as high as that of the original strain.Further the effects of carbon source,nitrogen source,C/N,the initial p H,the culture time and culture temperature on production of linoleic acid isomerase were studied.The conditions for production of linoleic acid isomerase were optimized by response surface.The optimal conditions for producing linoleic acid isomerase were as follows: 2% glucose,2% beef extract,C/N 1:1.5,cultivation time 36 h,cultivation temperature 36.4 ?,initial p H 5.6.Under above conditions,the enzyme activity of linoleic acid isomerase reached 1214.1 U/m L,which was 2.84-fold as high as that of the original strain.The dynamic changes of growth,substrate consumption and synthesis were studied.The kinetic model of mutant was constructed by mathematical fitting.The dynamics equation of growth,matrix consumption and synthesis on mutant were obtained.The nonlinear fitting of the 3 equations was found by using Origin8.6.It was found that the predicted values was similar to the actual observed values.The model could simulate the dynamic change of growth,consumption of substrate and the synthesis of linoleic acid isomerase in the process of producing enzymes.The effects of phosphate system,citrate system,acetate system,reaction p H,reaction temperature,reaction time and substrate concentrations on the conversion of LA to CLA were studied.The reaction conditions were optimized by response surface.The optimum reaction conditions were as follows: the concentration of substrate LA 15 mg/m L,the reaction temperature 37 ?,and the reaction p H 5.0.Under these conditions,the yield of CLA reached 31.86%.