Immobilization Of Lipase AYS And Its Application In Enzymatic Fractionation Of Conjugated Linoleic Acid

Conjugated linoleic acid(CLA)is a generic term for a class of linoleic acid isomers with conjugated double bonds.A large number of experiments show that CLA has anti-cancer,anti-atherosclerosis,enhance immunity,reduce fat deposition and other physiological activity,but at present researchers at home and abroad have not yet completely the CLA isomers and their biological activity of a A correspondence.Therefore,the resolution of CLA isomers to obtain a single component of great significance.Conjugated linoleic acid isomers of enzymatic resolution has been reported in some reports,the use of more Lipase AY30 catalytic reaction,and lipase AYS split rarely reported.After the lipase has been immobilized,it has the advantages of good operation stability,good storage stability and reusability.Therefore,this study decided to separate the conjugated linoleic acid by free lipase Lipase AYS and its immobilized enzyme to prepare high purity CLA isomers.The main findings are as follows:1.Enzymatic fractionation of conjugated linoleic acid using Lipase AYS.Conjugated linoleic acid was resolved using lipase AYS from Candida rugosa and the product was isolated.The optimal reaction conditions were as follows: methanol as reaction substrate,temperature of 40 ?,enzyme dosage of 180 U/ g,buffer p H of 6.5,molar ratio of CLA to methanol 1: 1.Under the optimal conditions,the esterification rate of CLA reached 49.5% after 8 h reaction.The resolution efficiency of c9,t11-CLA methyl ester and t10,c12-CLA were 86.2% and 80.0%,respectively.The recoveries were 72.4% and 54.2% respectively.2.Immobilization of Lipase AYS and its properties.Lipase AYS was immobilized and seven different types of resins were screened to determine the best effect when using epoxy resin ECR8285 immobilized Lipase AYS.The optimum immobilization conditions were as follows: the ratio of enzyme to carrier was 60 mg/ g and the optimum p H was 7.0.The immobilized enzyme obtained under this condition had a hydrolase activity of 1446 U / g.At the same time,some simple properties of the immobilized enzyme Lipase AYS were also studied.The experimental results show that the enzyme has the best catalytic temperature at 40 ?,and has good storage stability.After stored at 4 ? for 60 days,92% of the original enzyme activity;with good operational stability,after 5 batches of experiments still maintain the original enzyme activity of 80%.3.Enzymatic fractionation of conjugated linoleic acid using Immobilized Lipase AYS.The immobilized enzyme Lipase AYS was used to catalyze the resolution of conjugated linoleic acid.The optimal reaction conditions were: temperature of 40 ?,enzyme of dosage 150 U/g,reaction substrate CLA and methanol ratio of 1: 1,The resolution efficiency of c9,t11-CLA methyl ester and t10,c12-CLA were 86.0% and 82.9%,respectively,and the recoveries were 70.8% and 63.8% respectively.The reaction conditions were as follows:.Comparison of free enzyme and immobilized enzyme esterification results,we can conclude that the two catalyze the reaction of conjugated linoleic acid and methanol,the product of fatty acid composition gap is small,but the immobilized enzyme has a fast reaction time,the enzyme repeat stability high and conducive to recovery characteristics.