The Preparation Of Highly Active And Stable Immobilized Lipase And Its Application In Hydrolyzing Fish Oil

As one of the green biocatalysts,lipases are widely used in a variety of industrial applications,such as the oleochemical,food,pharmaceutical,detergents and biodiesel industries.However,the use of free lipase in industrial application was limited because of their high cost,instability and irrecoverability.Preparation of immobilized lipase with high activity and stability,has became an important subject in its applied research.The use of carrier-crossing linked enzyme aggregates can realize high-efficient immobilization,which were prepared by the combination of carrier material with cross-linked enzyme aggregates.This approach has dominant advantages including high recovery rate,simple preparation,easy recovery and repeated use.In this word the reusable immobilized lipase was prepared through method of carrier-crossing linked enzyme aggregates.The choice of carrier,preparation process,properties and biological transformation of fish oil were investigated.The main research contents and results are as follows:(1)WDA918 was used for the carrier of immobilized lipase.The lipase was prepared through carrier-crossing linked enzyme aggregates technology.The optimal preparation conditions were as follows:the volume ratio of settlement agent 1,4-dioxane to lipase was 4:1;settlement time was 180 min;crosslinking agent genipin concentration and crosslingking time was 30 mM and 60 min;the additive BSA was 0.04 mg/mL;temperature,pH and enzyme concentration was 25 ?,8.0 and 2.4 mg/mL.(2)The enzymatic properties of free lipase and immobilized lipase were studied in this word.The optimal reaction temperature go to 40 ?,which increased by 5 ?higher than free lipase.pH of immobilized lipase was 8.0.Compared with free lipase,temperature and pH stability has been widening.The stability and tolerance of immobilized lipase to temperature,pH,metal ions,chemical reagents,organic solvents and proteases has obviously enhanced.The halftime of free lipase and immobilized lipase enzyme were 31 days and 81 days respectively when they stored at room temperature.The catalytic activity of immobilized lipase retained 31%after 10 batches reuse and showed good storage stability and reusability.The Km value of immobilized and free enzyme were 1.034 mmol/L and 2.203 mmol/L,and the Vmax value of immobilized and free enzyme were 0.107 mmol/(L � min)and 0.393 mmol/(L � min),the immobilized enzyme indicated a higher substrate affinity than that of free lipase.(3)Biotransformation of fish oil catalyzed with immobilized lipase were optimized.The optimal technology was the 24 h reaction time,the 40 ? reaction temperature,oil-water ratio of 1:1,10 mg/mL enzyme quantity.The ethyl fish oil congversion rate was 83%.