Preparation And Characterization Of Immobilized Lipase Based On Macroporous Adsorbent Resin

Biocatalytic process has the advantages of environmental friendliness andmild reaction conditions,so its proportion in industrial applications has increased significantly.With the increasing application of immobilized lipase in industrial process,it is of great social and economic significance to develop new immobilized lipase with excellent catalytic performance.Based on the development trend of the application of biocatalysts in industrial production,more and more studies have been carried out on the design and characterization of new carrier materials for lipase immobilization.Therefore,based on macroporous resin as carrier,a new method of lipase immobilization was developed,and the catalytic performance and application of immobilized lipase were studied.This paper studies are carried out in the following three aspects:1.A fast and sensitive fluorescence method for the detection of lipase transesterification activity was established.Lipase catalyzed transesterification of butyric acid-4-methyl umbelliferone?Bu-4-Mu?with methanol in tert-butanol as a model reaction.The yield of 4-methyl umbelliferone?4-Mu?in the model reaction was determined by measuring the fluorescence intensity at?_ex=330 nm and?_em=390nm to determine the esterification activity of lipase.In order to determine the accuracy and efficiency of the proposed detection method,commercial immobilized lipase was used to verify the catalytic application.The apparent Michaelis constant?K_m?of lipase-catalyzed transesterification of Bu-4-Mu with methanol was calculated.The main advantages of this method include high sensitivity,low cost of reagents and simple operation.2.In this paper,a method of lipase immobilization by macroporous resin NKA adsorption combined with Lipozyme TL 100L cross-linked enzyme aggregates?CLEA-TLL?was developed.The key factors affecting the preparation of immobilized lipase CLEA-TLL@NKA,such as type of organic solvent,type and concentration of crosslinking agent,type and amount of resin,were studied.The optimum conditions for the preparation of immobilized enzyme were obtained.The esterification activity of immobilized enzyme reached 6.38 U/mg using isopropanol as precipitator,polyvinylimide and glutaraldehyde as crosslinking agent.The surface structure of the immobilized enzyme was characterized by scanning electron microscopy?SEM?.Scanning electron microscopy images showed that CLEA-TLL enzyme complex was evenly loaded on the surface of macroporous resin NKA.The immobilized enzyme CLEA-TLL@NKA was applied to methyl esterification of soybean oil deodorizer distillate.The catalytic performance and operational stability were studied.After 20 batches of immobilized enzymes catalyzed reaction,the enzyme activity remained above 90%of the original activity.The experimental results show that the immobilized enzyme CLEA-TLL@NKA has high catalytic activity and good operational stability,so it has good industrial application prospects in the field of oil processing.3.A method for immobilization of Candida antarctica lipase?CAL-B?on metal-organic framework?ZIF-8?and macroporous resin was developed.Metal organic frameworks have many advantages,such as porous and stable.As a carrier for enzyme immobilization,ZIF-8 can improve the stability and catalytic efficiency of immobilized enzyme.The experimental steps of enzyme immobilization are as follows:firstly,the molar ratio of 2-methylimidazole to zinc nitrate is 40:1,and the concentration of enzyme protein is 1.75 mg/mL.Then,CAL-B-ZIF-8 is immobilized by adding D101 macroporous resin and crosslinking agent.Finally,the immobilized enzyme CAL-B-ZIF-8@D101 is obtained by vacuum drying.The catalytic performance and surface properties of the immobilized enzyme CAL-B-ZIF-8@D101 were characterized.The results showed that the immobilized enzyme had good organic solvent tolerance and catalytic activity.