A Study On Immobilization Of Lipase And Trypsin And Their Co-Immobilization

The co-immobilization of lipase and trypsin on macro porous resin supports and theapplication was studied in the present paper. The effect of proteases involoving trypsin,papain and bromelain on lipase was also studied, and then trypsin was chose to beco-immobilized with lipase on macro porous resin HPD600as the support. The results showas followings:1: Sepharose CL-4B and seven kinds of macro porous resins were compared as thesupports for immobilizing lipase Palatase20000L and TL100L. The immobilizationconditions, such as temperature, pH, time and ratio of lipase/support were optimized, and thenthe stability and enzymatic properties of free lipase and immobilized lipase was compared.The results showed that the lipase immobilized on macro porous resins HPD600exhibitedhigher activity and better stability.2: Among bromelain, papain and trypsin, trypsin showed significant effect on the activityof lipase Palatase20000L. The activity of lipase Palatase20000L was found to be increasedabove30%by trypsin under optimal treatment conditions. The optimum pH of thetrypsin-treated lipase was kept unchanged at8.0, while the thermal stability of thetrypsin-lipase was lower than the native lipase, resulting in the decline of optimumtemperature. It was also found that the trypsin-treated lipase had lower Kmvalue(0.079g/mL) than the native lipase (0.100g/mL), indicating a higher affinity towards substrateolive oil. No appreciable change in the molecular was observed on the SDS-polyacrylamidegel electrophoresis chromatogram, indicating that the structural change in lipase associatedwith the treatment is considerably minor.3: Trypsin was chose to be co-immobilized with lipase20000L and lipase TL100L onmacro porous resin HPD600respectively. The co-immobilization conditions were optimized,and then the properties of co-immobilized trypsin were studied. The thermal stability andpH stability of trypsin were improved by immobilization, while the optimal reaction pH valueshowed unchanged, with extended range of optimal reaction temperature.4: The mixture of soybean oil and casein, soybean milk, peanut milk and pure milk werechose as substrates to study the application performance of the co-immobilized trypsin andlipase. The hydrolyzed protein degree was determined by the ninhydrin method. Highperformance liquid chromatography method was adopted for the determination of oilhydrolysis and alkali titration method was used for the determination of the fatty acidcontent. It was observed that the co-immobilized lipase and trypsin showed a high activity on the mixture of soybean oil and casein and the co-immobilized lipase and trypsin showed asynergistic effect. When soybean milk, peanut plasma and pure milk were chose as substrates,the co-immobilized enzymes also showed good hydrolysis activity.