Purification,characterization And Application Of Lipase From Golden Pompano(Trachinotus Ovatus) Viscera

Golden pompano is an important mariculture economic fish species in Southern China,which produces a large amount of scraps during processing.The viscera are the main by-product and rich in various enzymes.Due to special marine environment,fish have distinctive physiological functions and eating habits.Fish lipases may have higher activity under certain pH and temperature conditions or show strong specificity for certain fatty acids compared with microorganisms,terrestrial animal and plant-derived lipases.In order to improve the added value of golden pompano processing waste and explore a new source of lipase with specific activities and potential application value,a lipase was isolated and purified from golden pompano viscera,and its enzymatic properties,as well as applications in fish skin degreasing and ?-3 polyunsaturated fatty acids(?-3PUFA)enrichment in fish oil were studied to provide technical support for the development and utilization of the golden pompano viscera in the future.This research mainly includes the following contents and results:1.Isolation and purification of golden pompano viscera lipase.The lipase was extracted from the viscera and purified by organic reagent degreasing,buffer extraction,ammonium sulfate fractionation,anionic-exchange chromatography and gel filtration,and recorded as GPL.The specific activity of GPL reached 57.20 U/mg using p-nitrophenyl palmitate(p-NPP)as a substrate,a final purification factor of 27.50 was achieved,and the final recovery yield of enzymatic activity was 18.31%.2.Characterization of golden pompano viscera lipase.The molecular weight of GPL was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and then the effects of pH,temperature,metal ions,surfactants,enzyme inhibitors,bile acid,NaCl and organic reagents on GPL activity were determined using p-NPP as a substrate,and kinetic parameters of GPL were also measured.The results showed that the molecular weight of GPL was about 14 kDa.GPL was active within the pH range of 6.0?10.0,with an optimum pH of 8.0.GPL exhibited an optimum at 40 ? and was stable between 10?40 ?.The Km and vmax values had been identified as 40.16 ?M and 769.23 ?mol/(L min).The hydrolytic activity of GPL was enhanced by Mn2+,but inhibited by Cu2+,Zn2+,EDTA,PMSF,Triton X-100,and Tween-80.Ca2+ had no effect on GPL activity.It was worth to notice that bile salt acted as an activator of GPL.In addition,GPL was active in the presence of salt concentrations up to 1 M and relatively stable in methanol,ethanol,chloroform,and hexane.The inhibitory effect of Orlistat on lipase activity indicated that GPL was a serine enzyme,like all known lipases purified from different origins.3.Application of golden pompano viscera lipase.We discussed the applications of lipase in fish skin degreasing and enrichment of fish oil ?-3 PUFA.Tilapia skin is rich in lipids,and the majority are unsaturated fatty acids which are oxidative.Lipase partially purified from 75-90%ammonium sulfate precipitation was used for skin enzymatic degreasing to improve its processing adaptability,and the single factor test and response surface test were used to optimize the fish skin enzymatic degreasing process.The results showed that the degreasing rate of tilapia skin reached 67.02%under the conditions of enzyme addition of 90 U/g,pH value of 7.5,temperature of 37 ? for 60 min,indicating that the crude lipase could be effective in the removal of fish skin lipids in mild conditions.The content of EPA and DHA in golden pompano viscera oil is relatively low which cannot meet the demand of the production of fish oil health products.In this study,selective hydrolysis was used to hydrolyze golden pompano viscera oil by GPL.Under the conditions of enzyme addition of 3%(w/w),pH value of 7.0,temperature of 35 ? for 16 h,the content of PUFA in fish oil was enriched up to 56.99%,C20-22 ?-3 in particular,was enriched up to 40.9%,increased 3.9 times compared with that before enrichment.The content of EPA +DPA was enriched to 36.37%,matching the standard of SC/T 3503-2000 polyene fish oil.The consequences above showed that GPL could effectively enrich ?-3 PUFA in fish oil.