Study On The Construction And Catalytic Properties Of The Three-liquid Interfacial Enzyme Catalysis System

In order to improve the catalytic efficiency of enzymes,new reaction systems have been developed to improve the catalytic efficiency of enzymes since the enzyme has been found to have catalytic activity in the non aqueous phase and will change the catalytic properties of the enzyme.The three liquid phase enzyme catalytic system(Three-Liquid-Phase System,TLPS)is a newly developed enzyme catalysis system and there are few reports on the catalysis of this kind of system.The catalytic properties of the interface enzyme in TLPS have not been reported.In this paper,a Three-Liquid-Phase System(TLPS)was constructed,and the research on the hydrolysis of oil by the lipase and phospholipase in the three liquid phase system was studied.The main results were as follows:Taking lipase AYS as an example,the effect of three liquid phase system on the enzymatic properties and stability of the interfacial enzyme was investigated.It was found that different types of three liquid phase systems had great influence on the activity of enzymes.Compared to the ionic liquid and the short chain alcohol type three liquid phase system,the enzyme activity could be inhibited and the three liquid phase system of the polymer type could be active to the enzyme,The enzymatic properties of the enzyme in the buffer solution and TLPS were investigated and the results showed that the optimum pH of the enzyme in TLPS changed from 7 to 5 and was stable under the alkaline condition.Compared with the buffer solution,the enzyme decreased to half of the original Km in the TLPS,which was 0.47 mmol/L.The hydrolysis ability of the enzyme was evaluated by hydrolyzing the olive oil containing short chain fatty acids in the three liquid phase system and the oil-water system.The results showed that the hydrolysis efficiency in the three liquid phase system was higher than the oil-water system and the difference between the three liquid phase system and the oil-water system by hydrolyzing fish oil to enrich n-3 PUFA was compared.The results showed that the n-3 PUFA enrichment rate in TLPS was found 3.63 times higher than that of oil-water system.The effects of phase salts,adding amount of hydrophilic solvents,pH value,rotational speed and reaction time on enzyme catalytic behavior were investigated comprehensively.The reutilization of enzyme in three liquid phase system was investigated.The results showed that enzyme activity did not lose significantly after being used for eight times.The results of particle size analysis show that compared with water,fish oil has a larger specific surface area in the middle phase of TLPS.The laser confocal results show that the emulsion droplets have a unique Spherical shell structure in the three liquid phase system.The combined catalysis of double enzyme in the three liquid phase system was used to hydrolyze rice bran oil as an example,adding phospholipase and lipase to realize the degumming and hydrolysis of rice bran oil.The results of SDS-PAGE electrophoresis and enzyme activity analysis showed that the lipase and phospholipase could be enriched into the middle phase of TLPS.The effects of the amount of salt and hydrophilic solvent,the ratio of two enzymes and the speed of the enzyme on the double enzyme catalysis were investigated,and the reused of the double enzyme was also investigated.It was found that the activity of the double enzyme remained over 90% after 7 repeated use.