Study On Enzymatic Reactions In Subcritical R134a

1.The stability of lipases and proteinases in subcritical R134a (1,1,1,2-tetrafluoroethane) was investigated.The influence of operating variables, namely,pressure(2-8MPa),temperature(30-60℃),incubation time(1-12h),water content and depressurization rate,on the residual activity of enzymes was tested.The results indicated that subcritical R134a treatment led to significant increase of activities of lipase AY30G,lipase R,lipaseA6,lipase G50 and papain,and a loss of activity of bovine trypsin within the ranges studied.DSC and Raman spectra were used to study the structural changes of papain and trypsin after subcritical R134a treatment.The results indicated that subcritical R134a treatment led to no change of secondary structure.The activity changes of papain and trypsin may be resulted from modification of tertiary structures.The esterification of oleic acid with glycerol was studied as a model reaction for evaluation of activities of lipases in subcritical R134a. The influence of operating variables,namely,pressure,temperature and water content, on the activities of lipase was tested.The results indicated that lipases,namely,lipase AY30G,lipase R,lipaseA6 and lipase G50 exhibit high activities in subcritical R134a.2.Lipase-catalyzed esterification of glycerol with n-3 polyunsat urated fatty acid from fish oil to produce n-3 polyunsaturated fatty acid glyceride was performed in subcritical R134a.The stability and activity of commercial immobilized lipase from Candida antarctica(Novozym 435) in subcritical R134a was investigated.The esterification of oleic acid with glycerol was studied as a model reaction in subcritical R134a and in solvent-free conditions.The results indicated that subcritical R134a treatment led to significant increase of activity of Novozym 435.No loss of activity of Novozym435 treated with subcritical R134a under different operation factors (pressure 2-8 MPa,temperature 30-60℃,incubation time 1-12 h,water content 1:1, 1:2,1:5 enzyme/water) was observed.While the initial reaction rate was high in subcritical R134a,higher conversion was obtained in solvent-free conditions. Response surface methodology was used to model Novozym435-catalyzed esterification.The reaction factors investigated were reaction time,water content, substrate molar ratio,and enzyme amount.Well-fitting quadratic polynomial regression model for degree of esterification(DE) was established after regression analysis with backward elimination and verified by a F-test.Optimal reaction conditions were:in subcritical R134a,4MPa,40℃,reaction time 3 hours,enzyme amount 3%of substrates,substrate molar ratio 4:1(glycerol/n-3 polyunsat urated fatty acid,W/W),without additional water and DE was 84.06%under these conditions. Under optimal conditions,the rate of recovery of glyceride was 91.4%,and glyceride composition:monoglycerides:28.17%,diglycerides:68.31%,triglycerides:3.52%. The results indicated that Novozym435 exhibited 1,3-regioselectivity but no substrate selectivity..The property of repeated use of Novozym435 in subcritical R134a was investigated under optimal conditions.The residual activityof Novozym435 was 109.6%after 40 cycle uses.3.In subcritical R134a,commercial immobilized lipase Lipozyme TL IM was used as catalyst for preparation of EPA/DHA phospholipids.The stability of Lipozyme TL IM in subcritical R134a was investigated.The results indicated that subcritical R134a treatment led to significant increase of activity of Lipozyme TL IM.No loss of activity of Lipozyme TL IM treated with subcritical R134a under different operation factors(pressure 2-8 MPa,temperature 30-60℃,incubation time 1-12 h,water content 1:1,1:2,1:5 enzyme/water) was observed.The influences of factors on Lipozyme TL IM -catalyzed acidolysis between soybean phospholipids and free fatty acids was investigated.These factors include amount of enzyme,pressure, temperature,water content and molar ratio of substrates.The results indicated that Lipozyme TL IM exhibited high activity in subcritical R134a and higher conversion was obtained with lower amount of enzyme.Response surface methodology was used to model Lipozyme TL IM -catalyzed acidolysis.Optimal reaction conditions were:in subcritical R134a,6MPa,40℃,reaction time 1 hour,enzyme amount 14.77%%of substrates,substrate molar ratio 4:1(n-3 polyunsaturated fatty acid/ soybean phospholipids,mol/mol),water content of 1%and conversion was 52.16%under these conditions.Under optimal conditions,the rate of recovery of phospholipids was 90.4%,EPA:12.23%,DHA:39.83%in its fatty acid composition.4.Phospholipase D -catalyzed transphosphatidylation from phospholipids of egg of squid Symplectoteuthis oualaniensis to produce n-3 polyunsaturated fatty acidenriched phosphatidylserine(PS) was performed in subcritical R134a.The influence of several parameters(pressure,temperature,water addition) on the conversion was investigated.Response surface methodology was used to model the reaction. Optimal reaction conditions were:in subcritical R134a,6MPa,40℃,reaction time 3 hours,enzyme amount 9.6U,75mg PC,1.8 ml 0.2M acetate buffer(pH5.5) containing 85mg L-Serine and conversion was 89.06%under these conditions.The effect of resulted n-3 polyunsaturated fatty acid- enriched phosphatidylserine(PS) on cellular growth inhibition of Hela and Caco-2 cells was investigated.The results showed that growth inhibition of Hela and Caco-2 cells was apparent.