| Lipase(EC 3.1.1.3)is a ubiquitous hydrolase known to hydrolyze the ester bonds of water insoluble substrates in aqueous medium.Furthermore,in contrast to many other enzymes,lipase shows remarkable levels of activity and stability under non-aqueous conditions,which facilitates the catalysis of reversible reactions(such as esterification and transesterification reactions).Due to these unique properties,lipase is one of the most attractive enzymes for catalysis under non-aqueous conditions.However,lipase displays lower catalytic performance in non-aqueous conditions compared with aqueous solutions.Enzyme immobilization can overcome this problem.Siliceous mesocellular foams(MCFs)possess unique features such as high pore size,pore volume and specific surface area and have been prospected as a promising carrier for enzyme immobilization.Therefore,a series of MCFs materials modified with organo-functinal groups were synthesized for lipase immobilization.We selected Rhizopus chinensis Lipase(r27RCL)and Candida antarctica lipase B(CALB)as model enzymes and studied the effect of immobilized method on esterification activity,stability of immobilized lipases in non-aqueous conditions.(1)In view of“interfacial activation”of lipase,organo-functinal MCFs materials with various surface hydrophilicities/hydrophobicities were prepared to study the correlation between the catalytic performance of the immobilized lipase and the hydrophobicity of the support.The results showed that lipase loading,catalytic activity,thermal stability,organic solvents stability and reusability satbility of immobilized lipase were increased with the enhanced surface hydrophobicity of MCFs materials modified with organo-functinal groups.Furthermore,lipase immobilized onto octadecyl modified MCFs(MCFs-C18)showed remarkably increased activity for the esterification reaction between ethanol and n-Caprylic acid(The activities of MCFs-C18-r27RCL and MCFs-C18-CALB were about 15 and 20 times higher than those of r27RCL and CALB,respectively).Then immobilized lipase was used for the resolution of(R,S)-1-phenylethanol through acylation in n-hexane.Excellent enantioselectivity(E?400,with ee_p=99%)was obtained for MCFs-C18-r27RCL(E=4,with ee_p=56%for free r27RCL).(2)Lipase was immobilized via covalent bonding onto 3-aminopropyltriethoxysilane(APTES)modified MCFs(MCFs-NH2)with oxidized gum arabic(GA)as coupling agent.Under optimized conditions,the esterification activities of MCFs-NH2-GA-r27RCL and MCFs-NH2-GA-CALB were about 1.3 and 1.2 times higher than those of MCFs-NH2-G-r27RCL and MCFs-NH2-G-CALB,respectively.In addition,lipase immobilized onto MCFs-NH2 with oxidized gum arabic as coupling agent showed remarkably thermal stability,and reusability satbility compared to these with glutaraldehyde as coupling agent.For example,MCFs-NH2-GA-r27RCL retained about 45%initial activity by incubating immobilized enzyme in n-heptane at 70°C for 12 h(42%for MCFs-NH2-G-r27RCL).MCFs-NH2-GA-CALB retained about 60%initial activity by incubating immobilized enzyme in n-heptane at70°C for 12 h(56%for MCFs-NH2-G-CALB).(3)To combine the benefits of covalent binding and adsorption,chemical modification of MCFs materials was performed by the simultaneous use of two organic silican:glycidoxypropyltrimethoxylsilane for covalent linkage and octyltriethoxysilane for hydrophobic interaction.The results showed that immobilization of lipase onto hetero-functional MCFs(MCFs-epoxy-C8)obtained remarkably esterification activities,thermal stability,organic solvents stability and reusability stability.For example,MCFs-epoxy-C8-r27RCL retained about 42%initial activity by incubating immobilized enzyme in n-heptane at70°C for 12 h(40%for MCFs-C8-CALB)and about 34%initial activity by incubating immobilized enzyme in 30%(v/v)methanol for 2 h(20%for MCFs-C8-r27RCL).MCFs-epoxy-C8-CALB retained about 43%initial activity by incubating immobilized enzyme in n-heptane at 70°C for 12 h(40%for MCFs-C8-CALB)and about 98%initial activity by incubating immobilized enzyme in 30%(v/v)methanol for 96 h(81%for MCFs-C8-CALB).(4)Oxidized gum arabic(GA)and oxidized sodium alginate(SA)were used as cross-linkers to replace traditional glutaraldehyde.Supported cross-linked enzyme aggregates were prepared by immobilization of lipase onto hydrophobic surface of octyl-modified mesocellular foams(MCFs-C8).Supported cross-linked enzyme aggregates via oxidized gum arabic and oxidized sodium alginate exhibited significantly improved thermal stability and organic solvents tolerance compared to the free lipase,lipase adsorbed onto MCFs-C8 and supported cross-linked enzyme aggregates using glutaraldehyde(G-CLEAs@MCFs-C8).For example,supported cross-linked r27RCL aggregates using oxidized gum arabic(GA-CLEAs@MCFs-C8-r27RCL)produced esterification activity almost 9 folds greater than that of free lipase.In addition,GA-CLEAs@MCFs-C8-r27RCL maintained 48%of initial activity by incubating immobilized enzyme in n-heptane at 70°C for 12 h and 37%of initial activity by incubating immobilized enzyme in 30%(v/v)methanol for 2 h.However,G-CLEAs@MCFs-C8-r27RCL maintained 43%of initial activity by incubating immobilized enzyme in n-heptane at 70°C for12 h and 35%of initial activity by incubating immobilized enzyme in 30%(v/v)methanol for2 h.GA-CLEAs@MCFs-C8-CALB produced esterification activity almost 20 folds greater than that of free lipase.In addition,GA-CLEAs@MCFs-C8-CALB maintained 57%of initial activity by incubating immobilized enzyme in n-heptane at 70°C for 12 h and 99%of initial activity by incubating immobilized enzyme in 30%(v/v)methanol for 96 h.However,G-CLEAs@MCFs-C8-CALB maintained 42%of initial activity by incubating immobilized enzyme in n-heptane at 70°C for 12 h and 97%of initial activity by incubating immobilized enzyme in 30%(v/v)methanol for 96 h.(5)Regarding regiospecificity,lipase r27RCL was specific for the sn-1,3 positions.Enzymatic synthesis of 1,3-dioleoyl-2-palmitoylglycerol(OPO)from tripalmitin(PPP)with oleic acid(OA)was optimizing.Under optimized condition(30 mg immobilized lipase,4%water content,PPP/OA molar ratio 1:6,50°C,48 h),GA-CLEAs@MCFs-C8-r27RCL led to the highst oleic acid incorporation(37%)and could exhibit high convertion rate after being used for 5 cycles.Immobilized CALB was employed for biodiesel production by transesterification of soybean oil with methanol.In the optimization condition(30 mg immobilized lipase,12%water content,oil to methanol molar ratio 1:6,added 6 h intervals,40°C,24 h),GA-CLEAs@MCFs-C8-CALB were able to produce 91%fatty acid methyl esters(FAME)yield and still showed high FAME yield after 5 repeated cycles. |
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