Study On The Stereoselective Catalytic Performance And Purification Of Lipase From Aspergillus Oryzae

Lipases(triacylglycerol acyl hydrolase,EC 3.1.1.3)catalyze the hydrolysis of triacylglycerols to release free fatty acids and glycerol.Lipases usually display exquisite chemoselectivity,regioselectivity and stereoselectivity.The lipase-catalyzed reactions carry out under mild conditions and do not require cofactors.So lipases are very promising catalysts and are widely applied in chemical,pharmaceutical,food,detergent,biodiesel,leather,pulp and paper industries.In this paper,the lipase from Aspergillus oryzae WZ007(AOL)was demonstrated with high enantioselectivity in kinetic resolution of(RS)-?-lipoic acid,(DL)-menthol and(RS)-1-phenylethanol.And(RS)-1-phenylethanol was the best substrate.So the reaction conditions of AOL-catalyzed kinetic resolution of(RS)-1-phenylethanol were further investigated.Also,AOL was applied in the asymmetric catalysis of the aldol reaction of C-C bond formation.Then the purification and the properties of this lipase were studied.By screening the lipases for kinetic resolution of three substrates,including(RS)-?-lipoic acid,(DL)-menthol and(RS)-1-phenylethanol,AOL was finally selected as the favorable candidate because it showed good enantioselectivity for these substrates,especially for(RS)-1-phenylethanol.Then two types of AOL,such as mycelium-bound AOL and surfactant-modified extracellular free AOL(lipase-surfactant complex,LSC),were further investigated to kinetic resolution of(RS)-1-phenylethanol.The effects of acyl donor,type of solvent,substrate molar ratio and reaction temperature were studied when mycelium-bound AOL was served as a catalyst.The optimum reaction conditions were found to be transesterification with vinyl acetate at 30°C in methyl tert-butyl ether with a vinyl acetate:(RS)-1-phenylethanol molar ratio of1:1 and an enzyme concentration of 60 g/L.The conversion to(R)-1-phenylethyl acetate could reach above 46%with>99%ee_p when the substrate concentration was below to 1.4 M.The conversion could achieve 40%with>99%ee_p at the substrate concentration of 1.8 M.The volumetric productivity could achieve 7.5 mmol/L·h at the substrate concentration of 1.8 M with the reaction time of 96 h.The surfactant-modified extracellular free AOL(LSC)was also applied to kinetic resolution of(RS)-1-phenylethanol.The various modification conditions affecting the LSC yield and the performance for kinetic resolution of(RS)-1-phenylethanol were investigated,including type of the surfactant,organic cosolvent,buffer and surfactant amount.The optimum conditions were found to be modified with Tween-40 at the phosphate buffer(0.1 M,p H of 7.0)when tetrahydrofunan used as a cosolvent.The LSC precipitate was obtained by mixing 50 m L of crude enzyme solution and 0.3 g of Tween-40 dissolving in tetrahydrofunan.Freeze dried LSC showed good performance for kinetic resolution of(RS)-1-phenylethanol in methyl tert-butyl ether at the temperature of30°C.The conversion to(R)-1-phenylethyl acetate could reach above48%with>99%ee_p with an enzyme concentration of 10 g/L and a reaction time of 2 h when the substrate concentration was 0.1 M.The volumetric productivity of LSC could achieve 24 mmol/L·h at this reaction conditions,which was significantly improved compared with that of mycelium-bound AOL.AOL was then applied to catalyze aldol reaction of carbon-carbon bond formation,which demonstrated catalytic promiscuity with this lipase.Surprisingly,AOL also displayed enantioselectivity for the aldol reaction.The aldol reaction of p-nitrobenzaldehyde and cyclohexanone was selected as a model reaction.The various reaction parameters affecting the conversion and enantioselectivity were studied,including type of solvent,water content,substrate molar ratio,reaction temperature and enzyme concentration.The optimum reaction conditions were found with a solvent of acetonitrile,a water content of 5%,a cyclohexanone:p-nitrobenzaldehyde molar ratio of 35:1 and a reaction temperature of30°C.At the optimum reaction conditions,the conversion could reach above 95%after 60 h reaction with a diastereoselectivity(dr(anti/syn))of82:18 and an enantiomeric excess of anti-isomer product(ee(anti))of80%,when the substrate concentration was 0.1 M.AOL was purified by ammonium sulfate fractionation,hydrophobic interaction chromatography,anion-exchange chromatography,and gel filtration.The purified enzyme was homogeneous with a mass molecular of 25 k Da as judged by SDS-PAGE.MALDI-TOF/TOF mass spectrometry analysis of the purified enzyme showed the amino acid sequences of three peptide fragments were highly homologous with those of the lipase or cutinase from A.oryzae RIB410 and Aspergillus flavus NRRL3357.The purified enzyme showed high performance for the kinetic resolution of(RS)-1-phenylethanol and the aldol reaction of p-nitrobenzaldehyde and cyclohexanone.The optimum p H and optimum temperature of the enzyme were 7.5 and 40°C,respectively.The enzyme was stable over a p H range of 7.0–8.5 and up to 30°C.Mg²⁺,Na⁺,K⁺,Li⁺,NH₄⁺,Mn²⁺and Co²⁺had no effect on the enzyme activity.Ca²⁺and Cu²⁺had a slight inhibition on the enzyme activity.Both Ag⁺(10 m M)and sodium dodecyl sulfate(10 m M)completely inhibited the enzyme activity.The surfactant of Tween-80 significantly promoted the enzyme activity.Sodium deoxycholate slightly promoted the enzyme activity.Dithiothreitol,2-mercaptoethanol and EDTA had no effect on the enzyme activity.The enzyme showed high activities towards the short-chain p-nitrophenyl esters and long-chain triglycerides.Low enzymes activities were observed towards the long-chain p-nitrophenyl esters,short-chain triglycerides and some fatty acid ethyl esters.The enzyme kinetic parameters of K_mand V_mwith p-nitrophenyl acetate as substrate were25.33 mmol/L and 0.37 mmol/L·min,respectively.Ammonium salt was confirmed to be able to promote the spontaneous hydrolysis of p-nitrophenyl esters with a short-chain carboxylic group such as p-nitrophenyl acetate and p-nitrophenyl butyrate.The hydrolysis rate increased with the increase in the p H-value,the reaction temperature or the concentration of ammonium ion.The lipase from Candida antarctica(Nov 435)activities decreased about 13–40%when the concentration of ammonium sulfate increased from 40%to100%saturation.This work provides a useful reference for the assay of the lipase/esterase activity using p-nitrophenyl acetate or p-nitrophenyl butyrate as the substrate when ammonium salts exist in the enzyme samples.