Chiral Resolution Of(R,S)-1-phenylethanol With The Lipases In The Nonaqueous Phases

Lipase is the enzyme that degrades the fat.It was a kind of special ester-hydrolysis enzyme,found in most animals,plants and microorganisms.Lipase has widely been used in the synthesis of esters,resolution of racemic compounds,the selective protection of intermediates,and the synthesis of high polymers and peptides.1-Phenylethanol,including R and S enantiomers,is colorless liquid with the flowerlike aroma.It is one of the important chiral building blocks for organic synthesis in perfume industry and pharmaceutical industry.The preparation of chiral 1-phenylethanol can be divided into microbial and enzymatic methods.In this paper,one microorganism which owned high resolution activity for 1-phenylethanol was screened from soil.The microorganism was identified preliminary and the production conditions for lipase were optimized.The lipase was separated and purified.The kinetic parameters were determined,too.The resolution of 1-phenylethanol by transesterification with different lipases was compared in the organic solvents.And the process of hydrolysis with lipases was optimized in supercritical CO2.The obtained results were as follows:1.9 microorganisms with high lipase activity were screened from soil using olive oil as the sole C-source.In them,the lipase activity of 15-9(0.46 U/mL)was the highest.2.The isolated strain 15-9 was identified by internal transcribed spacer(ITS)region amplification and sequencing.Then,the evolutionary status and species of the strains were determined through sequence similarity analysis and construction of phylogenetic tree.The strain was identified as Rhizopus stolonifer.3.The media for the microorganism were optimized using Plackeet-Burman,Steepest Ascent and Box-Benhnken experiments.After optimization,the composition of the optimized medium used for lipase production was as follows:soluble starch 7.60 g/L,yeast extract 22.59 g/L,K2HPO45.55 g/L,NH4Cl 1.80 g/L,MgSO4·7H2O 2.40 g/L,NaCl 1.00 g/L,olive oil 25.00 mL/L,Tween-80 8.00 mL/L,pH 4.59,and the lipase activity increased nearly by 3.02-fold,from 0.46 to 1.40 U/mL.4.The lipase of R.stolonifer was separated and purified with pretreatment,(NH4)2SO4 salting-out,dialysis,concentration,n-hexane extraction and DEAE-Sepharose ion-exchanging.The electrophoresis-purified sample was prepared.The molecular weight was 35.2 KDa and lipase activity 5760 U/g.5.The kinetic parameters,Km and Vmax,were obtained as 0.84 mmol/L as 6.29?mol/(min·L),respectively,with Lineweaver-Burk plot using pNPP as the substrate.This indicated that the affinity between lipase and pNPP was great.6.The optimized process of R.stolonifer lipase by transesterification was:total volume 5 mL,n-heptane as the solvent,lipase amount 20 mg,water activity 0.35,temperature 45 ?,1-phenylethanol 0.08 mol/L and vinyl acetate 0.4 mol/L.After the optimization,the conversion rate and ee reached 49.88%and 99.53%,repectively,much higher than those before the optimization(15.97%and 19.0%,respectively).7.The process of hydrolysis with lipase was investigated and optimized.For the different lipases,Novozym 435 was the most suitable to resolute 1-phenylethanol,whose ee reached 100%.The optimized conditions in normal pressure were:substrate concentration 1.0%,solvent 40%tert-butanol and phosphate buffer,Novozym 435 amount 300 mg/mL substrate and reaction time 9 h.Under the conditions,ee value and hydrolysis rate reached 100%and 24.15%,respectively.The optimized conditions in supercritical CO2 were:substrate concentration 1.0%,temperature 40? pressure 10 MPa,Novozym 435 amount 300 mg/mL substrate and reaction time 75 min.Under the conditions,ee value and hydrolysis rate reached 100%and 28.92%.