| Optically pure chiral alcohols and their derivatives are important building blocks for thesynthesis of chiral pharmaceuticals, agrochemicals and functional materials. Among them,enantiopure1-(4-methoxyphenyl)ethanol (MOPE) is a key chiral building block.(R)-MOPEcan be employed for synthesis of chiral3-aryl-3-substituted propanoic acids with anti-inflammation activity, while (S)-MOPE is a key chiral synthon for the synthesis of cycloalkyl[b] indoles， which have the treatment function for general allergic response. Currently,enantiopure chiral alcohols could be prepared mainly through chemical or biological means.Compared with chemical methods, biological methods with advantages of mild reactionconditions, high entioselectivity and environmental friendly has attached much attention.Generally, whole-cell biocatalysts are preferable to isolated enzymes because they are morestable, with no need for enzyme purification and coenzyme addition. Additionally, the use ofimmobilized cells as biocatalysts can not only facilitate product separation, but also makecells recyclable and reusable, thus greatly simplifying the process and lowering the cost ofproduction. However, up to now the biocatalytic resolution of MOPE to get enantiomerpure(S)-MOPE using biocatalytic methods has remained largely unexplored, and the achievedresidual substrate e.e. and conversion of MOPE were unsatisfactory. In this dissertation, acomparative study was made of the biocatalytic asymmetric oxidation of racemic MOPE withimmobilized Acetobacter sp. CCTCC M209061cells in various reaction media [especially indeep eutectic solvents, DESs], and the effects of diverse reaction conditions on thebiocatalytic reduction of MOPE were explored systematically. The operational stability ofimmobilized Acetobacter sp. CCTCC M209061cells in various reaction media was examined.The efficient biocatalytic asymmetric oxidation of MOPE with immobilized Acetobacter sp.CCTCC M209061was evaluated on a preparative scale. Additionally, the novel biocatalyticreaction system used for highly efficient and enantioselective synthesis of enantiopure (S)-MOPE was established.Among the tested seven microbial strains, Acetobacter sp. CCTCC M209061, which wasisolated from Chinese kefir grains by our research group, showed to be the best biocatalyst forthe asymmetric oxidation of MOPE to get enantiopure (S)-MOPE. Then, Acetobacter sp.CCTCC M209061cells were entrapped by chitosan and then crosslinked with glyoxal.Compared with free cells, the immobilized cells showed better thermal stability, storabilityand operational stability. Additionally, the immobilized cells afforded the maximumconversion (around49.4%) which was close or comparable to that with the free cells, and more than99.0%of the residual substrate e.e., the residue substrate was (S)-MOPE and thesubstrate enatiometric ratio E value was above60. Consequently, the immobilizedAcetobacter sp. CCTCC M209061cells were chosen for subsequent investigations.The poor concentration of MOPE and the pronounced inhibitory of the reactants wereobserved in the aqueous monophasic system. The optimal substrate concentration, buffer pH,co-substrate concentration, reaction temperature, biocatalyst concentration and shaking speedwere30mmol/L,6.5,70mmol/L acetone,30oC,0.3g/mL and180r/min, respectively. Underthe optimized conditions, the initial reaction rate, the conversion and the residual substrate e.e.were102.1μmol/min,49.4%and above99.0%, the residue substrate was (S)-MOPE and thesubstrate enatiometric ratio E value was above60, respectively. The reaction efficiencyremains to be further improved.To solve the aforementioned problems, adding hydrophilic new media DESs to theaqueous buffer in a metabolic regulation way was thought to be able to make the cellmembrane more permeable and lower the product concentration in the cells, finallyimproving the reaction efficiency. Of these tested seven DESs,[ChCl][Gly] showed the bestbiocompatibility with immobilized cells. The cell membrane integrity was higher in the[ChCl][Gly]-buffer system to maintain the normal physiological activity, and the cellmembrane permeability was increased properly, thus easing the product inhibition andincreasing the initial reaction rate. In the [ChCl][Gly]-buffer system, the optimal content ofDES, reaction temperature, buffer pH, acetone concentration, shaking rate and substrateconcentration for the reaction were found to be10%,30oC,6.5,140mmol/L,220r/min and60mmol/L, the residue substrate was (S)-MOPE and the substrate enatiometric ratio E valuewas above60, respectively, under which for7h the initial reaction rate, the conversion andthe residual substrate e.e. were129.8μmol/min,51.5%and above99.9%.To further improve the reaction efficiency, using the hydrophobic solvents to extract thereactants in a phase transfer way was thought to be able to lower the reactants concentration.Hydrophobic organic solvents or ionic liquids/buffer biphasic system were common biphasicsystems. The effects of various organic solvents and hydrophobic ionic liquids (ethyl acetate,isopropyl ether, cyclohexane, n-hexane, isooctane, n-nonane, dodecane, C4MIM·PF6,C5MIM·PF6, C2MIM·Tf2N, C4MIM·Tf2N, PP14·Tf2N) on asymmetric oxidation of MOPE with immobilized Acetobacter sp. CCTCC M209061cells were studied here. Of all theexamined solvents, C4MIM·PF6was the best second phase. The optimal reaction condition forthe asymmetric oxidation of MOPE with immobilized Acetobacter sp. CCTCC M209061cells were as follow: the volume ratio of C4MIM·PF6was20%, co-substrate acetone, acetoneconcentration152mmol/L, substrate concentration65mmol/L, reaction temperature30oCand shaking speed220r/min. Under these conditions for10h, the initial reaction rate, theconversion and the residual substrate e.e. were97.8μmol/min,50.5%and above99.9%, theresidue substrate was (S)-MOPE and the substrate enatiometric ratio E value was above60,respectively. Further study had shown that the C4MIM·PF6gave the best biocompatibility andrevealed high partition coefficients on the reactants, thus illustrating the best reactionefficiency was observed in the C4MIM·PF6/buffer biphasic system.The pervious study had shown that hydrophobic organic solvents or ionic liquids/bufferbiphasic could effectively alleviate the substrate inhibition, but the initial reaction rate waslow. However, adding hydrophilic new media DESs to the aqueous buffer could improve theinitial reaction rate obviously and alleviate the product inhibition. So further study wasconducted in C4MIM·PF6/[ChCl][Gly]-buffer system to obtain the best catalytic efficiency.Reaction conditions were as follws: the volume ratio of C4MIM·PF6was20%,10%[ChCl][Gly] was adding to the buffer, buffer pH was6.5,30oC, acetone concentration was186mmol/L, shaking rate was220r/min and substrate concentration was80mmol/L, underwhich for7h the initial reaction rate, the conversion and the residual substrate e.e. were124.0μmol/min,51.3%and above99.9%, the residue substrate was (S)-MOPE and the substrateenatiometric ratio E value was above60.Among the above-mentioned three reaction systems examined, the best operationalstability of immobilized Acetobacter sp. CCTCC M209061cells was observed in the[ChCl][Gly]-containing system, the immobilized cells still retained more than71%of itsoriginal catalytic activity after being repeatedly used for10batches (7h per batch); in buffersystem, the relative activity of the immobilized cells was reduced to53.0%after beingrepeatedly used for10batches (9h per batch); in C4MIM·PF6/buffer biphasic system, therelative activity of the immobilized cells was65.0%after being repeatedly used for10batches (10h per batch) which was higher than buffer system; in C4MIM·PF6/[ChCl][Gly]-buffer biphasic system, the relative activity of the immobilized cells was67.0%after being repeatedly used for10batches (7h per batch)which was similar to C4MIM·PF6/bufferbiphasic system.The500-mL preparative scale under the optimized conditions was carried outof all the four reaction systems, the best reaction efficiency was observed inC4MIM·PF6/[ChCl][Gly]-buffer system, which the initial reaction rate, the conversion and theresidual substrate e.e. were113.3μmol/min,50.2%and99.9%, respectively. Henceasymmetric oxidation of MOPE to get enantiopure (S)-MOPE with immobilized Acetobactersp. CCTCC M209061cells is promising and competitive.This study provides not only a deeper understanding of immobilized cells catalysis inDESs, but also a novel and efficient route to the resolution of racemic aromatic alcohol. |
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