Biocatalytic Synthesis of Betahistine Chiral Intermediate by Asymmetric Reduction Using Kluweromyces sp.
|Course||Biochemistry and Molecular Biology|
|Keywords||(S)-(4-Chlorophenyl)-(pyridin-2-yl)methanol asymmetric reduction Kluyveromyces sp. aqueous two-phase system Betahistine carbonyl reductase purification|
Chiral aromatic alcohols are important intermediates for the synthesis of optically activemolecules and are widely applied in food, pharmaceutical, agricultural, fine chemicals areas.Biocatalytic synthesis is usually preferred to chemical synthesis due to number of advantagesincluding high enantioselectivity, mild reaction conditions, environmental friendly, and etc.The production of chiral alcohols by reducing prochiral ketones with microbial cells has beenapplied in a in a number of biocatalytic processes.（S）-（4-Chlorophenyl）-（pyridin-2-yl）methanol [（S）-CPMA] is an important chiralintermediate for the synthesis of antiallergic drug Betahistine. In this study, carbonylreductase-producing microorganisms was isolated from soil samples were screened for theasymmetric reduction of （4-chlorophenyl）-（pyridin-2-yl）methanone （CPMK） to （S）-CPMA.Among over400microorganisms isolated, one strain showed the highest activity was selectedand identified as Kluyveromyces sp based on its18S rRNA gene sequence. The reactionconditions of the asymmetric reduction of CPMK were optimized as follows:30oC,100g/Lcell concentration,2g/L substrate concentration,3%glucose in pH7.0potassium phosphatebuffer. Under above optimum conditions,（S）-CPMA in76.0%e.e. and77.0%yield wasobtained after48h of reaction.This study, for the first time, reported the asymmetric synthesisof （S）-CPMA using a microbial strain, which was newly isolated and identified asKluyveromyces sp.In this study, biocatalytic production of （S）-CPMA in an aqueous two-phase system（ATPs） using Kluyveromyces sp. was studied. In ATPs consisting of PEG4000（20%, w/w） andNa2HPO4（14%, w/w）, the product reached86.7%e.e. and92.1%yield at substrateconcentration of6g/L （in contrast with13.6%yield and77.4%e.e. in aqueous system）. Thesubstrate tolerance and the biocompatibility of cells are greatly improved in ATPs byaccumulating substrate/product in the upper PEG solution, and the two-phase partitioncoefficient was calculated to be K=6.57.An NADH-dependent carbonyl reductase was purified from cell-free extract ofKluyveromyces sp. using ammonium sulfate fractionation, HiPrep DEAE-Sepharose FF anionexchange chromatography and Sephadex G-100filtration, in which the activity of the enzymewas increased for326-fold （from0.072U/mg to23.5U/mg）. The molecular weight of theenzyme subunit was estimated to be around30kDa by SDS-PAGE. The purified carbonylreductase exhibited maximum activity at pH6.5and30℃, and retained over80%activityover a pH range of57.5and temperature range of2035℃. The carbonyl reductaseshowed high substrate specificity towards aromatic ketones substrates with phenyl group. Itwas observed that Ca2+, Mg2+, Ni2+exhibited certain different degree positive effect on theactivity of the enzyme while Ba2+, Cu2+, Ag+and Fe3+showed inhibitory effect.