Study On The Enzymatic Asymmetric Reduction Of Carbonyl Group In The Preparation Of(2S,3R)-2-benzamidomethyl-3-hydroxybutyrate

4-Acetoxy-2-azedinone,(3R,4R)-4-acetoxy-3-[(R)-1-(tert-butyldimeth-ysiloxy)ethyl]-2-acetidinone(4-AA)is the key intermediate for preparation of the penem and carbapenem antibiotics.(2S,3R)-2-benzamidomethyl-3-hydroxybutanoate is the important building block for preparation of 4-AA.However,the traditional chemical synthetic method is required by expensive metal ruthenium catalyst,which is conducted under high temperature and high pressure.Therefore,it’s very important to developed a successful biocatalytic process for the production of(2S,3R)-2-benzamidomethyl-3-hydroxybutanoate with high stereoselec-tivity,high activity and environmental friendliness.This paper was focused on the process,and investigated in terms of detection,characteristic,preparation and application of this biocatalyst.Firstly,a HPLC analytical method of substrate and product was established.Then the strain Burkholderia gladioli ZJB-12126,which could reduce methyl 2-benzamidomethyl-3-oxobutanoate to(2S,3R)－2-benzamidomethyl-3-hydroxybutanoate was isolated.Secondly,using one-factor-at-a-time experimental design,the medium components and cultivation conditions of B.gladioli ZJB-12126 were optimized,and the optimized conditions were as.follows:fructose 10.0 g/L,yeast extract 30.0 g/L,NaCI 4.0 g/L,pH 7.0,loading size 50 mL/250 mL,inoculum size 2%,temperature 28 ℃,150 rpm,culture time 40 h.Under the optimal conditions,the carbonyl reductase activity reached 31.25 U/L,the biomass reached 9.80 gDCW/L and e.e.value reached 62.70%,which improved 3.99 times,0.79 times,1.46 times,respectively.Further,the influences of factors on enzyme stereoselectivity were investigated.Results indicated that using DMF as cosolvent had significant effect not only on enzyme activity but also on enantioselect ivity,e.e.value increased from 48.36%to 62.59%.Thermal pretreatment could inhibit the activity of S-enzyme as an inhibitor.Addition of DMF as cosolvent,heat treatment under 45 ℃ for 5 min,e.e.value and yield were 81.92%and 82.47%,respectively.Using DMSO as cosolvent,and heat treatment under 45 ℃ for 15 min,e.e.value and yield were 80.51%and 66.26%,respectively,and heat treatment for 60 min,e.e.value and yield were 88.22%and 25.25%,respectively.Finally,influences of reaction conditions on activity and stereoselectivity were also investigated.Results indicated that optimal conditions was 35 ℃,pH7.0 phosphate buffer(100 mM),1 g wet cells in 10 mL reaction system,cosubstrate glucose concentration 5%(w/v),cosolvent DMF concentration 7.5%(v/v),thermal pretreatment under 45 ℃ for 5 min.After 24 hour-reaction with 10 g/L substrate under the optimal conditions,the yield and e.e.value of(2S,3R)-2-benzamidomethyl-3-hydroxyl-butanoate were 83.81%and 84.32%,which improved 47.55%and 64.78%,respectively.