Screening And Properties Of Lactobacillus Plantarum (R)-CHBE Dehydrogenase

(R)-Ethyl 4-chloro-3-hydroxybutyrate,abbreviated as（R）-CHBE,is a chiral organic compound according to biochemical standards.It is an indispensable intermediate in the synthesis of many chiral drug compounds,which can be synthesized by reducing the chlorine group,substitution or introducing other functional groups.Additionally,chiral substances synthesized using（R）-CHBE as an intermediate are widely used in pharmaceuticals and food industries.In recent years,（R）-CHBE has mainly been synthesized through kinetic resolution and asymmetric synthesis methods.The former has been gradually eliminated due to low substrate utilization and complicated product purification steps.The latter includes chemical and biological synthesis methods.However,the former is gradually being phased out due to its strict reaction conditions and low enantiomeric selectivity.The reaction conditions of biosynthesis of（R）-CHBE are mild,and the product enantio-selection rate is high,and it gradually enters industrial production.In addition,because the substrate 4-chloroethyl acetoacetate（COBE）and product（R）-CHBE are easily hydrolyzed under alkaline conditions or in environments with high temperatures.Therefore,this study aims to find enzymes that can stably synthesize（R）-CHBE under acidic conditions.First screening from the acid-resistant strain Lactobacillus plantarum DSM 20174（L.plantarum DSM 20174）to aldehyde ketone reductase（LPAKR5 and LPAKR9）capable of asymmetrically synthesized（R）-CHBE,and then constructing a co-expression system of LPAKR9 and glucose dehydrogenase（GDH）to achieve NADPH regeneration and（R）-CHBE synthesis.The main findings are as follows:（1）L.plantarum DSM 20174 with good acid tolerance and（R）-CHBE production ability was screened from laboratory-preserved strains.Based on the analysis of the complete genome of L.plantarum DSM 20174,14 LPAKRs genes were successfully cloned and connected to the p RSFDuet-1 plasmid,and then induced for expression in Escherichia coli BL21（DE3）.It was found that only LPAKR5 and LPAKR9 could catalyze COBE,and their protein molecular weight was about 31 k Da.（2）LPAKR5 and LPAKR9 were purified using a nickel column and desalination column,and their enzymatic properties were studied.the results of the experiment indicated that LPAKR5 and LPAKR9 had an optimal p H of 6.0,while exhibiting high stability for 12 hours at this p H level.Furthermore,LPAKR5 exhibited an optimal temperature of 30℃,whereas LPAKR9’s optimal temperature was 40℃.And they could maintain high stability at lower temperatures.The K_m values of LPAKR5 and LPAKR9 for COBE were 9.5 m M and 8.7 m M,the V_max values were 7.6 U·mg^-1 and8.59 U·mg^-1,and the k_cat/K_m values were 2.40 L·mmol^-1·s^-1 and 1.95 L·mmol^-1·s^-1,respectively.（3）A dual-enzyme co-expression system of E.coli BL21（DE3）/p RSFDuet-1-lpakr9-gdh was constructed based on LPAKR9 and glucose dehydrogenase（GDH）gene from Bacillus subtilis ATCC 13952 to regenerate NADPH.The optimal conditions to carry out whole-cell catalysis for synthesizing（R）-CHBE using COBE were obtained by optimizing the reaction parameters.these included:90 m M glucose,20 m M disodium hydrogen phosphate-citric acid buffer at p H 6.0,a concentration of 20 g·L^-1whole-cell catalyst,30 m M COBE,and a reaction temperature of 30℃with continuous shaking at 200 rpm for 4 hours.Under these optimized conditions,the yield is up to25.2 mM.