Cloning,Expression And Characterization Of Aldo-keto Reductases From Lactobacillus Reuteri

The aldo-keto reductases superfamily belongs to the class of oxidoreductases,which usually use NADP（H）as a coenzyme to catalyze the carbonyl groups to the alcohols.In vitro,aldo-keto reductases can catalyze oxidation reactions.Since aldo-keto reductases are widely present in various organisms and usually have broad substrate specificity,they perform complex physiological functions in living organisms,including detoxification,regulation of osmolality,and synthesis of secondary metabolites;in addition,they are often used in the catalytic synthesis of some chiral compounds.We comprehensively excavated the aldo-keto reductases in Lactobacillus reuteri DSM20016 and successfully cloned and expressed them in E.coli（DE3）.The molecular weights of these enzymes between 33.9 k Da to 38.1 k Da,and they all use NADPH as coenzyme.Their substrate specificity was studied using some flavor aldehydes and ketones,and these enzymes had a broad substrate spectrum and could convert a variety of flavor aldehydes to flavor alcohols,which let Lactobacillus reuteri had the ability to produce various volatile compounds,and could to impart complex aroma to some food;however,they did not catalyze the oxidation reactions.In addition,the AKR-3,AKR-4,and AKR-6 also had the ability to reduce 3-hydroxypropionaldehyde.In addition,we also investigated the enzymatic properties of aldo-keto reductases.The activity of AKRs was independent of metal ions.Fe²⁺,Zn²⁺,and Cu²⁺had a strong inhibitory effect on these AKRs,and the other metal ions showed a slight effect on the enzyme activity.The presence of EDTA had few effects on the enzyme activity,indicating that none of these AKRs were metal-dependent enzymes.The optimum reaction pH of these enzymes was acidic,and AKR-1 was the most stable at pH 5,and the residual enzyme activity was more than 50%when incubated at pH 4 for 1 h,AKR-2,AKR-3,and AKR-4 were most stable under neutral conditions,and AKR-6 and AKR-8 were most stable at pH 6.In addition,they have good thermal stability,except for AKR-2 and AKR-3,the other enzymes were completely inactivated at 70℃for 1 h incubation,and AKR-1 still had more than 60%activity after incubation at 60℃for one hour.However,the optimum reaction temperature of these aldo-keto reductases varied greatly,AKR-3 had an optimum reaction temperature of 25℃,AKR-1 and AKR-2 optimal reaction temperature was 45℃.The most affinity substrate of AKR-1was COBE.The V_m of AKR-2 for succinaldehyde reached 83.3μmol·L^-1·min^-1;The most affinity substrate of AKR-3 was succinaldehyde,and the V_m of AKR-3 for 3-（methylthio）propanal reached 429.5μmol·L^-1·min^-1.The most affinity substrate of AKR-4 was succinaldehyde（K_m=2.0 m M）,and the V_m was 44.5μmol·L^-1·min^-1.AKR-6 was the most affinity for hexanal with a K_m of 0.4 m M and the conversion rate k_cat/K_mwas 63.4 s^-1·m M^-1.AKR-8 had a V_m of 145.8μmol·L^-1·min^-1 for COBE.