Screening Of Cyclic Amino Alcohol Specific Deaminase And Its Application In Synthesis Of Chiral Cyclic β-1,2-Amino Alcohol

Chiral cyclicβ-1,2-amino alcohol is a key component in drug synthesis,and research on chiral drugs is of great significance.Traditional chemical synthesis of chiral cyclicβ-1,2-amino alcohols has high catalyst toxicity,low selectivity,harsh reaction conditions,and requires complex protection and deprotection steps for the groups.Biocatalysis has the advantages of high catalytic efficiency,high selectivity,mild reaction conditions,green and sustainable,et,which has attracted more and more attention.In this subject,a new high-throughput solid-phase screening method was constructed for the screening of cyclic amino alcohol deaminase.And successfully screened a high activity and high enantioselective cyclic amino alcohol deaminase producing strain Arthrobacter sp.TYUT010-15;the strain was identified and characterized;the substrate specificity of the strain was studied,and various amino alcohol substrates were subjected to substrate kinetic resolution to synthesize enantiomerically pure cyclic amino alcohol.Finally,the related cyclic amino alcohol deaminase in this strain was cloned and expressed,and a new cyclohexylamine oxidase was successfully cloned.The enzymatic properties of this enzyme were characterized and applied to the kinetic resolution of amino alcohol.This subject provides a novel and rapid method for screening cyclic amino alcohol deaminase-producing bacteria,and has potential application value for the biological synthesis of chiral cyclic amino alcohols.First,using the principle of the reaction of the product2-hydroxycyclohexanone with colorless 2,3,5-triphenyl-2H-tetrazole chloride（TTC）to produce red TPF（color reaction for short）,a high-throughput solid-phase screening method was constructed for screening of bacteria with cyclic amino alcohol deamination activity,and successfully screened a highly viable and highly selective strain Arthrobacter sp.TYUT010-15.The growth characteristics of this strain were studied.After culturing for 12 h in a basic salt containing trans-2-aminocyclohexanol（MBSM）medium,the OD₆₀₀reached 1.0,and the deamination activity reached 126 U/g cdw.The optimum reaction conditions for deamination of this strain were optimized,and results showed that the optimum reaction p H was 8.0 and the optimum reaction temperature was 30^oC.The deamination activity of this strain was relatively stable in low temperature and alkaline conditions.Kinetic resolution of racemic trans-2-aminocyclohexanol and trans-2-aminocyclopentanol with this strain,After 3 hours of reaction,the conversion rate was 50%and the ee value reached99%.At the same time,the other 7 kinds of amino alcohol compounds and 2kinds of amine compounds were subjected to kinetic resolution.The results showed that the strain had no activity against chain amine alcohols,and had higher activity against aromatic amine alcohols and amine substrates.（1R,2S）-cis-1-amino-2-indananol,β-amino-4-fluoro-benzeneethanol,β-amino-4-chloro-benzeneethanol,β-amino-4-bromo-benzeneethanol,phenylglycinol,phenethylamine and 1,2,3,4-tetrahydro-1-naphthyl amine was used for kinetic resolution.After a certain period of time,the substrate conversion rate could reach 49.6-49.9%,and the ee value reached 99%.Racemic trans-2-aminocyclohexanol（200 m M）was subjected to kinetic resolution in a 50m L reaction system.After 42 h of reaction,the conversion reached 49.6%and the ee value was greater than 99%.（1S,2S）-trans-2-amino cyclohexanol was successfully prepared,and the product separation yield was 40%（464.0 mg）.Secondly,by analyzing the whole genome sequencing results of strain Arthrobacter sp.TYUT010-15,a new cyclohexylamine oxidase with deamination activity on cyclic amino alcohol was obtained through screening.The enzyme was cloned and recombinantly expressed in E.coli.The target protein was purified by nickel column affinity chromatography,and target protein band was observed by SDS-PAGE electrophoresis.Finally,the enzymatic properties of the cloned cyclohexylamine oxidase were characterized.And the results showed that the enzyme activity was higher under neutral conditions,the optimal p H of the reaction was 7.0,and the optimal temperature of the reaction was 30 ^oC.The kinetic parameters of racemic cyclicβ-1,2-amino alcohol at different concentrations of 5-30 m M were measured with cyclohexylamine oxidase,the results showed that the K_Mvalues for cyclohexylamine oxidase toward（1R,2R）-trans-2-aminocyclopentanol and（1S,2S）-trans-2-aminocyclopentanol were 1.311 m M and 0.244 m M,and the V_maxvalues were 6.173 U/mg and 2.519 U/mg.The K_Mvalues for cyclohexylamine oxidase toward（1R,2R）-trans-2-aminocyclohexanol and（1S,2S）-trans-2-aminocyclohexanol were 1.382 m M and 0.61 m M,and the V_maxvalues were 6.373 U/mg and 2.68 U/mg.And the K_Mvalues for cyclohexylamine oxidase toward（1R,2S）-cis-1-amino-2-indanol and（1S,2R）-cis-1-amino-2-indanol were 0.375 m M and 0.627 m M,and the V_maxvalues were 4.958 U/mg and 2.263 U/mg.And the kinetic resolution of different amine alcohols and amine compounds was carried out.The results showed that the activity was lower for chain amine alcohols,but higher for cyclic amine alcohols and amine substrates.Among them,racemic trans-2-amino cyclopentanol,trans-2-aminocyclohexanol and cis-1-amino-2-indanol showed high activity,their activities were 6.53 U/mg,6.62 U/mg,5.27 U/mg,the conversion rate reaches 53.4-58.1%,and the ee value is greater than 99%.In the50 m L reaction system,400 m M racemic cyclicβ-1,2-amino alcohol was kineticly resolved,after 30 hours of reaction,the conversion rate reached 58.3%and the ee value was greater than 99%.By simple extraction and purification,the final product can obtain（1S,2S）-trans-2-aminocyclohexanol with a yield of26.34%（790.2 mg）and a purity greater than 99%.