Characterization And Molecular Modification Of Secondary Alcohol Dehydrogenase

Secondary alcohol dehydrogenase（SADH）,with nicotinamide adenine dinucleotide（phosphate）（NAD（P）⁺）as cofactor,is capable of reversibly catalyzing the conversion between the secondary alcohol and the corresponding ketone.Secondary alcohol dehydrogenase with the advantages of high regioselectivity,chemical selectivity,enantioselectivity and wide substrate spectrum,has an important application value in the fields of fine chemical,pharmaceutical and food.Hydroxyacetone is an important intermediate in the synthesis of organic products such as spices,drugs and dyes.However,there has been few reports on the production of hydroxyacetone from 1,2-propanediol through the catalyzed oxidation by secondary alcohol dehydrogenase.In this paper,Hansenula polymorpha secondary alcohol dehydrogenase was screened and expressed,its enzymatic properties and molecular dynamics were analyzed.Moreover,molecular modification was carried through semi-rational and rational design to improve its catalytic activity of oxidizing 1,2-propanediol to hydroxyacetone.The main research results are as follows:（1）The screened secondary alcohol dehydrogenase DH-2 from Hansenula polymorpha was successfully cloned and expressed in E.coli.The activity of recombinant DH-2 on 1,2-propanediol was 1.28 U/mg,the optimum p H was 9.0,and the optimum temperature was30℃.DH-2 was non-metal ion-dependent.The single chain of DH-2 was composed of 382amino acid residues with the theoretical molecular weight of 40.82 k Da.Its molecular formula was C₁₈₀₉H₂₈₃₇N₄₇₇O₅₅₅S₂₁,the total number of atoms and the theoretical isoelectric point are 5699 and 5.34,respectively.DH-2 was a hydrophilic protein without transmembrane region and signal peptide,The secondary and tertiary structures of DH-2were predicted by computer,which provided the structural basis for molecular modification.（2）SWISS-MODEL and Alpha Fold were used to model structures of DH-2.The quality assessment of the structural model of DH-2 showed that the monomer structure obtained by Alpha Fold had a higher quality and a higher confidence than the homodimer obtained by SWISS-MODEL.The DH-2 monomer from Alpha Fold was docked with coenzyme NAD⁺and the substrate 1,2-propanediol,and then designed amino acid mutation sites to perform molecular modification,which were based on a sensitive PMA high-throughput fluorescence screening method..（3）In the semi-rational design,24 mutation sites near 4（?）of the substrate were selected to construct a mutant library,and an effective mutant Y319Q was screened with about 2 fold enzymatic activity of the wild-type enzyme.In the rational design,the amino acids in the range of 6（?）at the coenzyme and substrate binding sites were selected,and the selected amino acids with a predicted low binding free energy in virtual mutation were mutated,and the dominant single-site mutant S222T and double-site mutant S222T/S316A were obtained,with about 8 fold and 14 fold enzymatic activity of wild-type enzyme respectively.（4）The kinetics analysis of the mutant showed that the catalytic efficiency k_cat/K_m of double mutant S222T/S316A for 1,2-propanediol was significantly higher than that of wild type,and the binding free energy of double mutant with substrate and coenzyme was lower than that of wild type,which indicated that the affinity of mutant for 1,2-propanediol and coenzyme NAD⁺was enhanced,thus improving the efficiency of enzymatic synthesis of hydroxyacetone.The screening,recombinant expression,molecular modification of the secondary alcohol dehydrogenase and its functional characterization of hydroxyacetone by oxidation of1,2-propanediol can provide guidance to the biotransformation application of alcohol dehydrogenase and the green biomanufacture of hydroxyacetone.