The Study On Purification And Characterization Of (R)-1-phenylethanol Dehydrogenase And Its Immobilization

(R)-1-phenylethanol dehydrogenase((R)-PEDH)with nicotinamide adenine dinucleotide(NAD+)as cofactor specifically catalyzes the formation of acetophenone by(R)-1-phenylethanol,to resolve racemic 1-phenylethanol and chiral(S)-1-phenylethanol was sobtained.The purpose of this study was to obtain purified(R)-PEDH and to study the enzymatic properties of(R)-PEDH.The regeneration of(R)-PEDH cofactor NAD+ was achieved by enzymatic catalysis and photocatalysis coupling.Employing metal-organic framework ZIF-8 for(R)-PEDH immobilization can effectively improve the enzyme activity stability and reusability.A strain of producing(R)-PEDH bacteria was screened from the soil.The strain was identified as Lysinibacillus sp.by 16S rDNA sequence analysis.The bacterial cells which were harvested from liquid culture is treated by sonication,ammonium sulfate precipitation and preparative PAGE,finally obtained(R)-PEDH which is purified 60 folds with a specific activity of 4.44 U/mg.SDS-PAGE showed that the purified(R)-PEDH was a single band with a molecular weight of 28 kDa.The enzymatic properties of(R)-PEDH were studied experimentally,the optimum pH and temperature of(R)-PEDH were 9.5 and 70?,respectively,and the optimum pH and temperature for the catalytic reduction reaction were 6.5 and 65?,respectively.Under optimal conditions,the Km of(R)-PEDH for(R)-1-phenylethanol was 0.78 mM,the kcat was 123 s-1,the Km of(R)-PEDH for acetophenone was 0.56 mM,and the kcat was 125 s-1.The regeneration of NAD+ was carried out by coupled the reaction of(R)-PEDH catalysis and photocatalysis.VB2 was used as the photosensitizer and LED was used as the light source.The coupling reaction was carried out at pH 8.0 and 37?,after 48 h,2.7 mM(R)-1-phenylethanol was completely converted to acetophenone and the reaction system had only 0.17 mM NAD+.The entire reaction system does not require the additional substrate as an electron acceptor,producing only water without other byproducts.(R)-PEDH was immobilized by ZIF-8.The average particle diameter of ZIF-8@(R)-PEDH was about 500 nm,and the size of particles was uniform.The experiment explored that 20%glycerol has no effect on the formation of ZIF-8,while NaCl affects the morphology and structure of ZIF-8,and then affects the rate of ZIF-8@(R)-PEDH encapsulation.The immobilization enzymatic activity of ZIF-8@(R)-PEDH was 44%,the theoretical protein yield was 76%,the Km value of ZIF-8@(R)-PEDH against(R)-1-phenylethanol was 0.046 mM.The appropriate reaction pH and temperature for ZIF-8@(R)-PEDH catalyze oxidation reaction were 9.5 and 65?.ZIF-8@(R)-PEDH enzymatic properties compared with the free enzyme,the affinity with the substrate stronger,resistant to trypsin decomposition and can be recycled.