Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

Abstract

ABSTRACT A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 ( Tk ADH). The gene, tk0845 , which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli . The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90�C and a half-life of 4.5 h at 95�C. The apparent K m values for the cofactors NAD(P) + and NADPH were similar within a range of 66 to 127 μM. Tk ADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding ( R )-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). Tk ADH could also reduce 2,2,2-trifluoroacetophenone to ( R )-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H 2 O–20% 2-propanol and H 2 O–50% n -hexane or n -octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.