Thermostable alcohol dehydrogenase from Thermococcus kodakarensis KOD1 for enantioselective bioconversion of aromatic secondary alcohols.
A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). 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. TkADH 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). TkADH 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 H2O-20% 2-propanol and H2O-50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.
Department of Chemical Engineering, Tsinghua University, Beijing, China.
Xi
X
Wu
Chong
C
Zhang
Izumi
I
Orita
Tadayuki
T
Imanaka
Toshiaki
T
Fukui
Xin-Hui
XH
Xing
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000438:Alcohols / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D000438:Alcohols; D003067:Coenzymes; D009249:NADP; D000426:Alcohol Dehydrogenase