Cloning and characterization of a NADH-dependent aldo-keto reductase from a newly isolated Kluyveromyces lactis XP1461
•Kluyveromyces lactis XP1461 was screened, harbouring the aldo-keto reductase activity.•KlAKR was cloned and expressed in E. coli and the enzyme was purified.•The KlAKR has broad substrate specificity to carbonyl compounds.•The KlAKR provided optically pure chiral alcohol for the majority of test substrates.
An aldo-keto reductase gene (klakr) from Kluyveromyces lactis XP1461 was cloned and heterologously expressed in Escherichia coli. The aldo-keto reductase KlAKR was purified and found to be NADH-dependent with a molecular weight of approximately 36 kDa. It is active and stable at 30 °C and pH 7.0. The maximal reaction rate (vmax), apparent Michaelis–Menten constant (Km) for NADH and t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) and catalytic number (kcat) were calculated as 7.63 U mg−1, 0.204 mM, 4.42 mM and 697.4 min−1, respectively. Moreover, the KlAKR has broad substrate specificity to a range of aldehydes, ketones and keto-esters, producing chiral alcohol with e.e. or d.e. >99% for the majority of test substrates.
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Journal: Enzyme and Microbial Technology - Volume 77, September 2015, Pages 68–77