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.

Figure optionsDownload as PowerPoint slide