화학공학소재연구정보센터
Enzyme and Microbial Technology, Vol.125, 29-36, 2019
Structure-guided engineering of ChKRED20 from Chryseobacterium sp. CA49 for asymmetric reduction of aryl ketoesters
ChKRED20 is a robust NADH-dependent ketoreductase identified from the genome of Chryseobacterium sp. CA49 that can use 2-propanol as the ultimate reducing agent. The wild-type can reduce over 100 g/l ketones for some pharmaceutical relevant substrates, exhibiting a remarkable potential for industrial application. In this work, to overcome the limitation of ChKRED20 to aryl ketoesters, we first refined the X-ray crystal structure of ChKRED20/NAD(+) complex at a resolution of 1.6 angstrom, and then performed three rounds of iterative saturation mutagenesis at critical amino acid sites to reshape the active cavity of the enzyme. For methyl 2-oxo-2-phenylacetate and ethyl 3-oxo-3-phenylpropanoate, several gain-of-activity mutants were achieved, and for ethyl 2-oxo-4-phenylbutanoate, improved mutants were achieved with k(cat)/K-m increasing to 196-fold of the wild-type. All three substrates were completely reduced at 100 g/l loading catalyzed with selected ChKRED20 mutants, and deliver the corresponding chiral alcohols with > 90% isolated yield and 97 - > 99%ee.