Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomycesârouxii
2000
Costello, Colleen A. | Payson, Robert A. | Menke, Michael A. | Larson, Jeffrey L. | Brown, Keith A. | Tanner, Joseph E. | Kaiser, Raymond E. | Hershberger, Charles L. | Zmijewski, Milton J.
A novel ketoreductase isolated from Zygosaccharomycesârouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8âkDa ketoreductase was purified more than 300âfold to >â95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4âmethylenedioxyphenyl acetone of 2.9âmm and a Km for NADPH of 23.5âµm. The enzyme is able to effectively reduce αâketolactones, αâketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0âkb ketoreductase gene was cloned and sequenced from a Z.ârouxii cDNA library using a degenerate primer to the Nâterminal sequence of the purified protein. Furthermore, it was expressed in both Escherichiaâcoli and Pichiaâpastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.
Mostrar más [+] Menos [-]Palabras clave de AGROVOC
Información bibliográfica
Este registro bibliográfico ha sido proporcionado por National Agricultural Library