Purification and characterization of NADPH-dependent aldo-keto reductase specific for beta-keto esters from Penicillium citrinum, and production of methyl (S)-4-bromo-3-hydroxybutyrate.
A novel beta-keto ester reductase (KER) was purified to homogeneity from recombinant Escherichia coli (pTrcKER) cells, which efficiently expressed the ker gene cloned from Penicillium citrinum IFO4631. The enzyme was monomeric and had a molecular mass of 37 kDa. It catalyzed the reduction of some beta-keto esters, especially alkyl 4-halo-3-oxobutyrates. However, it did not catalyze the reverse reaction, the dehydrogenation of alkyl 4-halo-3-hydroxybutyrates and other alcohols. The enzyme required NADPH as a cofactor and showed no activity with NADH. Therefore, it was defined as a NADPH-dependent aldo-keto reductase (AKR3E1), belonging to the AKR superfamily. The enzyme stereospecifically produced methyl (S)-4-bromo-3-hydroxybutyrate from its keto derivative with high stereospecificity (97.9% enantiomer excess). E. coli cells expressing KER and glucose dehydrogenase in the water/butyl acetate two-phase system achieved a high productivity of (S)-4-bromo-3-hydroxybutyrate (277 mM, 54 mg/ml) in the organic solvent layer.
Itoh
N
N
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa Kosugi, 939-0398 Toyama, Japan. itoh@pu-toyama.ac.jp
Asako
H
H
Banno
K
K
Makino
Y
Y
Shinohara
M
M
Dairi
T
T
Wakita
R
R
Shimizu
M
M
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D001407:Bacillus / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000378:metabolism*; D042843:Glucose 1-Dehydrogenase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006885:Hydroxybutyrates / Q000378:metabolism*; D007659:Ketones / Q000378:metabolism; D030781:Organisms, Genetically Modified; D010407:Penicillium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D004952:Esters; D006885:Hydroxybutyrates; D007659:Ketones; D011994:Recombinant Proteins; C496653:methyl-4-bromo-3-hydroxybutyrate; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D042843:Glucose 1-Dehydrogenase