title: Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature.
abstract: The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta™(DE3)pLysS by induction with 1 mM of isopropyl-β-D-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0-8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH 6.5-7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.
authors.affiliation: Graduate School of Bioscience and Biotechnology, Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 2268501, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Nakata
authors.forename: Chen
authors.initials: C
authors.lastname: Cao
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Sugiyama
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Asanuma
authors.forename: Shuji
authors.initials: S
authors.lastname: Kanamaru
authors.forename: Tomoko
authors.initials: T
authors.lastname: Matsuda
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D005848:Geotrichum / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009930:Organic Chemicals / Q000378:metabolism*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D009930:Organic Chemicals; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C021869:nitroacetophenone reductase