3 years ago

Purification and Characterization of an Enone Reductase from Sporidiobolus salmonicolor TPU 2001 Reacting with Large Monocyclic Enones

Purification and Characterization of an Enone Reductase from Sporidiobolus salmonicolor TPU 2001 Reacting with Large Monocyclic Enones
Yasuhisa Asano, Yuko Oku, Syuji Ebata, Kazunori Yamamoto, Masaharu Doya, Atsushi Izumi, Atsutoshi Ina
We discovered a novel enone reductase from Sporidiobolus salmonicolor TPU 2001 (SsERD) and expressed the gene in Escherichia coli. The enzyme catalyzed the reduction of (E)-3-methylcyclopentadec-2-en-1-one (E-2), cyclopentadec-2-en-1-one (3), and cyclododec-2-en-1-one (4) to (S)-muscone (S-1), cyclopentadecan-1-one (5), and cyclododecan-1-one (6), respectively. The apparent Km and Vmax values for E-2 were estimated to be 4.9±0.4 μm and 100±1.4 nmol min−1 mg−1, respectively. The enzyme was specific to NADPH, and cysteine residues strongly affected the enzyme activity. The enzyme exhibited the highest activity at pH 8.0 and high stability in the pH range from 4.5 to 11.0. Using 10 mU of the enzyme, S-1 was synthesized from 0.1 mm E-2 with 94.8 % yield and 100 % enantiomeric excess by incubation at pH 7.0 and 30 °C for 60 min. We further successfully constructed an enone reductase with high specific activity by mutation of SsERD. The Y67A variant from SsERD exhibited 4.5 times higher specific activity and 3 times higher catalytic efficiency toward E-2. This is the first report of an enzyme catalyzing the reduction of carbon–carbon double bond of large monocyclic enones. No scent here: This is the first report of an enone reductase reducing carbon–carbon double bond of large monocyclic enones. The enone reductase is available for asymmetric synthesis of (S)-muscone from (E)-3-methylcyclopentadec-2-en-1-one, which has so far only been reported by chemical reduction using Ru-BINAP catalyst.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cctc.201700244

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