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Purification and Characterization of an Enone Reductase from Sporidiobolus salmonicolor TPU 2001 Reacting with Large Monocyclic Enones
Author(s) -
Yamamoto Kazunori,
Oku Yuko,
Ina Atsutoshi,
Izumi Atsushi,
Doya Masaharu,
Ebata Syuji,
Asano Yasuhisa
Publication year - 2017
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201700244
Subject(s) - chemistry , enzyme , reductase , enantiomeric excess , enone , stereochemistry , enzyme assay , biocatalysis , double bond , yield (engineering) , catalysis , organic chemistry , reaction mechanism , enantioselective synthesis , materials science , metallurgy
We discovered a novel enone reductase from Sporidiobolus salmonicolor TPU 2001 ( Ss ERD) 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 K m and V max 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 Ss ERD. 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.