Expression of a novel epoxide hydrolase of Aspergillus usamii E001 in Escherichia coli and its performance in resolution of racemic styrene oxide.
The full-length cDNA sequence of Aueh2, a gene encoding an epoxide hydrolase of Aspergillus usamii E001 (abbreviated to AuEH2), was amplified from the total RNA. Synchronously, the complete DNA sequence containing 5', 3' flanking regions, eight exons and seven introns was cloned from the genomic DNA. In addition, a cDNA fragment of Aueh2 encoding a 395-aa AuEH2 was expressed in Escherichia coli. The catalytic activity of recombinant AuEH2 (re-AuEH2) was 1.44 U/ml using racemic styrene oxide (SO) as the substrate. The purified re-AuEH2 displayed the maximum activity at pH 7.0 and 35 °C. It was highly stable at a pH range of 5.0-7.5, and at 40 °C or below. Its activity was not obviously influenced by β-mercaptoethanol, EDTA and most of metal ions tested, but was inhibited by Hg(2+), Sn(2+), Cu(2+), Fe(3+) and Zn(2+). The K m and V max of re-AuEH2 were 5.90 mM and 20.1 U/mg towards (R)-SO, while 7.66 mM and 3.19 U/mg towards (S)-SO. Its enantiomeric ratio (E) for resolution of racemic SO was 24.2 at 10 °C. The experimental result of re-AuEH2 biasing towards (R)-SO was consistent with the analytical one by molecular docking (MD) simulation.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People's Republic of China.
Die
D
Hu
Cun-Duo
CD
Tang
Biao
B
Yang
Jia-Chi
JC
Liu
Tao
T
Yu
Chao
C
Deng
Min-Chen
MC
Wu
D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D062105:Molecular Docking Simulation; D013237:Stereoisomerism; D013696:Temperature
D004852:Epoxy Compounds; C013690:styrene oxide; D004851:Epoxide Hydrolases