Cloning, expression, purification, and characterization of a novel epoxide hydrolase from Aspergillus niger SQ-6.
A novel epoxide hydrolase from Aspergillus niger SQ-6 has now been cloned by inverse PCR. Its gene shows eight exons including a non-coding exon at its 5'-terminal (GenBank Accession No. AY966486). Phylogenetic analysis using deduced amino acid sequence (395 aa) confirms it as an epoxide hydrolase and shares 58.3% identity with that of A. niger LCP521 (GenBank Accession No. AF238460). The predicted catalytic triad is composed of Asp(191), His(369) and Glu(343). Active recombinant epoxide hydrolase has been successfully expressed in Escherichia coli as protein fusions with a poly-His tail. Scale-up fermentation can yield 2.5g/L of recombinant protein. The electrophoretic pure recombinant protein, which shows similar characterization as natural enzyme purified from A. niger SQ-6, can be easily purified by Ni(2+)-chelated affinity and gel-filtration chromatography. Optimal pH and temperature for purified enzyme are pH 7.5 and 37 degrees C, respectively. The K(m), k(cat) and maximal velocity (V(max)) for p-nitrostyrene oxide are determined to be 1.02mM, 172s(-1) and 231micromol min(-1)mg(-1), respectively. The enzyme can be inhibited by oxidant (H(2)O(2)), solvent (Tetrahydrofuran) and several metal ions including Hg(2+), Fe(2+) and Co(2+). This (R)-stereospecific epoxide hydrolase exhibits high enantioselectivity (enantiomeric excess value, 99%) for the less hindered carbon atom of epoxide. It may be an industrial biocatalyst for the preparation of enantiopure epoxides or vicinal diols.
Liu
Yanbin
Y
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China. yanbin@tll.org.sg
Wu
Sheng
S
Wang
Jianjun
J
Yang
Liu
L
Sun
Wanru
W
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D015870:Gene Expression; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012997:Solvents; D013379:Substrate Specificity; D013696:Temperature
D004271:DNA, Fungal; D008670:Metals; D011994:Recombinant Proteins; D012997:Solvents; D004851:Epoxide Hydrolases