A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization

Protein Expr Purif. 2013 Oct;91(2):175-83. doi: 10.1016/j.pep.2013.08.001. Epub 2013 Aug 21.

Abstract

A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/β hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495μM and 0.24μM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspergillus / enzymology*
  • Aspergillus / genetics
  • Chromatography, Affinity
  • Circular Dichroism
  • Epoxide Hydrolases / chemistry*
  • Epoxide Hydrolases / genetics
  • Epoxide Hydrolases / isolation & purification
  • Epoxide Hydrolases / metabolism
  • Epoxy Compounds / chemistry
  • Escherichia coli / genetics
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism
  • Histidine / genetics
  • Hydrolysis
  • Molecular Sequence Data
  • Oligopeptides / genetics
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • Stereoisomerism

Substances

  • Epoxy Compounds
  • Fungal Proteins
  • His-His-His-His-His-His
  • Oligopeptides
  • Recombinant Fusion Proteins
  • Histidine
  • styrene oxide
  • Epoxide Hydrolases