Abstract
A highly enantioselective, soluble epoxide hydrolase from Nocardia sp. EH1 was purified to homogeneity via a four-step procedure: (i) hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, (ii) anion exchange chromatography on SOURCE 30Q, followed by (iii) a second hydrophobic interaction chromatography on Phenyl Sepharose HP, and finally (iv) gel-filtration on Superdex 75 HR 10/30. The pure protein was shown to be a monomer of ∼ 34 kDa possessing an optimum pH of 8–9. Neither UV-absorbing cofactors nor metal ions were required for activity. In contrast to whole-cell activity, the partially purified enzyme proved to be considerably less stable. Stabilization was achieved by addition of non-ionic detergents such as Tween 80 or Triton X-100, causing a shift of the temperature optimum from 35 to 40°C. Both effects combined led to an enhancement of the relative activity of up to ∼ 150% of that of the native enzyme.
Published Version
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