Soluble epoxide hydrolase (EH) from the potato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were purified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2-ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselectivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethanediol (2.0 +/- 0.2 micromol/min/mg), and the potato EH converts (S)-styrene oxide primarily to the same enantiomer, (R)-1-phenyl-1,2-ethanediol (22 +/- 1 micromol/min/mg), with an enantiomeric ratio of 40 +/- 17 (based on substrates). By mixing these two purified enzymes, inexpensive racemic styrene oxide (5 mM) was converted at 100% yield to 98% enantiomeric excess (R)-1-phenyl-1,2-ethanediol at 4.7 +/- 0.7 micromol/min/mg. Hence, at least 99% of substrate is converted into a single stereospecific product at a rapid rate.

Saturation mutagenesis was used to generate 10 mutants of toluene-o-xylene monooxygenase (ToMO) at alpha subunit (TouA) positions A101 and A110: A101G, A101I, A101M, A101VE, A101V, A110G, A110C, A110S, A110P, and A110T; by testing the substrates toluene, o-cresol, m-cresol, p-cresol, phenol, naphthalene, o-methoxyphenol, m-methoxyphenol, p-methoxyphenol, o-xylene, and nitrobenzene, these positions were found to influence the regiospecific oxidation of aromatics. For example, compared to wild-type ToMO, TouA variant A101V produced threefold more 3-methoxycatechol from m-methoxyphenol as well as produced methylhydroquinone from o-cresol whereas wild-type ToMO did not. Similarly, variant A110C synthesized 1.8-fold more o-cresol from toluene and 1.8-fold more 3-methoxycatechol from m-methoxyphenol, and variant A110G synthesized more m-nitrophenol and twofold less p-nitrophenol from nitrobenzene. The A101V and A110C mutations did not affect the rate of reaction with the natural substrate toluene, so the variants had high activity. This is the first report that these or analogous residues influence the catalysis with this class of enzymes. Wild-type ToMO was found to oxidize o-methoxyphenol to methoxyhydroquinone (60%) and 4-methoxyresorcinol (40%), m-methoxyphenol to 4-methoxycatechol (96%) and 3-methoxycatechol (4%), and p-methoxyphenol to 4-methoxycatechol (100%).