Enantioselective Hydrolysis of Racemic and <i>Meso</i>-Epoxides with Recombinant <i>Escherichia coli</i> Expressing Epoxide Hydrolase from <i>Sphingomonas</i> sp. HXN-200: Preparation of Epoxides and Vicinal Diols in High <i>ee</i> and High Concentration

A unique epoxide hydrolase (SpEH) from <i>Sphingomonas</i> sp. HXN-200 was identified and cloned based on genome sequencing and expressed in <i>Escherichia coli</i>. The engineered <i>E. coli</i> (SpEH) showed the same selectivity and substrate specificity as the wild type strain and 172 times higher activity than <i>Sphingomonas</i> sp. HXN-200 for the hydrolysis of styrene oxide <b>1</b>. Hydrolysis of racemic styrene oxide <b>1</b>, substituted styrene oxides <b>3</b>, <b>5</b>–<b>7</b>, and <i>N</i>-phenoxycarbonyl-3,4-epoxypiperidine <b>8</b> (200–100 mM) with resting cells of <i>E. coli</i> (SpEH) gave (<i>S</i>)-epoxides <b>1</b>, <b>3</b>, <b>5</b>–<b>7</b> and (−)-<b>8</b> in 98.0–99.5% enantiomeric excess (<i>ee</i>) and 37.6–46.5% yield. Hydrolysis of cyclopentene oxide <b>9</b>, cyclohexene oxide <b>10</b>, and <i>N</i>-benzyloxycarbonyl-3,4-epoxypyrrolidine <b>11</b> (100 mM) afforded the corresponding (<i>R</i>, <i>R</i>)-vicinal <i>trans</i>-diols <b>12</b>–<b>14</b> in 86–93% <i>ee</i> and 90–99% yield. The <i>ee</i> of (1<i>R</i>, 2<i>R</i>)-cyclohexane-1,2-diol <b>13</b> was improved to 99% by simple crystallization. These biotransformations showed high specific activity (0.28–4.3 U/mg cdw), product concentration, product/cells ratio, and cell-based productivity. Hydrolysis at even higher substrate concentration was also achieved: (<i>S</i>)-<b>1</b> was obtained in 430 mM (51 g/L_org) and 43% yield; (1<i>R</i>, 2<i>R</i>)-<b>13</b> was obtained in 500 mM (58 g/L) and >99% yield. Gram-scale preparation of epoxides (<i>S</i>)-<b>1</b>, (<i>S</i>)-<b>3</b>, (<i>S</i>)-<b>6</b> and diols (1<i>R</i>, 2<i>R</i>)-<b>12</b>, (1<i>R</i>, 2<i>R</i>)-<b>13</b>, (3<i>R</i>, 4<i>R</i>)-<b>14</b> were also demonstrated. <i>E. coli</i> (SpEH) cells showed the highest enantioselectivity to produce (<i>S</i>)-<b>1</b> (<i>E</i> of 39) among all known EHs in the form of whole cells or free enzymes and the highest enantioselectivities to produce (<i>S</i>)-<b>3</b>, <b>5</b>, <b>6</b>, <b>7</b>, (−)-<b>8</b>, and (<i>R</i>, <i>R</i>)-<b>14</b> (<i>E</i> of 36, 35, 28, 57, 22, and 28) among all known EHs. The easily available and highly active <i>E. coli</i> (SpEH) cells are the best biocatalysts known thus far for the practical preparation of these useful and valuable enantiopure epoxides and vicinal diols via hydrolysis