A novel epoxide hydrolase (BMEH) with unusual (R)-enantioselectivity and very high activity was cloned from Bacillus megaterium ECU1001. Highest enantioselectivities (E > 200) were achieved in the bioresolution of ortho-substituted phenyl glycidyl ethers and para-nitrostyrene oxide. Worthy of note is that the substrate structure remarkably affected the enantioselectivities of the enzyme, as a reversed (S)-enantiopreference was unexpectedly observed for the ortho-nitrophenyl glycidyl ether. As a proof-of-concept, five enantiopure epoxides (> 99% ee) were obtained in high yields, and a gram-scale preparation of (S)-ortho-methylphenyl glycidyl ether was then successfully performed within a few hours, indicating that BMEH is an attractive biocatalyst for the efficient preparation of optically active epoxides.
Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. On the basis of computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1-F92C/F94W variant was obtained that exhibited higher activity toward all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg −1 (54−775 U mg −1 ). Using CgKR1-F92C/F94W as a catalyst, five substrates at high loading (>100 g −1 L −1 ) were reduced completely in gramscale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g −1 L −1 day −1 ). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity improvement. Our findings provide useful guidance for engineering other ketoreductases, especially those possessing a similar active pocket to that in CgKR1.
A new and efficient sulfide monooxygenase-producing strain, ECU0066, was isolated and identified as a Rhodococcus sp. that could transform phenylmethyl sulfide (PMS) to (S)-sulfoxide with 99% enantiomeric excess via two steps of enantioselective oxidations. Its enzyme activity could be effectively induced by adding PMS or phenylmethyl sulfoxide (PMSO) directly to a rich medium at the early log phase (6 h) of fermentation, resulting in over 10-times-higher production of the enzyme. This bacterial strain also displayed fairly good activity and enantioselectivity toward seven other sulfides, indicating a good potential for practical application in asymmetric synthesis of chiral sulfoxides.Because of the high configurational stability of the sulfinyl group as well as their synthetic versatility, chiral sulfoxides are powerful stereodirecting groups (18), valuable asymmetric starting materials (19), and chiral auxiliaries (7). The value of chiral sulfoxide functionality is further illustrated by their diverse biological activities and pharmaceutical uses (17). In many cases, only one enantiomer of a sulfur-containing drug exhibits the desired biological activity (10); therefore, it is necessary and important to prepare enantiopure sulfoxides.The asymmetric oxidation of a prochiral sulfide is undoubtedly a more direct and economical method for the synthesis of enantiomerically pure sulfoxides than the kinetic resolution of racemic sulfoxides. Asymmetric sulfoxidations mediated by either metal catalysts or isolated enzymes have received considerable attention over the past several years (1,9,14,20,24). However, the transformations with metal catalysts or isolated enzymes (such as peroxidases, haloperoxidases, and monooxygenases) are tedious and expensive, which are major disadvantages for preparative applications. And only very few of the enzymes used for sulfoxidation, such as the cyclohexanone monooxygenase from Acinetobacter NCBI 9871, have been isolated and characterized (8). By contrast, asymmetric sulfoxidations catalyzed by whole-cell systems (e.g., fungi and bacteria) are much cheaper and more convenient, avoiding the involvement of expensive cofactors (NADH/NADPH). Although a few microorganisms have so far been successfully used for such a biocatalytic sulfoxidation, the most frequently used cultures for this purpose were fungi (12,21,22). For the bacteria, there have been only a few brief reports regarding the oxidation of sulfides with whole cells (2, 15), but the results were unsatisfactory because of low enantioselectivity or poor substrate tolerance. Therefore, we decided to screen for new bacterial strains with higher enzyme activity and better enantioselectivity for asymmetric oxidation of sulfides.In this article, we report the catalytic performance of the newly isolated bacterium Rhodococcus sp. strain ECU0066 for asymmetric oxidation of sulfides. This bacterium displayed pretty high activity and satisfactory enantioselectivity for most of the sulfides examined, indicating that this bacterium is very ...
The upgrading of biomass-derived furfural into high-value bio-based chemicals has attracted interest.
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