Enzymatic asymmetric sulfoxidation using molecular oxygen as the oxidant is a promising green chemistry approach to chiral sulfoxide production. Despite the broad substrate spectrum of cyclohexanone monooxygenases (CHMOs), some unnatural substrates with bulky functional groups, such as the pharmaceutically relevant omeprazole sulfide, cannot be effectively accepted by CHMOs. Herein, we describe a set of variants derived from an Acinetobacter calcoaceticus CHMO (AcCHMO), whose active sites adjacent to the substrate tunnel were altered to shift the substrate specificity from cyclohexanone monooxygenation toward omeprazole sulfide sulfoxidation. We performed homologous modeling and molecular docking to identify key residues that might affect the substrate specificity. Two libraries of residues lining the active center of AcCHMO were then constructed and screened by an effective halo-based selection method using the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole). Functional evaluation of the resultant variants showed that the substrate specificity of AcCHMO was markedly altered from the small natural substrate (cyclohexanone) toward the desired bulky substrate (omeprazole sulfide) despite the extremely poor activity detected even for the best variant, M2 (0.61 U/ g prot ). The crystal structure of M2 complexed with a flavin adenine dinucleotide (FAD) prosthetic group was determined, which provided insight into the altered substrate specificity. To improve the activity of enzyme M2 toward pharmaceutical precursor omeprazole sulfide, we performed both local and global protein engineering among the two CASTing libraries surrounding FAD + and NADP + prosthetic groups and an error-prone PCR library of the full-length AcCHMO. As a result, variant M6 was obtained, giving a 50-fold higher activity compared to M2. This structure-guided protein engineering of AcCHMO provided a promising candidate for converting omeprazole sulfide into (S)-omeprazole using a green biocatalytic method.

Esomeprazole is the most popular proton pump inhibitor (PPI) for treating gastroesophageal reflux disease. Enzymatic asymmetric sulfoxidation is a green approach to produce chiral sulfoxides. In this report, we focused on optimizing asymmetric sulfoxidation catalyzed by prazole sulfide monooxygenase (AcPSMO). The costly redox cofactor NADPH utilized by AcPSMO was regenerated by formate dehydrogenase with CO 2 as the coproduct, which can be removed easily. During the scale-up process, oxygen supply was found to be the main limiting factor during the early phase of the reaction, while the instability of AcPSMO and the lack of the cofactor NADPH hindered progress during the middle and late phases of the 0.6 L reaction. Finally, by adjusting oxygen mass transfer and increasing the dissolved oxygen, the enzymatic reaction was stepwise amplified to a 120 L scale using a 300 L thermostatic stirred reactor, affording 95.9% conversion and 99.9% enantiomeric excess after 12 h. Extraction and refinement of the product resulted in 0.39 kg of the isolated esomeprazole (sodium salt), with 57.8% overall yield (73.4% before the salt-forming reaction) and 99.1% purity. Thus, a green-by-design system was constructed for the efficient and precise oxidation of omeprazole sulfide into esomeprazole with molecular O 2 as the green cosubstrate and CO 2 and H 2 O as byproducts.

The fatty acid photodecarboxylase from Chlorella variabilis NC64 A (CvFAP) catalyses the light-dependent decarboxylation of fatty acids. Photoinactivation of CvFAP still represents one of the major limitations of this interesting enzyme en route to practical application. In this study we demonstrate that the photostability of CvFAP can easily be improved by the administration of medium-chain length carboxylic acids such as caprylic acid indicating that the best way of maintaining CvFAP stability is 'to keep the enzyme busy'.

Light‐driven biocatalytic processes are notoriously hampered by poor penetration of light into the turbid reaction media. In this study, wirelessly powered light‐emitting diodes are found to represent an efficient and scalable approach for process intensification of the photobiosynthetic production of diesel alkanes from renewable fatty acids.

Two Baeyer-Villiger monooxygenases (BVMOs), designated BVMO andBVMO, were discovered from and, respectively. Both monooxygenases displayed novel features for catalyzing the asymmetric sulfoxidation of bulky and pharmaceutically relevant thioethers. Evolutionary relationship and sequence analysis revealed that the two BVMOs belong to the family of typical type I BVMOs and the subtype ethionamide monooxygenase. Both BVMOs are active toward medium- and long-chain aliphatic ketones as well as various thioether substrates but are ineffective toward cyclohexanone, aromatic ketones, and other typical BVMO substrates. BVMO andBVMO showed the highest activities (0.117 and 0.025 U/mg protein, respectively) toward thioanisole among the tested substrates. Furthermore, these BVMOs exhibited distinct activity and excellent stereoselectivity toward bulky and prochiral prazole thioethers, which is a unique feature of this family of BVMOs. No native enzyme has been reported for the asymmetric sulfoxidation of bulky prazole thioethers into chiral sulfoxides. The identification of BVMO andBVMO provides an important scaffold for discovering enzymes capable of asymmetrically oxidizing bulky thioether substrates by genome mining. Baeyer-Villiger monooxygenases (BVMOs) are valuable enzyme catalysts that are an alternative to the chemical Baeyer-Villiger oxidation reaction. Although BVMOs display broad substrate ranges, no native enzymes were reported to have activity toward the asymmetric oxidation of bulky prazole-like thioether substrates. Herein, we report the discovery of two type I BVMOs from (BVMO) and (BVMO) which are able to catalyze the asymmetric sulfoxidation of bulky prazole thioethers (proton pump inhibitors [PPIs], a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production). Efficient catalysis of omeprazole oxidation by BVMO was developed, indicating that this enzyme is a promising biocatalyst for the synthesis of bulky and pharmaceutically relevant chiral sulfoxide drugs. These results demonstrate that the newly identified enzymes are suitable templates for the discovery of more and better thioether-converting BVMOs.