Immobilization of Baeyer-Villiger monooxygenase from acetone grown Fusarium sp.

OBJECTIVE: A novel biocatalyst for Baeyer-Villiger oxidations is necessary for pharmaceutical and chemical industries, so this study aims to find a Baeyer-Villiger monooxygenase (BVMO) and to improve its stability by immobilization. RESULTS: Acetone, the simplest ketone, was selected as the only carbon source for the screening of microorganisms with a BVMO. A eukaryote, Fusarium sp. NBRC 109816, with a BVMO (FBVMO), was isolated from a soil sample. FBVMO was overexpressed in E. coli and successfully immobilized by the organic-inorganic nanocrystal formation method. The immobilization improved the thermostability of FBVMO. Substrate specificity investigation revealed that both free and immobilized FBVMO were found to show catalytic activities not only for Baeyer-Villiger oxidation of ketones to esters but also for oxidation of sulfides to sulfoxides. Furthermore, a preparative scale reaction using immobilized FBVMO was successfully conducted. CONCLUSIONS: FBVMO was discovered from an environmental sample, overexpressed in E. coli, and immobilized by the organic-inorganic nanocrystal formation method. The immobilization successfully improved its thermostability.

Impact and relevance of alcohol dehydrogenase enantioselectivities on biotechnological applications.

Alcohol dehydrogenases (ADHs) catalyze the reversible reduction of a carbonyl group to its corresponding alcohol. ADHs are widely employed for organic synthesis due to their lack of harm to the environment, broad substrate acceptance, and high enantioselectivity. This review focuses on the impact and relevance of ADH enantioselectivities on their biotechnological application. Stereoselective ADHs are beneficial to reduce challenging ketones such as ketones owning two bulky substituents or similar-sized substituents to the carbonyl carbon. Meanwhile, in cascade reactions, non-stereoselective ADHs can be utilized for the quantitative oxidation of racemic alcohol to ketone and dynamic kinetic resolution.

Organic-inorganic nanocrystal reductase to promote green asymmetric synthesis.

An acetophenone reductase from Geotrichum candidum (GcAPRD) was immobilized by the organic-inorganic nanocrystal method. The GcAPRD nanocrystal presented improved stability and recyclability compared with those of the free GcAPRD. Moreover, the GcAPRD nanocrystal reduced broad kinds of ketones with excellent enantioselectivities to produce beneficial chiral alcohols such as (S)-1-(3',4'-dichlorophenyl)ethanol with >99% yield and >99% ee. The robust and versatile properties of the GcAPRD nanocrystal demonstrated an approach to promote green asymmetric synthesis and sustainable chemistry.

Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction.

Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.

Correction to: Structural basis for a highly (S)-enantioselective reductase towards aliphatic ketones with only one carbon difference between side chain.

The original version of this article contains error for some of the authors corrections were not included during correction stage.

Structural basis for a highly (S)-enantioselective reductase towards aliphatic ketones with only one carbon difference between side chain.

Aliphatic ketones, such as 2-butanone and 3-hexanone, with only one carbon difference among side chains adjacent to the carbonyl carbon are difficult to be reduced enantioselectively. In this study, we utilized an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD) to reduce challenging aliphatic ketones such as 2-butanone (methyl ethyl ketone) and 3-hexanone (ethyl propyl ketone) to their corresponding (S)-alcohols with 94% ee and > 99% ee, respectively. Through crystallographic structure determination, it was suggested that residue Trp288 limit the size of the small binding pocket. Docking simulations imply that Trp288 plays an important role to form a C-H⋯π interaction for proper orientation of ketones in the pro-S binding pose in order to produce (S)-alcohols. The excellent (S)-enantioselectivity is due to a non-productive pro-R binding pose, consistent with the observation that the (R)-alcohol acts as an inhibitor of (S)-alcohol oxidation.