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Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products ("normal" and "abnormal") when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (kcat /KM ) 8.9-fold to 484 s-1 mM-1 for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMOC308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L-1 d-1 . This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.

Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. The mutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening

A novel (R)-imine reductase (PlRIR) from Paenibacillus lactis was heterologously overexpressed in Escherichia coli, purified and characterized. The purified PlRIR exhibited relatively high catalytic efficiency (kcat/Km=1.58 s−1 mm−1) towards 2,3,3-trimethylindolenine. A panel of 3H-indoles and 3H-indole iodides were reduced by PlRIR to yield the corresponding products with good-to-excellent enantioselectivity (66–98 % ee). In addition, PlRIR also possesses good activities toward other types of imines such as pyrroline, tetrahydropyridine, and dihydroisoquinoline, indicating a reasonably broad substrate acceptance. In a 100 mg scale preparative reaction, 100 mm 2,3,3-trimethylindolenine was converted efficiently to afford (R)-2,3,3-trimethylindoline with 96 % ee and 81 % yield.

In order to search for oxidoreductases suitable for the preparation of methyl (R)-o-chloromandelate [(R)-CMM], the key intermediate for clopidogrel, the homologous proteins of Gre2p were expressed in Escherichia coli, among which CgKR1 showed the most satisfactory activity and stereoselectivity towards methyl o-chlorobenzoylformate (CBFM). Using the crude enzyme of CgKR1 and glucose dehydrogenase (GDH), as much as 300 g⋅L−1 of CBFM was almost stoichiometrically converted to (R)-CMM with excellent enantiomeric excess (98.7% ee). More importantly, the reaction could be performed without external addition of an expensive cofactor. The substrate profile indicates that keto esters serve as the most suitable substrate, which was confirmed by gram-scale preparations. Homology modeling and docking analysis revealed the molecular basis for the high stereoselectivity of CgKR1. These demonstrate not only the feasibility of in silico mining of novel enzymes based on sequence homology but also the applicability of this new reductase for the practical production of optically active (R)-CMM.

In the last 50 years the number of known natural organo-halogen compounds has grown from a dozen to more than 4000 today. The most studied group of halogenase biocatalysts are haloperoxidases, which are able to accelerate a wide range of halogenation reactions following the mode of action represented in Scheme 11.1. More recently new halogenases have been identified such as 2-oxoglutarate irondependent halogenase, nucleophilic halogenases that can introduce fluorine and FADH2 dependant halogenases. The FADH2 cofactor enzymes can halogenate substrates with excellent regioselectivity. A good example of this reaction is the synthesis of isolated 7chlorotriptophan by Walsh et al. a target synthesis that would involve many steps using traditional organic synthesis methods (Scheme 11.2). Hence this group of enzymes offers the chemical industry significant improvements in synthetic methodologies as low regio-selectivities are usually obtained in traditional halogenation reagents and these reagents are limited to reaction at specific activated sites. Halogenases are now known that regioselectively halogenate all four positions of the indole benzene ring which is not possible with chemical reagents. By replacing chloride salts by bromide salts in the reaction media bromination is possible in many cases. Dehalogenases have been known to efficiently dehalogenate activated halogen compounds and are applied on the large scale for the synthesis of (S)-chloropropionic acid as

A β-ketoacyl-ACP reductase (FabG) gene from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli and the encoded protein was purified to homogeneity. The recombinant reductase could reduce a broad spectrum of prochiral ketones including aromatic ketones and keto esters and showed the highest activity in the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE). Using E. coli cells coexpressing both FabG and glucose dehydrogenase (GDH) genes, as much as 620 g·L ―1 of OPBE was almost stoichiometrically converted to ethyl (S)-2-hydroxy-4-phenylbutyrate [(S)-HPBE] with excellent (>99%) enantiomeric excess. More importantly, the process could be performed smoothly without external addition of an expensive cofactor as usually done and could be scaled up very easily. All these positive features demonstrate the applicability of this reductase for the large-scale production of optically active α-hydroxy acids/esters.