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Although biocatalysis has garnered widespread attention in both industrial and academic realms, the enzymatic synthesis of chiral oxetanes remains an underdeveloped field. Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that have been engineered to accomplish the reversible transformation of epoxides. In this study, a biocatalytic platform was constructed for the stereoselective kinetic resolution of chiral oxetanes and formation of 1,3‐disubstituted alcohols. HheC from Agrobacterium radiobacter AD1 was engineered to identify key variants capable of catalyzing the dehalogenation of γ‐haloalcohols (via HheC M1‐M3) and ring opening of oxetanes (via HheC M4‐M5) to access both (R)‐ and (S)‐configured products with high stereoselectivity and remarkable catalytic activity, yielding up to 49 % with enantioselectivities exceeding 99 % ee and E>200. The current strategy is broadly applicable as demonstrated by expansion of the substrate scope to include up to 18 examples for dehalogenation and 16 examples for ring opening. Additionally, the functionalized products are versatile building blocks for pharmaceutical applications. To shed light on the molecular recognition mechanisms for the relevant variants, molecular dynamic (MD) simulations were performed. The current strategy expands the scope of HHDH‐catalyzed chiral oxetane ring construction, offering efficient access to both enantiomers of chiral oxetanes and 1,3‐disubstituted alcohols. [ABSTRACT FROM AUTHOR] more...

Biocatalysts that are eco-friendly, sustainable, and highly specific have great potential for applications in the production of fine chemicals, food, detergents, biofuels, pharmaceuticals, and more. However, due to factors such as low activity, narrow substrate scope, poor thermostability, or incorrect selectivity, most natural enzymes cannot be directly used for large-scale production of the desired products. To overcome these obstacles, protein engineering methods have been developed over decades and have become powerful and versatile tools for adapting enzymes with improved catalytic properties or new functions. The vastness of the protein sequence space makes screening a bottleneck in obtaining advantageous mutated enzymes in traditional directed evolution. In the realm of mathematics, there are two major constraints in the protein sequence space: (1) the number of residue substitutions (M); and (2) the number of codons encoding amino acids as building blocks (N). This feature review highlights protein engineering strategies to reduce screening efforts from two dimensions by reducing the numbers M and N, and also discusses representative seminal studies of rationally engineered natural enzymes to deliver new catalytic functions. [ABSTRACT FROM AUTHOR] more...

Reductive amination by amine dehydrogenases is a green and sustainable process that produces only water as the by-product. In this study, a continuous flow process was designed utilizing a packed bed reactor filled with co-immobilized amine dehydrogenase wh84 and glucose dehydrogenase for the highly efficient biocatalytic synthesis of chiral amino alcohols. The immobilized amine dehydrogenase wh84 exhibited better thermo-, pH and solvent stability with high activity recovery. (S)-2-aminobutan-1-ol was produced in up to 99% conversion and 99% ee in the continuous flow processes, and the space-time yields were up to 124.5 g L-1 d-1. The continuous reactions were also extended to 48 h affording up to 91.8% average conversions. This study showcased the important potential to sustainable production of chiral amino alcohols in continuous flow processes. [ABSTRACT FROM AUTHOR] more...