Enzymatic approaches to challenges in chemical synthesis are increasingly popular and very attractive to industry given their green nature and high efficiency compared to traditional methods. In this historical review we highlight the developments across several fields that were necessary to create the modern field of biocatalysis, with enzyme engineering and directed evolution at its core. We exemplify the modular, incremental, and highly unpredictable nature of scientific discovery, driven by curiosity, and showcase the resulting examples of cutting-edge enzymatic applications in industry.

The identification and 3D structural characterization of a homolog of the (R)-selective transaminase (RTA) from Aspergillus terreus (AtRTA), from the thermotolerant fungus Thermomyces stellatus (TsRTA) is here reported. The thermostability of TsRTA (40% retained activity after 7 days at 40 • C) was initially attributed to its tetrameric form in solution, however subsequent studies of AtRTA revealed it also exists predominantly as a tetramer yet, at 40 • C, it is inactivated within 48 h. The engineering of a cysteine residue to promote disulfide bond formation across the dimer-dimer interface stabilized both enzymes, with TsRTA_G205C retaining almost full activity after incubation at 50 • C for 7 days. Thus, the role of this mutation was elucidated and the importance of stabilizing the tetramer for overall stability of RTAs is highlighted. TsRTA accepts the common amine donors (R)-methylbenzylamine, isopropylamine, and D-alanine as well as aromatic and aliphatic ketones and aldehydes.

Chiral amines are a common feature of many active pharmaceutical ingredients. The synthesis of very small chiral amines is particularly challenging, even via biocatalytic routes, as the level of discrimination between similarly sized R-groups must be exceptional. Yet, their synthesis creates attractive building blocks that may then be used to prepare diverse compounds in further steps. Herein, the synthesis of one of the smallest chiral amines, 2-aminobutane, using transaminases, is being investigated. After screening a panel of mainly wild-type transaminases, two candidates were identified: an (S)-selective transaminase from Halomonas elongata (HEwT), and a pre-commercial (R)-selective transaminase from Johnson Matthey (*RTA-X43). Notably, a single strategic point mutation enhanced the enantioselectivity of HEwT from 45% ee to >99.5% ee. By covalently immobilizing these candidates, both enantiomers of 2-aminobutane were synthesized on a multi-gram scale, and the feasibility of isolation by distillation without the need for any solvents other than water was demonstrated. The atom economy of the process was calculated to be 56% and the E-factors (including waste generated during enzyme expression and immobilization) were 55 and 48 for the synthesis of (R)-2-aminobutane and (S)-2aminobutane, respectively.

Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications.

6-Aminocaproic acid (6ACA) is a key building block and an attractive precursor of caprolactam, which is used to synthesize nylon 6, one of the most common polymers manufactured nowadays. (Bio)-production of platform chemicals from renewable feedstocks is instrumental to tackle climate change and decrease fossil fuel dependence. Here, the cell-free biosynthesis of 6ACA from 6-hydroxycaproic acid was achieved using a coimmobilized multienzyme system based on horse liver alcohol dehydrogenase, Halomonas elongata transaminase, and Lactobacillus pentosus NADH oxidase for in-situ cofactor recycling, with > 90 % molar conversion (m.c.) The integration of a step to synthesize hydroxy-acid from lactone by immobilized Candida antarctica lipase B resulted in > 80 % m.c. of ɛ-caprolactone to 6ACA, > 20 % of δ-valerolactone to 5-aminovaleric acid, and 30 % of γ-butyrolactone to γ-aminobutyric acid in one-pot batch reactions. Two serial packed-bed reactors were set up using these biocatalysts and applied to the continuous-flow synthesis of 6ACA from ɛ-caprolactone, achieving a space-time yield of up to 3.31 g 6ACA h À 1 L À 1 with a segmented liquid/air flow for constant oxygen supply.