Biocatalysis is one of the most promising technologies for the sustainable synthesis of molecules for pharmaceutical, biotechnological and industrial purposes. From the gram to the ton scale, biocatalysis is employed with success. This is underpinned by the fact that the global enzyme market is predicted to increase from $7 billion to $10 billion by 2024. This review concentrates on showing the strong benefits that biocatalysis and the use of enzymes can provide to synthetic chemistry. Several examples of successful implementations of enzymes are discussed highlighting not only high-value pharmaceutical processes but also low-cost bulk products. Thus, biocatalytic methods make the chemistry more environmentally friendly and product specific.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L^–1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H₂O₂ feed rate, linear product formation was observed for 7 days, producing more than 5 g L^–1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO).

Epimerization of cholic and chenodeoxycholic acid (CA and CDCA, respectively) is a notable conversion for the production of ursodeoxycholic acid (UDCA). Two enantiocomplementary hydroxysteroid dehydrogenases (7α‐ and 7β‐HSDHs) can carry out this transformation fully selectively by specific oxidation of the 7α‐OH group of the substrate and subsequent reduction of the keto intermediate to the final product (7β‐OH). With a view to developing robust and active biocatalysts, novel NADH‐active 7β‐HSDH species are necessary to enable a solely NAD⁺‐dependent redox‐neutral cascade for UDCA production. A wild‐type NADH‐dependent 7β‐HSDH from Lactobacillus spicheri (Ls7β‐HSDH) was identified, recombinantly expressed, purified, and biochemically characterized. Using this novel NAD⁺‐dependent 7β‐HSDH enzyme in combination with 7α‐HSDH from Stenotrophomonas maltophilia permitted the biotransformations of CA and CDCA in the presence of catalytic amounts of NAD⁺, resulting in high yields (>90 %) of UCA and UDCA.

A sequential‐type as well as a tandem‐type chemoenzymatic flow cascade combining an organocatalytic aldol reaction and a biocatalytic reduction to form stereoselectively a 1,3‐diol with two stereogenic centers were developed. Initially, a comprehensive screening of 24 alcohol dehydrogenases was carried out and the identified candidates were applied in different multi‐step flow cascades. All four stereoisomers of the desired 1,3‐diol product are accessible via a sequential flow approach with product formation‐related conversions of up to 76 % over two steps, isolated yields of up to 64 % and enantiomeric excess of >99 % in all cases. In addition, a tandem‐type flow process, performing both reaction steps simultaneously, was established leading to 51 % conversion with >99 % ee and 8 : 1 d.r. and representing a combination of the fields of asymmetric chemocatalysis, biocatalysis and flow chemistry.

The specific oxidation of 12α-OH group of hydroxysteroids is required for the preparation of cheno- and ursodeoxycholic acid (CDCA and UDCA, respectively). The C₁₂ oxidation of hydroxysteroids into their 12-oxo derivatives can selectively be performed by employing 12α-hydroxysteroid dehydrogenases. These enzymes use NAD(P)⁺ as an electron acceptor, which has to be re-oxidized in a so-called “regeneration system”. Recently, the enzyme NAD(P)H oxidase (NOX) was applied for the regeneration of NAD⁺ in the enzymatic preparation of 12-oxo-CDCA from cholic acid (CA), which allows air to be used as an oxidant. However, the NOX system suffers from low activity and low stability. Moreover, the substrate loading is limited to 10 mM. In this study, the laccase/mediator system was investigated as a possible alternative to NOX, employing air as an oxidant. The laccase/mediator system shows higher productivity and scalability than the NOX system. This was proven with a preparative biotransformation of 20 g of CA into 12-oxo-CDCA (92% isolated yield) by employing a substrate loading of 120 mM (corresponding to 50 g/L). Additionally, the performance of the laccase/mediator system was compared with a classical ADH/acetone regeneration system and with other regeneration systems reported in literature.

Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD⁺-dependent oxidation of 10-hydroxystearic acid.