Water addition reactions to (un)-activated double bonds are very rewarding reactions as they elegantly introduce a hydroxyl-group thereby often adding value to the generated product by establishing a novel stereocentre in tertiary, chiral alcohols. However, performing selective water addition reactions is an extremely challenging task using classical, chemical approaches. Next to overall unfavourable reaction equilibria, the unreactive water molecule is a poor nucleophile and therefore requires activation. Furthermore, due to its small size, a controlled, stereo- and regioselective addition is difficult to achieve. Consequently, establishing straightforward processes with a preferably high selectivity under reaction conditions as environmentally benign as possible is of high interest to both industry and academia...@en

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.@en

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.@en

The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research.@en

The implementation of a stereoselective Michael addition with water as substrate is still a major challenge by classical, chemical means. Inspired by nature's ability to carry out this attractive reaction with both high selectivity and efficiency, the interest in hydratases (EC 4.2.1.x) to accomplish a selective water addition is steadily rising. The gram-positive bacterial genus Rhodococcus is known as biocatalytic powerhouse and has been reported to hydrate various Michael acceptors leading to chiral alcohols. This study aimed at the in-depth re-investigation of the hydration potential of Rhodococcus whole-cells towards Michael acceptors. Here, two concurrent effects responsible for the hydration reaction were found: while the majority of substrates was hydrated in an oxygen-independent manner by amino-acid catalysis, an enzyme-catalysed water addition to (E)-4-hydroxy-3-methylbut-2-enoic acid was proven to be oxygen-dependent. 18O2-labelling studies showed that no 18O2 was incorporated in the product. Therefore, a novel O2-dependent hydratase distinct from all characterised hydratases so far was found.@en

The first enantioselective synthesis was the selective addition of cyanide to benzaldehyde catalysed by a hydroxynitrile lyase (HNL). Since then these enzymes have developed into a reliable tool in organic synthesis. HNLs to prepare either the (R)- or the (S)-enantiomer of the desired cyanohydrin are available and a wide variety of reaction conditions can be applied. As a result of this, numerous applications of these enzymes in organic synthesis have been described. Here the examples of the last decade are summarised, the enzyme catalysed step is discussed and the follow-up chemistry is shown. This proves HNLs to be part of main stream organic synthesis. Additionally the newest approaches via immobilisation and reaction engineering are introduced.@en

Enzymes are supreme catalysts when it comes to high enantiopurities and their immobilization will pave the way for continuous operation. In this context, we show the covalent immobilization of hydroxynitrile lyases HbHNL (from Hevea brasiliensis) and MeHNL (from Manihot esculenta) in a siliceous monolithic microreactor for continuous operation. A thorough characterization of the immobilized HNLs on mesoporous silicates indicated the conditions essential for a successful immobilization. Their application in a continuous flow system enabled a remarkably fast (3.2 min) production of chiral cyanohydrins with high conversion (97%) and high ee (98%) using minimal enzyme loading (STY = 71 g L -1 h -1 mg protein -1 ). MeHNL showed increased operational stability, possibly due to a structural difference. The continuous flow microreactor outperformed batch systems, demonstrating the advantage of the mesoporous/macroporous environment for the expression of enzyme activity and the favorable characteristics of the microreactor. Overall, the system shows great potential for future industrial application of biocatalytic asymmetric syntheses. @en