Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.

Biocatalysis has emerged as a powerful alternative to traditional chemistry, especially for asymmetric synthesis. One key requirement during process development is the discovery of a biocatalyst with an appropriate enantiopreference and enantioselectivity, which can be achieved, for instance, by protein engineering or screening of metagenome libraries. We have developed an in silico strategy for a sequence-based prediction of substrate specificity and enantiopreference. First, we used rational protein design to predict key amino acid substitutions that indicate the desired activity. Then, we searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases, which catalyzed the synthesis of several (R)-amines with excellent optical purity up to >99% enantiomeric excess.

For the biocatalytic preparation of optically active amines, omega-transaminases (omega-TA) are of special interest since they allow the asymmetric synthesis starting from prostereogenic ketones with 100% yield. To facilitate the purification and characterization of novel omega-TA, a fast kinetic assay was developed based on the conversion of the widely used model substrate alpha-methylbenzylamine, which is commonly accepted by most of the known omega-TAs. The product from this reaction, acetophenone, can be detected spectrophotometrically at 245 nm with high sensitivity (epsilon = 12 mM(-1) cm(-1)), since the other reactants show only a low absorbance. Besides the standard substrate pyruvate, all low-absorbing ketones, aldehydes, or keto acids can be used as cosubstrates, and thus the amino acceptor specificity of a given omega-TA can be obtained quickly. Furthermore, the assay allows the fast investigation of enzymatic properties like pH and temperature optimum and stability. This method was used for the characterization of a novel omega-TA cloned from Rhodobacter sphaeroides, and the data obtained were in excellent accordance with a standard capillary electrophoresis assay.

Two is better than one (enzyme). The combination of ω‐transaminase with pyruvate decarboxylase (PDC) allowed for the efficient asymmetric synthesis of chiral amines, because the unfavorable equilibrium could be shifted by removal of pyruvate by using PDC.

Seven (R)-selective amine transaminases (R-ATAs) recently discovered by an in silico-based approach in sequence databases were produced recombinantly in Escherichia coli and subjected to partial purification by ammonium sulfate precipitation. A range of additives and various buffers were investigated to identify best conditions to ensure good storage stability and stable activity during biocatalysis. All enzymes show pH optima between pH 7.5-9. These R-ATAs were then applied in the asymmetric synthesis of twelve aliphatic, aromatic and arylaliphatic (R)-amines starting from the corresponding prochiral ketones using a lactate dehydrogenase/glucose dehydrogenase system to shift the equilibrium. For all ketones, at least one enzyme was found that allows complete conversion to the corresponding chiral amine having excellent optical purities > 99% ee. Variations in substrate profiles are also discussed based on the phylogenetic relationships between the seven R-ATAs. Thus, we have identified a versatile toolbox of (R)-amine transaminases showing remarkable properties for application in biocatalysis.