For biocatalytic production of pharmaceutically important chiral amines the.-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum omega-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (similar to 5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a well-known enzyme to achievechiral amines of high enantiomeric excess in laboratory scales. However, the low operational stabilityof Cv-ATA limits the enzyme applicability on larger scales. In order to improve the operational stabilityof Cv-ATA, and thereby extending its applicability, factors (additives, co-solvents, organic solvents anddifferent temperatures) targeting enzyme stability and activity were explored in order to find out how tostore and apply the enzyme. The present investigation shows that the melting point of Cv-ATA is improvedby adding sucrose or glycerol, separately. Further, by storing the enzyme at higher concentrations and inco-solvents, such as; 50% glycerol, 20% methanol or 10% DMSO, the active dimeric structure of Cv-ATAis retained. Enzyme stored in 50% glycerol at −20◦C was e.g., still fully active after 6 months. Finally,the enzyme performance was improved 5-fold by a co-lyophilization with surfactants prior to usage inisooctane.

The use of enzymes, nature´s own catalysts, both isolated or as whole cells to perform chemical transformations is called biocatalysis. As a complement to classical chemical catalysis, biocatalysis can be an environmentally friendly and more economical option in the production and synthesis of chemicals. Research on the application of amine transaminases in synthesis of chiral amines have exploded over the last two decades and interest from the industry is increasing. Amine transaminases are promising catalysts due to their ability to perform reductive amination of ketones with excellent enantioselectivity.

For a process to be efficient, high substrate specificity of the applied enzyme is an important factor. A variant of Chromobacterium violaceum amine transaminase that was obtained through rational design has an increased specific activity toward (S)-1-phenylethylamine and a set of 4´-substituted acetophenones. This result makes this variant a promising catalyst for the asymmetric synthesis of similar amines.

Amine transaminase catalyzed asymmetric synthesis of amines generally suffers from unfavorable equilibrium. Two methods that include spontaneous tautomerization and biocatalytic amidation for equilibrium displacement have therefore been developed.

Efficient assays and screening methods are demanded for the discovery and development of novel amine transaminases. For this purpose, a sensitive fluorescence-based assay that holds promise as a high-throughput screening method was developed.

One of the major obstacles for application of enzymes in industrial processes is the instability of the enzyme toward harsh conditions. The stability of Chromobacterium violaceum amine transaminase was investigated and improved using co-solvents and other additives. Co-lyophilization with surfactants was also applied to improve the performance of the same enzyme in organic solvents.

Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. We hereby present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60°C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3°C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the Proline rule was deemed successful at increasing the stability of this enzyme.

An efficient one-pot one-step biocatalytic amine transaminase/acyl transferase cascade for the formation of amides from the corresponding aldehydes and ketones in aqueous solution has been developed. N-benzyl-2-methoxyacetamide has been synthesized utlilizing the developed cascade in conversions up to 97%. The cascade was also evaluated for the synthesis of chiral amides.

omega-Transaminases are a valuable class of enzymes for the production of chiral amines with either (R)- or (S)-configuration in high optical purity and 100% yield by the biocatalytic reductive amination of prochiral ketones. A versatile new assay was developed to quantify omega-transaminase activity for the kinetic characterization and enantioselectivity typing of novel or engineered enzymes based on the conversion of 1-(6-methoxynaphth-2-yl)alkylamines. The associated release of the acetonaphthone product can be monitored by the development of its bright fluorescence at 450 nm with very high sensitivity and selectivity. The assay principle can be used to quantify omega-transaminase catalysis over a very broad range of enzyme activity. Because of its simplicity and low substrate consumption in microtiter plate format the assay seems suitable for liquid screening campaigns with large library sizes in the directed evolution of optimized transaminases. For assay substrates that incorporate structural variations, an efficient modular synthetic route was developed. This includes racemate resolution by lipase-catalyzed transacylation to furnish enantiomerically pure (R)and (S)-configured amines. The latter are instrumental for the rapid enantioselectivity typing of omega-transaminases. This method was used to characterize two novel (S)-selective taurine-pyruvate transaminases of the subtype 6a from thermophilic Geobacillus thermodenitrificans and G. thermoleovorans.

In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof.

An efficient single-enzymatic cascade approach for the asymmetric synthesis of chiral amines has been developed, which applies the amino donor 3-aminocyclohexa-1,5-dienecarboxylic acid spontaneously tautomerizing to reach reaction completion with excellent ee values.