Improving protein stability is an important goal for basic research as well as for clinical and industrial applications but no commonly accepted and widely used strategy for efficient engineering is known. Beside random approaches like error prone PCR or physical techniques to stabilize proteins, e.g. by immobilization, in silico approaches are gaining more attention to apply target-oriented mutagenesis. In this review different algorithms for the prediction of beneficial mutation sites to enhance protein stability are summarized and the advantages and disadvantages of FoldX are highlighted. The question whether the prediction of mutation sites by the algorithm FoldX is more accurate than random based approaches is addressed.

Optically pure β-amino acids constitute interesting building blocks for peptidomimetics and a great variety of pharmaceutically important compounds. Their efficient synthesis still poses a major challenge. Transaminases (also known as aminotransferases) possess a great potential for the synthesis of optically pure β-amino acids. These pyridoxal 5'-dependent enzymes catalyze the transfer of an amino group from a donor substrate to an acceptor, thus enabling the synthesis of a wide variety of chiral amines and amino acids. Transaminases can be applied either for the kinetic resolution of racemic compounds or the asymmetric synthesis starting from a prochiral substrate. This review gives an overview over microbial transaminases with activity towards β-amino acids and their substrate spectra. It also outlines current strategies for the screening of new biocatalysts. Particular emphasis is placed on activity assays which are applicable to high-throughput screening.

In this study, we investigated the possibility of using a modified hydantoinase process for the production of optically pure β-amino acids. Two aryl-substituted dihydropyrimidines D,L-6-phenyl-5,6-dihydrouracil (PheDU) and para-chloro-D,L-6-phenyl-5,6-dihydrouracil (pClPheDU) were synthesized. Hydrolysis of these novel substrates to the corresponding N-carbamoyl-β-amino acids by three recombinant D-hydantoinases and several bacterial strains was tested. All applied recombinant D-hydantoinases and eight bacterial isolates catalyzed the conversion of PheDU to N-carbamoyl-β-phenylalanine (NCβPhe). Some of these biocatalysts showed an enantioselectivity for either the D- or the L-PheDU enantiomer. The second dihydropyrimidinase substrate pClPheDU was hydrolyzed by all three recombinant D-hydantoinases and six of the wild-type strains. To our knowledge, this is the first dihydropyrimidinase activity reported with this aryl-substituted dihydropyrimidine. For selected biocatalysts, hydantoinase activity towards aryl-substituted hydantoins was demonstrated as well. However, none of the bacterial strains tested so far exhibited any carbamoylase activity towards NCβPhe.

ω-Transaminases (ω-TAs) are important biocatalysts for the synthesis of active, chiral pharmaceutical ingredients containing amino groups, such as β-amino acids, which are important in peptidomimetics and as building blocks for drugs. However, the application of ω-TAs is limited by the availability and stability of enzymes with high conversion rates. One strategy for the synthesis and optical resolution of β-phenylalanine and other important aromatic β-amino acids is biotransformation by utilizing an ω-transaminase from Variovorax paradoxus. We designed variants of this ω-TA to gain higher process stability on the basis of predictions calculated by using the FoldX software. We herein report the first thermostabilization of a nonthermostable S-selective ω-TA by FoldX-guided site-directed mutagenesis. The melting point (T ) of our best-performing mutant was increased to 59.3 °C, an increase of 4.0 °C relative to the T value of the wild-type enzyme, whereas the mutant fully retained its specific activity.

The successful synthesis of chiral amines from ketones using ω-transaminases has been shown in many cases in the last two decades. In contrast, the amination of β-keto acids is a special and relatively new challenge, as they decompose easily in aqueous solution. To avoid this, transamination of the more stable β-keto esters would be an interesting alternative. For this reason, ω-transaminases were tested in this study, which enabled the transamination of the β-keto ester substrate ethyl benzoylacetate. Therefore, a ω-transaminase library was screened using a coloring o-xylylenediamine assay. The ω-transaminase mutants 3FCR_4M and ATA117 11Rd show great potential for further engineering experiments aiming at the synthesis of chiral (S)- and (R)-β-phenylalanine esters. This alternative approach resulted in the conversion of 32% and 13% for the (S)- and (R)-enantiomer, respectively. Furthermore, the (S)-β-phenylalanine ethyl ester was isolated by performing a semi-preparative synthesis.