Transaminases evolution

I 2 Directed Evolution as a Means to Engineer Enantioseleaive Enzymes (S)-transaminase... 

A more recent study focused on the directed evolution of the co-transaminase from Vibrio fiuvialis JS17, specifically with the aim to eliminate product inhibition by aliphatic ketones while maintaining high enantioselectivity. This was achieved by screening 85 000 clones produced by epPCR [72]. 

Aspartate aminotransferase is the prototype of a large family of PLP-dependent enzymes. Comparisons of amino acid sequences as well as several three-dimensional structures reveal that almost all transaminases having roles in amino acid biosynthesis are related to aspartate aminotransferase by divergent evolution. An examination of the aligned amino acid sequences reveals that two residues are completely conserved. These residues are the lysine residue that forms the Schiff base with the pyridoxal phosphate cofactor (lysine 258 in aspartate aminotransferase) and an arginine residue that interacts with the a-carboxylate group of the ketoacid (see Figure 23.11). 

Tn amino acid production, we encounter an important problem in bio.synthesis—namely, stereochemical control. Because all amino acids except glycine are chiral, biosynthetic pathways must generate the correct isomer with high fidelity. In each of the 19 pathways for the generation ofchiral amino acids, the stereochemistry at the a-carbon atom is established by a transamination reaction that includes pyridoxal phosphate (PEP). Almost all the transaminases that catalyze these reactions descend from a common ancestor, illustrating once again that effective solutions to biochemical problems are retained throughout evolution. 

The evolution of a transaminase from Arthrohacter citreus to a thermostable transaminase with increased specific activity and decreased inhibition by the amine product was accomplished using error prone polymerase chain reaction (PCR) [64] The reaction of substituted tetralone 75 and isopropylamine to produce substituted (S) aminotetralin 76 was carried out at greater than 50 °C to facilitate the removal of the acetone by product and drive reaction equilibrium (Figure 14.43). 

This enzyme is a pyridoxal phosphate protein, which catalyses the formation of 7-aminobutyric acid, a possible chemical transmitter in the CNS. It can be used in an indicator reaction for the radiochemical assay of transaminases. It is assayed radiochemically using Clabelled glutamate and COj evolution [391]. 

The best method for the assay of pyridoxal phosphate is the use of tyrosine decarboxylase as described by Gunsalus, Bellamy, and Umbreit. The enzyme is prepared from a dried powder of cells of S. faecalis R. which has been grown deficient in vitamin Be by growth In a vitamin-Be-free alanine-rich medium. Thus, the decarboxylase is obtained almost completely resolved. This is a convenient preparation, since such a powder is stable for long periods and since the resolution of transaminases, decarboxylases, and tryptophanases isolated from tissues is a rather difficult task. The assay is performed manometrically by measuring the rate of CO2 liberation from tyrosine by the dried powder in the presence of pyridoxal phosphate. The rate of CO2 evolution is a function of the concentration of pyridoxal phosphate. 

To develop a biocatalytic process, screening of commercially available transaminases by Merck and Codexis provided no enz3une with detectable activity for amination of tile prositagliptin ketone 6 [28]. They therefore applied a combination of in silico design and directed evolution in an effort to confer such an enzyme. 

Transaminases are important enzymes in the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter the properties of transaminases, the reaction mechanisms, and their selectivity and substrate specificity are presented. The synthesis of chiral building blocks for pharmaceutically relevant substances and fine chemicals with transaminases as biocatalysts is discussed. Enzymatic asymmetric synthesis and dynamic resolution are discussed using transaminases. Protein engineering by directed evolution as well as rational design of transaminases under process condition is presented to develop efficient bioprocesses. 

Another approach in directed evolution was the evolution of an aspartate transaminase to an enz3une possessing the properhes of the closely related tyrosine transaminase [128]. Eight roxmds of DNA shuffling led to mutants with 100- to 270-fold increase of for phenylalanine and a 40- to 150-fold increase for tyrosine. 

The development of rapid HTS assays is important to test the substrate scope, suitable amino donors/acceptors, and the stability under different reaction conditions, like temperature, pH, different solvents, and immobilization methods. Furthermore the rapid progress in protein engineering like directed evolution requires fast selection methods. This subject was extensively reviewed by Mathew et al. [148]. In the following a colorimetric, photometric, and kinetic assay for rapid transaminase activity screening is described and illustrated in Scheme 29.17. 

Transaminases are most powerful tools for the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter several approaches for tiie preparation of fine chemicals or building blocks for pharmaceuticals were discussed, like asymmetric synthesis or kinetic resolution. The main limitations of transaminase-catalyzed reactions are the need to shift the equihbrium to the product side and substrate and product inhibition. Some solutions to overcome such inhibition were presented here for example, multienzyme cascades or biphasic extraction of the product. Protein engineering by directed evolution or rational enzyme design is a promising option to find transaminases with different substrate specificities and enantiopreferences. This is becoming more and more important for the pharmaceutical industry. Furthermore, it is a way to alter enzyme properties known so far, like thermostability and solvent and pH stability. Protein engineering has been assisted by the recently solved structures of certain transaminases. 

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