Transaminases directed 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]. 

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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