Transaminases substrate scope

Very recently, Kroutil and coworkers extended the substrate scope also to primary alcohols in a sequence of ADH-catalyzed oxidation and co-transaminase-catalyzed reductive amination of the intermediate aldehyde. Both reactions were connected via alanine dehydrogenase, which mediated the regeneration of both NAD" and alanine (as amine donor for the reductive amination, Scheme 8.17) [82]. 

Since their discovery, first published by Needham et al, transaminases or aminotransferases (EC2.6.1.X) have received much attention as biocatalysts for the transformation of a keto acid to the corresponding amino acid/amine or vice versa [1]. Transaminases play an important role in amino acid metabolism and are ubiquitous in microbes and eukaryotic cells. They are pyridoxal-5 -phosphate (PLP)-dependent enzymes and are qualified as biocatalysts, due to their wide substrate scope, high enantio- and regioselectivity, high reaction rates, and stability [2]. As pictured in Scheme 29.1, the amino group of the amino donor is transferred to the carbonyl group of the amino acceptor. 

Another possible classification of transaminases is based on their differing substrate scope [16,17]. The amino donor for the transamination, catalyzed by transaminases, can be classified in three different chemical groups, differing in the presence and position of the functional groups, usually a negatively charged carboxylate group. 

Due to benefits like the wide substrate scope and their role in the biodegradation process of p-amino acids by microbes, the focus for the synthesis of optically pure p-amino acids has been relied recently on transaminases. 

In the previous sections, transaminases were referred as a suitable biocatalysts for the synthesis of chiral amines and amino acids. Their benefits are their wide substrate scope, high activity, and high enanho- and stereoselectivity. But some limitations in transaminase-catalyzed reachons occur as well, like product and substrate inhibition. To overcome these limitahons and to improve or alter the enzyme s properhes, enzyme immobilizahon can be the method of choice. Furthermore, to use enzymes in industrial processes, enzyme immobilizahon can... 

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. 

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