Biocatalyst format regeneration

Biocatalysts based on hydrolases (E.C. class 3, Table 5.2) ate mostly used as (purified) enzymes since they are cofactor independent, since these preparations are commercially available and because a number of hydrolases can be applied in organic solvents. Oxidoreductases (E.C. class 1) however, are relatively complex enzymes, which require cofactors and frequently consist of more than one protein component. Thus, despite the fact that efficient cofactor regeneration systems for NADH based on formate dehydrogenase (FDH) have been developed (Bradshaw et al, 1992 Chenault Whitesides, 1987 Wandrey Bossow, 1986, chapter 10) and that also an NADPH dependent FDH has been isolated (Klyushnichenko, Tishkov Kula, 1997), these enzymes are still mostly used as whole-cell biocatalysts. 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

Toxic substrates and products to whole-cell biocatalysts. Finally, in whole-cell format, the substrate and/or product of the bioreduction can be toxic to the cells, preventing cofactor regeneration. Such irreversible loss of regeneration capacity is, of course, catastrophic for the process. In principle, this can be overcome by maintaining a low substrate concentration, but this will ultimately prevent a sufficiently high product concentration for an effective process. In some cases, dependent upon the water-solubility (and if the substrate is a liquid), it may be possible to feed the substrate, such that a low concentration is provided to the cells in the reactor, but at the end of the reaction a high product concentration is achieved. However, in nearly all cases at the required concentration for an... 

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