Biocatalyst cofactor regeneration

In some cases it is more attractive to use whole microbial cells, rather than isolated enzymes, as biocatalysts. This is the case in many oxidative biotransformations where cofactor regeneration is required and/or the enzyme has low stability outside the cell. By performing the reaction as a fermentation, i.e. with growing microbial cells, the cofactor is continuously regenerated from the energy source, e.g. glucose. 

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. 

Since cinnamyl aldehyde is the main component of cassia oil (approximately 90%) and Sri Lanka cinnamon bark oil (approximately 75%) [49], it is industrially more important to generate cinnamyl alcohol, which is less abundantly available from nature but is important as cinnamon flavour, by biotransformation of natural cinnamyl aldehyde than vice versa. Recently, a whole-cell reduction of cinnamyl aldehyde with a conversion yield of 98% at very high precursor concentrations of up to 166 g L was described [136]. Escherichia coli DSM 14459 expressing a NADPH-dependent R alcohol dehydrogenase from Lactobacillus kefir and a glucose dehydrogenase from Thermoplasma acidophilum for intracellular cofactor regeneration was applied as the biocatalyst (Scheme 23.8). 

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. 

The use of isolated enzymes to form or cleave P-O bonds is an important application of biocatalysts. Restriction endonucleases, (deoxy)ribonucleases, DNA/ RNA-ligases, DNA-RNA-polymerases, reverse transcriptases etc. are central to modern molecular biology(1). Enzyme catalyzed phosphoryl transfer reactions have also found important applications in synthetic organic chemistry. In particular, the development of convenient cofactor regeneration systems has made possible the practical scale synthesis of carbohydrates, nucleoside phosphates, nucleoside phosphate sugars and other natural products and their analogs. This chapter gives an overview of this field of research. 

The problem of cofactor regeneration is an important limitation in the use of many biocatalysts, and hence requires specific consideration. 

Mouri T, Michizoe J, Ichinose H, Kamiya N, Goto M (2006) Arecombinant c/reric/rra coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration. Appl Microbiol Biotechnol 2 514-520... 

Economic benefits Reduction in unit operations (including downstream) More compact process units Cofactor regeneration Biocatalyst recycling... 

An E. coli cell biocatalyst that overproduced the KER-L54Q mutant and glucose dehydrogenase (GDH) as a cofactor regeneration enzyme [(iii) in Eigure 6.13b the... 

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