In cofactor regeneration

The enzyme hydrogenase (hydrogen dehydrogenase EC 1.12.1.2) is able to reduce electron acceptors by molecular hydrogen. When it is used in cofactor regenerating systems, consumed NADH can be regenerated directly by molecular hydrogen. 

Diliberto E) Ir, Dean G, Carter C, and Allen PL (1982) Tissue, subcellular, and submito-chondrial distributions of semidehydroascorbate reductase possible role of semide-hydroascorbate reductase in cofactor regeneration. Journal of Neurochemistry 39, 563-8. 

Rodriguez, C., Lavandera, I., and Gotor, V. (2012) Recent advances in cofactor regeneration systems applied to biocatalyzed oxidative processes. Curr. Org. Chem.. 16 (21), 2525-2541. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

Biocatalytic ledox reactions offer great synthetic utility to organic chemists. The majority of oxidase-catalyzed preparative bioconversions are still performed using a whole-ceU technique, despite the fact that the presence of more than one oxidoreductase in cells often leads to product degradation and lower selectivity. Fortunately, several efficient cofactor regeneration systems have been developed (160), making some cell-free enzymatic bioconversions economically feasible (161,162). 

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. 

Wichmann, R. and Vasic-Racki, D. (2005) Cofactor regeneration at the lab scale. Advances in Biochemical Engineering/Biotechnology, 92, 225-260. 

Cofactor regeneration is a necessary prerequisite for an in-vitro application of oxidoreductase enzymes, as the cofactors are too expensive to be used in stoichiometric amounts (Fig. 43.2) [17, 18]. 

The principal strategies of cofactor regeneration - namely the enzymatic, chemical and electrochemical approach - are presented in Scheme 43.2 and have been reviewed recently [17, 21-23]. This chapter does not intend to be exhaustive rather, it focuses on the systems where a transition-metal complex and... 

Enzymatic cofactor regeneration can be subdivided into two categories the enzyme-coupled approach, where two different enzymes are used (one for the production reaction, and one for the regeneration reaction) and the substrate-coupled approach, where one and the same enzyme is used for both production and regeneration (E = E2). The most convenient and commonly used enzymatic regeneration systems are summarized in Table 43.1. 

To circumvent the cofactor regeneration problem, redox biotransformations are also carried out in whole cells - for example, baker s yeast [28, 29] or engineered Escherichia coli cells [30] - using the intracellular cofactor pool and inherent or recombinant regeneration systems. 

Considering that these two transition-metal complexes are the only ones reported for the electrochemical cofactor reduction, the results are quite promising and show the need for further research in this field to identify new catalysts. In addition to the use of soluble redox mediators in electrochemical cofactor regeneration, modified electrodes have also been used. Details on these systems can also be found in the above-mentioned reviews [31, 32]. 

The use of redox enzymes in organic synthesis, while having a large potential for broad application in the selective formation of high-value compounds, has been limited by the necessity of cofactor regeneration or enzyme reactivation. Electrochemistry offers an attractive and, in principle, simple way to solve this problem because the mass-free electrons are used as regenerating agents. No... 

Phenazine methosulfate, cofactor regeneration using, 3 673 Phenazocine, 4 359t Phendimetrazine (Bontril), 3 91 Phenethanolamines, 13 14 Phenethyl alcohol, antimicrobial used in cosmetics, 7 831t... 

As most enzymes function under compatible ambient conditions, bio-bio cascades had already been successfully developed by the 1970s. By far, most examples have been reported in the field of carbohydrates, using combinations of enzymatic conversions (up to eight enzymes in one-pot), as well as for the in situ cofactor regeneration of enzymatic redox reactions towards amino and hydroxy acids. 

An impressive one-pot six-step enzymatic synthesis of riboflavine from glucose on the laboratory scale has been reported with an overall yield of 35-50%. Six different enzymes are involved in the various synthesis steps, while two other enzymes take care for the in situ cofactor regenerations [12]. This example again shows that many more multi-enzyme cascade conversions will be developed in the near future, as a much greater variety of enzymes in sufficient amounts for organic synthetic purposes will become available through rapid developments in genomics and proteomics. 

Unlike the whole-cell system, enzymatic reductions require the addition of a hydride donating cofactor to regenerate the reduced form of the enzyme. Depending on the chosen ADH, the cofactor is usually NADH or NADPH, both of which are prohibitively expensive for use in stoichiometric quantities at scale. Given the criticality of cofactor cost, numerous methods of in situ cofactor regeneration, both chemical and biocatalytic, have been investigated. However, only biocatalytic regeneration has so far proven to be sufficiently selective to provide the cofactor total turnover numbers of at least 10 required in production. 

Biocatalytic approaches to cofactor regeneration can be divided into coupled-enzyme methods and coupled-substrate methods.In the coupled-enzyme method, the oxidized cofactors (NAD+ and NADP+) are recycled in situ by performing an oxidation reaction using a second enzyme and an inexpensive auxiliary substrate. This second enzyme must employ the same cofactor, but neither enzyme should be able to accept the same substrate. 

In an extensive study, Okamoto and co-workers [76-86] introduced a biochemical switching device based on a cyclic enzyme system in which two enzymes share two cofactors in a cyclic manner. Cyclic enzyme systems have been used as biochemical amplitiers to improve the sensitivity of enzymatic analysis [87-89], and subsequently, this technique was introduced into biosensors [90-93], In addition, cyclic enzyme systems were also widely employed in enzymic reactors, in cases where cofactor regeneration is required [94-107], Using computer simulations, Okamoto and associates [77,80-83] investigated the characteristics of the cyclic enzyme system as a switching device, and their main model characteristics and simulation results are detailed in Table 1.1, as is a similar cyclic enzyme system introduced by Hjelmfelt et al. [109,116], which can be used as a logic element. 

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