Formate regeneration system

The DDI conventional inhibition screen is moderate throughput and can range from a few compounds per week to a few hundred per week depending on the amount of inhibitor concentrations, inhibition curve replicates and analytical methods used. This screen typically uses 96- or 384-well formats. The reaction components (HLM, 100 mM potassium phosphate buffer, specific probe substrate, NCE, NADPH cofactor or NAD PH-regenerating system) are pre-warmed to 37 °C and are mixed together to initiate the reaction, then incubated at 37 °C for the appropriate length of... 

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. 

In the emulsion process, a high 3-phenylcatechol concentration in the organic phase and the same or higher productivities (up to 0.45 g L-1 h 1) as in the in vivo process were achieved1401. Here, formate dehydrogenase and formate served as the coenzyme regeneration system (Fig. 16.3-10 A). The benefits of this regeneration... 

One of our troubles was deactivation, in fact, the pilot plant operation did show that we did accumulate carbon on the catalyst. If the initial rate of carbon formation were maintained, the run would not last more than a month or so. This would mean regeneration, and instead of a relatively simple operation, we would have to go to a more complex plant involving a cyclic regeneration system and all its difficulties that, at that time, were most formidable. Fortunately, as we learned, the carbon formation did not continue but leveled out at an acceptable value. 

Several cofactor regeneration systems were based on FDH. The substrate formate is an inexpensive, stable, and innocuous compoimd, while CO2, which is produced by FDH, can be easily removed from the reaction by evaporation. A general drawback of FDH is, however, its low specific activity [262]. More stable FDH variants have been engineered and successfully applied... 

In recent years, these facts have significantly prompted the research toward the development of new and more efficient in situ regeneration systems of the NAD(P)(H) cofactors [2, 3], which allow their use in catalytic instead of stoichiometric amounts, thus making the dehydrogenase-catalyzed processes acceptable from an economical point of view. Moreover, the recycling reactions can be also used to shift the equilibrium of thermodynamically unfavorable transformations toward product formation. 

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