Cofactor recycling

Many procedures have been suggested to achieve efficient cofactor recycling, including enzymatic and non-enzymatic methods. However, the practical problems associated with the commercial application of coenzyme dependent biocatalysts have not yet been generally solved. Figure A8.18 illustrates the continuous production of L-amino adds in a multi-enzyme-membrane-reactor, where the enzymes together with NAD covalently bound to water soluble polyethylene glycol 20,000 (PEG-20,000-NAD) are retained by means of an ultrafiltration membrane. 

Figure A8.18 A racemic mixture of a-hydroxyacids (like L, D-lactate) can be transformed via the corresponding a-ketoacid (pyruvate) to the desired L-amino acid (L-alanine) with cofactor recycling.

Recent studies on isolated BVMOs using Rh-complexes as NADPH substitutes for facile cofactor recycling suggested a pivotal role of the native cofactor to generate the proper environment within chiral induction in sulfoxidation reactions. While biooxidation was still observed in the presence of the metal complex, stereoselectivity of the enzyme was lost almost completely [202]. 

In respect of designing an economic production process, the stoichiometric cofactor required in carbonyl reductions or the respective oxidation reactions needs to be minimized that is, enabled by recycling of the cofactor. The measure for the efficiency of the recycling process is the total turnover number (TTN), which describes the moles of product synthesized in relation to the moles of cofactor needed. The different approaches in cofactor recycling were recently reviewed by Goldberg et at. [12]. 

Groger, H., Hummel, W., Rollmann, C. et al. (2004) Preparative asymmetric reduction of ketones in a biphasic medium with an (5)-alcohol dehydrogenase under in f/w-cofactor-recycling with a formate dehydrogenase. Tetrahedron, 60 (3), 633-640. 

Each reaction was performed with a CYP biocatalyst concentration of 1 pM (1000 nmol L 1), in the presence of a corresponding CYP reaction mix containing reduced nicotinamide cofactor and a cofactor recycling system at 30 °C, with agitation to promote oxygen transfer to the reaction solution. 

The asymmetric reduction of prochiral functional groups is an extremely useful transformation in organic synthesis. There is an important difference between isolated enzyme-catalyzed reduction reactions and whole cell-catalyzed transformations in terms of the recycling of the essential nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] cofactor. For isolated enzyme-catalyzed reductions, a cofactor recycling system must be introduced to allow the addition of only a catalytic amount (5% mol) of NAD(P)H. For whole cell-catalyzed reductions, cofactor recycling is automatically achieved by the cell, and the addition of a cofactor to the reaction system is normally not required. 

It is only recently that isolated enzymes have been used in the presence of appropriate cofactor recycling systems.14 Not long ago, application of the whole-cell system was the only way to get high yields and high ee in enzyme-catalyzed organic synthesis. 

Thus a number of enzymes have been shown to be able to control the oxidation of sulfides to optically active sulfoxides most extensive investigations have concentrated on mono-oxygenases (e.g. from Acinetobacter sp., Pseudomonas putida) and haloperoxidases1 071 (from Caldariomyces fumago and Coral I ina officinalis). A comparison of the methodologies11081 led to the conclusion that the haloperoxidase method was more convenient since the catalysts are more readily available (from enzyme suppliers), the oxidant (H2O2) is cheap and no cofactor recycling is necessary with the haloperoxidases. Typical examples of haloperoxidase-catalysed reactions are described in Scheme 24. 

Biosynthetic production of thymidine is overall a complex process combining the controlled introduction of a novel biotransformation step into a biological system with selective enhancement or knock-out of a series of existing metabolic steps. Metabolic engineering to enhance cofactor recycling at both ribonucleotide reduction and dUMP methylation steps has important parallels in other systems, as whole-cell biotransformations are frequently employed as a means to supply, in situ, high-cost and usually labile cofactors. 

The use of water-miscible organic solvent-water mixtures is a particularly attractive method for use with cofactor-dependent enzymes due to its simphcity. The high water content can allow dissolution of both enzyme and cofactor, whilst the water-miscible solvent can provide a dual role in both substrate dissolution and as a cosubstrate for cofactor recycling (substrate-coupled cofactor recycling).The asymmetric reduction of a ketone intermediate of montelukast using an engineered ADH in the presence of 50 % v/v isopropanol offers a powerful demonstration of this methodology (Scheme 1.55). 

Two groups in particular have pioneered the practical application of PheDH from various bacterial species, namely those of Asano etal in Japan and Hummel etal. in Germany. Asano and his colleagues initially explored the application of PheDH to the chiral synthesis of the physiological substrate L-phenylalanine. The PheDH of Bacillus sphaericus was overexpressed in E. coll The issue of cofactor recycling was tackled by using the FDH of Candida hoidinil Importantly, these authors showed that both catalytic activities could be successfully... 

Cofactor Recycle in Multi-Step Oxidizing Biocatalytic Systems... 

A key consideration in development of all multi-step bioprocesses is the type of bioreactor it may be necessary to accommodate a range of conditions including compartmentalization of the enzymes, cofactor recycle, adequate oxygen supply, variable temperature and pH requirements, and differential substrate feed rates. Examples described below include a range of different reactors, of which membrane bioreactors are clearly often particularly useful. 

Good reactor productivities and cofactor recycling efficiencies with reuse of the... 

Reactor productivities of 640 g.l day and a cofactor recycling efficiency of 130,000 (mol product formed/mol cofactor used) are achieved making cofactor costs very low. 

Cofactor recycling required required not required not required... 

The potential of lyases for the synthesis of optically active compounds are of commercial interest, because these enzymes are stereospecific and do not require complicated cofactor recycling procedures. What types of reactions are catalyzed by lyases Lyases typically catalyze reversible reactions. How can you push the equilibrium in the desired direction ... 

Ketoreductases (KREDs) are dependent on nicotinamide cofactors NADH or NADPH. Due to the reaction mechanism, these rather costly cofactors are needed in stoichiometric amounts, disclosing an economic problem that has to be dealt with when using these enzymes. Many different possibilities for cofactor recycling have been established with three major approaches finding application in research and industry (Fig. 13). Further regeneration systems, such as electrochemical methods, are not discussed within this review [22-24, 37, 106-108],... 

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