Co-factor recycling

K. F. Gu and T. M. S. Chang, Conversion of ammonia or urea into L-leucine, L-valine and L-isoleucine using artificial cells containing an immobilized multienzyme system and dextran-NAD+, glucose dehydrogenase for co-factor recycling, A 4/0, 11(1), 24-28 (1988). 

Leucine dH is the enzyme used as the biocatalyst in the process commercialized by Degussa AG (Hanau, Germany) to produce L-tert-leucine (L-Tle).28-60 This UAA has found widespread use in peptidomimetic drugs in development, and the demand for this unique amino acid continues to increase.61-62 This process, which has been the subject of much study, requires a co-factor recycling system (Scheme 19.4, R = Me3C).63-64 Similar to phenylalanine dH, leucine dH has been used to prepare numerous UAAs because of its broad substrate specificity.43-65-66... 

Use of Enzyme Membrane Reactors (EMR) the method of choice for systems with co-factor recycling or for reactions with expensive enzymes. 

A more recent EMR-process for the enantioselective reduction of 2-oxo-4-phenylbutyric acid (OPBA) to (R)-2-hydroxy-4-phenylbutyric acid (HPBA, e.e.>99.9%) with NADH-de-pendent D-lactate dehydrogenase (D-LDH) from Staphylococcus epidermidis and FDH/for-mate for co-factor recycling uses free NADH instead of PEG-NADH [100, 101] (Fig. 3). Within the selected residence time of 4.6 h, a cycle number for the co-factor of 1000 can easily be achieved. Instead of NADH, the less expensive NAD+ is used. A second reason for the application of the native co-enzyme is the fact that the activity of D-LDH is reduced to one tenth when PEG-enlarged co-enzyme is used instead of the native co-enzyme. HPBA produced by this method with a space-time yield of 165 g 1 d-1 is of considerably higher enantiomeric purity and the process shows competitive economy in comparison with chemical... 

However, attempting to use a co-factor-dependent enzyme outside whole cells means either using expensive co-factors as co-substrates in stoichiometric amounts or developing some form of compatible in vitro co-factor recycling system. Although this has been achieved for some isolated enzymes (notably some oxidoreductases), this is not always possible and/or cost-worthy. 

The question of co-factor recycling has been addressed by a number of scientists, and several convenient solutions to the problem have been found. One of these solutions is shown in Figure 4.4, which features the working head group of the co-factor the figure demonstrates the use of a second enzyme, formate dehydrogenase, which takes the co-factor back to the reduced form, with the evolution of carbon dioxide. 

Figure 5.22 Biocatalytic oxidation of alcohol using acetone for co-factor recycling.

W. Hussain, D.l. Pollard, M. Tmppo, GJ. Lye, Enzymatic ketone reductions with co-factor recycling Improved reactions with ionic liquid co-solvents. Mol. Cat. B Enz. 55 (2008) 19-29. 

A recently characterized class of dehydrogenases are the quinoproteins which contain a pyrroloquinolene quinone prosthetic group and do not require a separate co-factor Electron transfer mediators such as phenazine ethosulphate 2,6-dichloroindophenol and ferricenium ions have been used to recycle the quinoprotein the reduce mediator is detected amperometrically. 

Another major factor when considering whole-cell versus cell-free reactions are the overall reaction kinetics. Some enzymatic reactions utilize a complex multicomponent enzyme system. Reconstitution of the crude or purified enzyme components are not usually as effective in vitro as they are when they remain in the intracellular milieu. Whole cells have often been called little bags of enzymes. Although this is an oversimplification, it is a useful concept to consider. Whole cells sequester the enzyme components in a small but concentrated form, which is usually optimal for high efficiency. Whole cells also contain co-factors, including the systems that recycle them, and control pH and ionic strength. Altogether these factors combine to make whole cells a very useful form for the presentation and use of sensitive enzyme catalysts. 

Transaminases possess many features appropriate for effident biocatalysts, such as high turnover numbers and no requirement for external recycling of the co-factor. Because of the wide substrate tolerance of many amino transferases such as tyrosine amino transferase and branched-chain amino transferases from E. coU, these enzymes have been largely employed in the enantiospecific preparation of non-proteinogenic amino acids. These include straight-chain alkyl, diadd, branched-chain, aromatic, and bifunctional amino adds [65]. 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

Reagents Pure enzymes normally need no more than pH adjustment though some, say enzymes that catalyse redox reactions, need expensive co-factors. These can normally be supplied by a second enzyme the recycles the co-factor. Organisms need feeding - this is cheap but may require a lot of material. 

Biotechnology has had a significant effect on the flavour industry but two factors have limited its application to fragrance. The first is cost, as biotechnological processes are usually quite expensive. The second is selectivity. Individual enzymic reactions are very selective, but biochemical redox reactions require expensive co-factors and so the usual technique is to run whole cell fermentations so as to allow the cell s chemical factory to recycle the co-factors. However, the cell does much chemistry in addition to the reaction we wish it to do and the result is a horrendous effluent problem. In flavours, the problem is often simpler as the whole cell, e.g. a yeast cell, can be used as the product. 

Multienzymatic approaches of special interest for co-factor-dependent systems with recycling enzymes and multistep one-pot enzymatic synthesis FDH, kinases, glycosyltransferases... 

An important technical issue is the large-scale applicability of co-factor-dependent enzymatic systems. It is generally accepted that, e.g., NADH-requiring oxidoreductases can easily be used in whole-cell biocatalysis such as baker s yeast-mediated reductions, where the cofactor recycling step is simultaneously performed within the intact cell, driven by the reduction equivalents introduced via the external carbon and energy source (glucose). 

Occasionally, enzymes require coenzymes, also known as co-factors, to take part in a reaction. For example, in a reduction or oxidation reaction, the coenzyme may provide the reducing or oxidising power to drive the reaction. The coenzyme is then recycled by being re-oxidised or re-reduced, as appropriate, in a subsequent cycle or even in a different enzyme system. There are three coenzymes that are particularly important in the biosynthesis of terpenoids and it is worth looking at them in a little more detail before we move on to the main topic. 

Isolated enzymes Simple apparatus Simple work-up Specific for selected reaction Co-solvents better tolerated Expensive Addition of enzyme co-factors required or enzyme cofactor recycling necessary... 

Such co-factors are recycled by complementary enzyme-catalysed... 

The enzyme-catalysed oxidation of alcohols to carbonyl compounds is not as attractive as the reverse reaction discussed earlier. This is because the oxidation often removes a chiral centre from the substrate, and the recycling of oxidized co-factors NAD(P) can be problematic. However, there are some instances where the enzyme technology has an advantage over conventional chemical oxidants. For example, the polyol D-sorbitol is oxidised by the micro-organism Acetobacter suboxydans to give L-sorbose (Scheme 4.14) (see also Chapter 1, Section 1.7). 

D-glycerol-3-phosphate (s -glycerol-3-phosphate) (47X which is used in phospholipid manufacture, is another synthon whose production requires the recycling of a co-factor. It is synthesized from glycerol and ATP, with... 

Homocysteine aminothiol biosynthesized from methionine by removal of its terminal C methyl group can be recycled into methionine or converted into cysteine with B vitamin co-factors. Deficiencies of vitamin Bg, Bg or Bn and/or genetic mutations can lead to hyperhomocysteinemia. 

Recycle of the co-factor NAD(P)H by using formate dehydrogenase and formate or glucose dehydrogenase and glucose... 

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