Co-factor regeneration

Biocatalyst regeneration can be envisioned to be more than just co-factor regeneration. This may include induction and production of desulfurization enzymes during the desulfurization process or reproduction of new cells. Neither of these issues has been looked... 

The biological methods class of reagents holds the most promise for rapid development in the near future because most reactions are asymmetric. The problems that are being overcome are the tight substrate specificity of many enzymes and the need for co-factor regeneration. Systems are now being developed for asymmetric synthesis rather than resolution approaches. Some of these reactions are discussed in Chapters 19 through 21, but see also Chapters 2 and 3. 

One of the major disadvantages of using these enzymes to reduce ketones is generally the very poor aqueous solubility of the substrates (usually <5-10 mM) and the fact that the co-factor regeneration system is sensitive to organic solvents. To address this limitation, biphasic and membrane bioreactors have been used that allow for moderate to high conversions with often >99% ee for selected products. This has been demonstrated with the (.S )-Adi I from Rhodococcus erythropolis,36-37... 

Scheme 4.23 The use of glycosyl transferases in combination with in situ co-factor regeneration. E —galactosyl tranferase E2 — pyruvate kinase E3 = UDP-Glc-pyrophosphorylase E4 = galactose-1-phosphate uridyl transferase E5 = galactokinase.

To date, only a few examples of laboratory preparative-scale processes based on purified enzyme have been reported. Several studies have focused on the small-scale implementation of processes associating a new co-factor regenerating system, enzyme immobilization, membrane reactor, continuous substrate feeding, or resin-based SFPR with various results [110], Using the outstanding stabihty of PAMO, a 200 ml biotransformation of 5g/l phenyl cyclohexanone by an engineered mutant under two-Hquid phase conditions using methyl tert-butyl ether as solvent was described [102]. 

At present, studies to extend this strategy to other enzymes are being conducted in our groups and we hope that this concept may become an appealing solution to the problem of co-factor regeneration in redox biocatalysis. 

Screening for the optimum reaction conditions with whole cells, crude or purified enzymes (including technological aspects such as co-factor regeneration, immobilization, type of reactor, etc.). 

More essential amino acids are produced by fermentation for the pharmaceutical markets - as ingredients in infusion solutions or as a precursor for pharmaceutical amino acid derivatives. The latter requires extremely ambitious bio-catalytic processes including co-factor regeneration. Evonik Industries developed a process to the pharma-building block L-tert. leucine based on formate-dehydrogenase and NAD+-regeneration (Figure 12.3). Today this process runs on a tons scale. 

Figure 12.3 1-Step bio-catalysis including co-factor regeneration.

Other enzymes, the enoate reductases, reduce a, -unsaturated esters, aldehydes, and so forth. These enzymes have been isolated, and some impressive transformations have been recorded (e.g. Scheme 4.13). However, co-factor regeneration and enzyme stability can be problematic, and this sort of biotransformation is not yet at a stage at which it can be used on a substantial scale by a non-expert. 

Disadvantages Addition of co-factor regeneration system necessary Many enzymes not commercially available in isolated form or isolation from whole cells necessary Unwanted side reactions possible Mass-transfer limitations possible Substrate or product inhibition possible... 

SCHEME 36.2. Commonly used co-factor regeneration systems for the regeneration of NAD(P) and NAD(P)H. 

When biocatalytic redox processes are introduced, the problems of the co-factor regeneration must be addressed to provide an economic way of using these enzymes (see previous section and Scheme 36.3). In this respect, as described previously, industrial applications of... 

SCHEME 36.4. Enzyme-catalyzed production of (S)-3,5-bistrifluoromethylphenyl ethanol 5, an intermediate in the synthesis of MK-869 6, by using glucose dehydrogenase as a couple enzyme for co-factor regeneration. ... 

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