Cofactor recycling, biocatalytic

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

Much higher productivities can be obtained using isolated enzymes or cell extracts [118]. This approach is therefore highly preferred. Because of the importance of whole cell technology for biocatalytic reduction a few examples will be given. However the main part of this chapter will be devoted to industrial examples of bioreduction involving isolated enzymes and cofactor recycling. 

Fig. 4.75 Biocatalytic oxidation of alcohol using acetone for cofactor recycling.

Simultaneous biocatalytic oxidation and reduction was also reported for the resolution of secondary alcohols through combination of molecular oxygen and stereoselective reducing agent (alcohol dehydrogenase) with a cofactor-recycling... 

In 2006, Kosjek et al. reported a similar methodology for the biocatalytic reduction of a,jS-unsaturated ketones, providing the corresponding chiral allyhc alcohols in both high enantio- and diastereoselectivities, as depicted in Scheme 3.10. The method employed the enzyme KRED 108 including an NADPH cofactor recycling system using KRED 104/2-propanol. 

An alternative was provided by Codexis that developed a route to a chiral precursor to ezetimibe based on the asymmetric biocatalytic reduction of 5-[(4S)-2-oxo-4-phenyl(l,3-oxazolidin-3-yl)]-l-(4-fluorophenyl)pentane-l,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxy-pentanoyl]-4-phenyl-l,3-oxazolidin-2-one (Figure 13.5a). Ketoreductases from LactohaciUus sp. identified as potential catalysts were improved via protein engineering and the best mutant was implemented in a process running at 100 g/1 with a coupled-enzyme cofactor recycling system (GDH and glucose), allowing formation of the alcohol with >99.9% ee [20]. 

A rare case of enzyme catalyzing imine reduction reaction (see also Section 13.4.4) is the stereoselective reduction of dihydrofolic acid to (6S)-tetrahydrofolic acid by dihydrofolate reductase (DHFR) at the expense of NADPH. This biocatalytic step was employed in the synthesis of (S)-leucovorin [(6S)-5-formyl-5,6,7,8-tetrahydro-folate], a drug used in cancer chemotherapy. DHFR produced by E. coli was combined with a GDH/glucose cofactor recycling system and yielded (6S)-tetrahy-drofolic acid, which upon formylation furnished L-leucovorin with >99.5% de (Figure 13.31) [37-39]. 

Nakamura K, Yamanaka R. Light mediated cofactor recycling system in biocatalytic asymmetric reduction of ketone. /. Chem. Soc. Chem. Commun. 2002 16 1782-1783. 

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. 

Technologies for recycle of cofactors in single-electron biocatalytic processes have been extensively studied and many reviews have been published (e.g. [50] and references therein). Clearly, if several redox enzymes are incorporated into... 

The highly selective biocatalytic reactions afford a substantial reduction in waste. The overall isolated yield is greater than 90%, and the product is more than 98% chemically pure with an enantiomeric excess of >99.9%. All three evolved enzymes are highly active and are used at such low loadings that counter-current extraction can be used to minimize solvent volumes. Moreover, the butyl acetate solvent is recycled with an efficiency of 85%.The E factor (kgs waste per kg product) for the overall process is 5.8 if process water is excluded (2.3 for the reduction and 3.5 for the cyanation) [47]. If process water is included, the E factor for the whole process is 18 (6.6 for the reduction and 11.4 for the cyanation). The main contributors to the E factor are solvent losses which accounted for 51% of the waste, sodium gluconate (25%), NaCl and Na2SO4 (combined circa. 22%). The three enzymes and the NADP cofactor account for <1% of the waste. The main waste streams are aqueous and directly biodegradable. 

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