Oxidoreductase cofactor immobilization

Glucose-grown cells of G. candidum SC 5469 have also catalyzed die stereoselective reduction of ethyl, isopropyl, and terdary butyl esters of 4-chloro-3-oxobutanoic acid and both methyl and ethyl esters of 3-bromo-3-oxobutanoic acid. A reaction yield of more than 85% and e.e. s of more than 94% were obtained. NAD -dependent oxidoreductase responsible for the stereoselective reduction of P-keto esters of 4-chloro- and 4-bromo-3-oxobutanoic acid was purified 100-fold. The molecular weight of purified enzyme is 950,000. The purified oxidoreductase was immobilized on Eupergit C and used to catalyze the reduction of 52 to 5-(—)-53. The cofactor NAD required for the reduction reaction was regenerated by glucose dehydrogenase. 

Other oxidoreductases (dehydrogenases) use electron acceptors other than oxygen, such as the cofactor NAD or NAD(P)". These cofactors maybe advan-t eously combined with electrochemical sensors, provided that the cofactor is externally supplied or immobilized and regenerated. More than 250 highly specific dehydrogenases are commercially available. Hydrolases are another class of readily available enzymes that can be combined with potentiometric sensors. Hydrolases catalyse the hydrolytic cleavage of C-O, C-N, C-C, and other bonds. Other biosensors can be fabricated from the new generation of hybrid and synthetic enzymes (17). 

Together with enantioselective hydrolysis/acylation reactions, enantioselective ketone reductions dominate biocatalytic reactions in the pharma industry [10], In addition, oxidases [11] have found synthetic applications, such as in enantioselective Baeyer-Villiger reactions [12] catalyzed by, for example, cyclohexanone monooxygenase (EC 1.14.13) or in the TEMPO-mediated oxidation of primary alcohols to aldehydes, catalyzed by laccases [13]. Hence, the class of oxidoreductases is receiving increased attention in the field of biocatalysis. Traditionally they have been perceived as difficult due to cofactor requirements etc, but recent examples with immobilization and cofactor regeneration seem to prove the opposite. 

Oxidoreductase enzymes include dehydrogenases, reductases, and oxidases that typically contain flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NAD(P)" ), or pyrroloquinohne quinone (PQQ) as redox cofactors. Although some specific enzymes possess cofactors with theoretically low redox potentials, this does not always translate to efficient performance when immobilized onto electrodes for BFCs. The caveat provides a need to study... 

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