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Regeneration of oxidized form of coenzyme with addition of co-substrate

Dehydrogenases are enzymes, which need so called "free" coenzyme for their catalytic activity. The coenzyme acts as a second substrate in the catalytic reaction [1]. These biocatalysts are used to obtain chiral products such as amino acids [2] or alcohols [3], from their non-chiral precursors, as well as to obtain aldehydes in the first step of the cascade reactions to obtain chiral 2-hydroxy ketones or precursors for iminocyclitols. To use the coenzyme economically and successfully in the biocatalytic reaction, which also implies shifting of the reaction equilibrium towards formation of the product ; it is necessary to regenerate the coenzyme in situ. From the literature [1] that describes a range of methods of coenzyme regeneration, it is clear that the problem of regeneration of oxidized form of coenzyme (NAD+) is still not successfully resolved. This study used a method of regeneration of NAD+ by adding co-substrate in the reaction medium. Acetone was used as a co-substrate. As a model reaction oxidation of hexanol was carried out. Alcohol dehydrogenase (YADH), which was isolated from baker's yeast, was used as a biocatalyst. This enzyme needs coenzyme: nicotinamide adenine dinucleotide NAD(H) for its catalytic activity. The enzyme was isolated by the method described in the literature [4] and kinetically characterized by initial reaction rate method. The kinetic parameters were estimated from the experimental data for this enzyme using nonlinear regression. According to the estimated kinetic parameters, it can be concluded that the reaction product: hexanal significantly inhibits the reaction rate and acts as inhibitor of the enzyme. Using experimental data, a formal kinetic model and the model of batch reactor were developed. Using the model, the process in the batch reactor was simulated, and the amount of biocatalyst required for the implementation of reaction was estimated. Experimental results of the implementation of biocatalytic oxidation of hexanol are in good agreement with the results of simulation of processes by model, which indicates the validity of the model and estimation of kinetic parameters. Oxidation of hexanol was carried out in a reactor volume of 10 ml, temperature 30 ° C and pH 8.8 in 0.1 M pyrophosphate buffer.

alcohol dehydrogenase; hexanol oxidation; yeast

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