engleski

Biocatalytic synthesis of an aldol product, a precursor of D-fagomine

Iminosugars have attracted a great interest among scientists due to their efficient inhibition of various glycosidases involved in the intestinal degradation of carbohydrates. D- Fagomine is a naturally occurring iminosugar which showed inhibitory activity towards mammalian glucosidase and galactosidase. This compound also reduces the risk of developing insulin resistance and of becoming overweight. Small quantities of D-fagomine can be found in plants, but higher quantities are obtained by chemoenzymatic synthesis. One of the precursors of D-fagomine is (3S, 4R)-6-[(benzyloxycarbonyl)amino]-5, 6- dideoxyhex-2-ulose and its synthesis was studied in this dissertation. This precursor of D-fagomine can be synthesized by aldol addition of dihydroxyacetone to N-Cbz-3-aminopropanal catalyzed by D-fructose-6-phosphate aldolase. Two different aldolase variants, FSA A129S i FSA A129S/A165G, were tested as biocatalysts in the reaction of aldol addition. Enzymes were kinetically characterized and aldol addition was carried out in different reactor types ; batch reactor, ultrafiltration membrane reactor and microreactor to determine the most efficient reactor type. The use of microreactors in this field presents a novelty, since aldol addition of DHA to N-Cbz-3- aminopropanal catalyzed by D-fructose-6- phosphate aldolase has not been carried out in microreactor until now. Considering that the aminoaldehyde N-Cbz-3- aminopropanal is unstable, its synthesis from the corresponding aminoalcohol, N-Cbz-3- aminopropanol, was investigated. This reaction was catalyzed by alcohol dehydrogenase. It is an oxidoreductase which catalyzes the reversible oxidation of alcohols to corresponding aldehydes or ketones. This enzyme requires coenzyme NAD+ for its catalytic function, and therefore, in situ regeneration system was studied since the use of coenzymes in stoichiometric quantities is too expensive. Various methods can be used for the regeneration of coenzymes, and a simple approach is an enzyme-coupled regeneration in which a second enzyme is added to the reaction system. An example of this method, for the regeneration of NAD+, is the use of NADH oxidase which was studied in this dissertation. One of the main advantages of using NADH oxidase for coenzyme NAD+ regeneration is that it catalyzes an irreversible reaction which then shifts the equilibrium of the primary reaction to the side of the product formation. Alcohol dehydrogenase and NADH oxidase were isolated and partially purified for the use in the cascade reaction of alcohol oxidation with coenzyme regeneration and aldol addition. Alcohol dehydrogenase was isolated from baker’s yeast and optimization of cell disruption process was carried out using a statistical method. Cell disruption time, glass beads diameter, glass beads mass and ultrasound amplitude were optimized to achieve maximum volume activity of alcohol dehydrogenase. NADH oxidase was isolated from Lactococcus lactis. After isolation and partial purification of enzymes, cascade reaction of alcohol oxidation and aldol addition with coenzyme regeneration was carried out. Aldol product was not obtained in the reaction with alcohol dehydrogenase from baker’s yeast and hence alcohol dehydrogenase from horse liver was used. With this enzyme as biocatalyst for the reaction of N-Cbz-3- aminopropanol oxidation in the cascade reaction, aldol product was formed. Since the yield on aldol product was low (25%), optimization of initial conditions of cascade reaction was carried out using a statistical method. Concentrations of coenzyme NAD+, alcohol dehydrogenase and D-fructose-6- phosphate aldolase were optimized to achieve maximum yield on aldol product. Yield on aldol product was increased to 79%.

alcohol dehydrogenase; aldolase; aldol product; cascade reaction; enzyme isolation; NADH oxidase; optimization of initial process conditions

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