Biotechnical production and use of pyruvic acid with special reference to coenzyme regeneration: Dissertation

Pyruvic acid is a key intermediate of microbial carbohydrate metabolism. It can be used as a starting material for production of amino acids, pharmaceuticals and pesticides. It is also possible to utilize pyruvic acid itself as a flavoring agent. In this work the enhancement of pyruvic acid secretion by Candida utilis IFO 0396 was studied with reference to coenzyrne regeneration and metabolic modifications. The coenzyme dependent conversion of pyruvate to L alanine was investigated with immobilized and free enzymes. High glucose concentration was shown to be beneficial for pyruvic acid production. Oxygen availability, affecting the redox balance in the cells, was a critical factor. In microbial metabolism pyruvic acid is formed at the end of the glycolytic sequence, which operates only in the presence of sufficient amounts of the oxidized coenzyrne NAD+. The pathway of coenzyme regeneration corresponded with the extracellular redox potentials measured. The highest pyruvic acid yield observed in this study, 0.33 g (g glucose)~l, was obtained using a decreasing aeration profile. In these conditions pyruvic acid was also produced independently of cell growth. Ammonium was shown to be a better nitrogen source for pyruvic acid production than nitrate. By addition of optimal concentrations of arsenate or dinitrophenol (uncouplers of oxidation and phosphorylation) or arsenite (blocking pyruvate degradation by the pyruvate dehydrogenase complex) pyruvic acid production could be enhanced. The most efficient metabolic modifier was acetoin, which was stoichiometrically reduced to 2,3 butanediol. Acetoin reductase, which catalyzes this reaction, was shown to be predominantly NADH dependent. The highest pyruvic acid productivity, 0.5 g l lh ', was obtained by acetoin addition. Ethanol was the dominant by product in pyruvic acid production. Glycerol was produced under strongly reducing conditions. Minor amounts of other carboxylic acids, amino acids and volatile compounds were also produced. Pyruvate decarboxylase, catalyzing the first step of pyruvate degradation to ethanol, was shown to be present even in conditions where no ethanol could be detected. Clear correlation between ethanol concentration and the specific activity of pyruvate decarboxylase was not observed. The amination of pyruvate to L alanine, catalyzed by alanine dehydrogenase, requires NADH. NADH could be oxidized in the production of carbon dioxide from formate by formate dehydrogenase. Two different methods, droplet gel entrapment and covalent binding to tresyl chlorideactivated agarose, were examined for the coimmobilization of these two dehydrogenases. The kinetic constants as well as optimal conditions for continuous reactors applying these coimmobilized enzymes were determined. In long term operation (30 days) the highest L alanine productivity per unit of alanine dehydrogenase, conversion and total turnover number of the coenzyme (TIN) were obtained using free enzymes. However, using the droplet gel immobilized biocatalyst the decrease of both the conversion ratio and the TTN value were smallest during long term operation indicating enchanced stability of the enzymes. In this system 60 % of the maximum production of L alanine remained after 30 days, in comparison with 10 % when covalently boud enzymes were employed. Using free enzymes 80 % of the alanine produced was obtained during the first five days, after which the production rate slowed down considerably.