Kinetic characterization of two neuraminic acid synthases and evaluation of their application potential.

Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH5, obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co2+, Mn2+ and Mg2+, was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH5, some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids. KEY POINTS: • A novel neuraminic acid synthase was characterized. • The effect of cofactors on NeuS activity was elucidated. • Kinetic and stability characterization of two neuraminic acid synthases was performed.

Mathematical model of the MenD-catalyzed 1,4-addition (Stetter reaction) of α-ketoglutaric acid to acrylonitrile.

The Stetter reaction, a conjugate umpolung reaction, is well known for cyanide-catalyzed transformations of mostly aromatic aldehydes. Enzymatic Stetter reactions, however, have been largely unexplored, especially with respect to preparative transformations. We have investigated the kinetics of the MenD-catalyzed 1,4-addition of α-ketoglutaric acid to acrylonitrile which has shown that acrylonitrile, while an interesting candidate, is a poor substrate for MenD due to low affinity of the enzyme for this substrate. The kinetic model of the reaction was simplified to double substrate Michaelis-Menten kinetics where the reaction rate linearly depends on acrylonitrile concentration. Experiments at different initial concentrations of acrylonitrile under batch, repetitive batch, and fed-batch reactor conditions were carried out to validate the developed mathematical model. Thiamine diphosphate dependent MenD proved to be quite a robust enzyme; nevertheless, enzyme operational stability decay occurs in the reactor. The spontaneous reactivity of acrylonitrile towards polymerization was also taken into account during mathematical modeling. Almost quantitative conversion of acrylonitrile was achieved in all batch reactor experiments, while the yield of the desired product was dependent on initial acrylonitrile concentration (i.e., the concentration of the stabilizer additive). Using the optimized reactor parameters, it was possible to synthesize the product, 6-cyano-4-oxohexanoic acid, in a concentration of 250 mM. The highest concentration of product was achieved in a repetitive batch reactor experiment. A fed-batch reactor experiment also delivered promising results, especially regarding the short reaction time needed to achieve a 200 mM concentration of product. Hence, the enzymatic Stetter reaction with a highly reactive acceptor substrate can be performed on a preparative scale, which should enable similar transformations with acrylate, methacrylate, and methyl vinyl ketone.

Complete starch hydrolysis by the synergistic action of amylase and glucoamylase: impact of calcium ions.

Starch hydrolysis was performed by the synergistic action of amylase and glucoamylase. For that purpose glucoamylase (Dextrozyme) and two amylases (Liquozyme and Termamyl) in different combinations were investigated. Experiments were carried out in the repetitive- and fed-batch modes at 65 °C and pH 5.5 with and without the addition of Ca(2+) ions. 100 % conversion of starch to glucose was achieved in batch experiments. Calcium ions significantly enhanced stability of the amylase Termamyl. The intensity of synergism between amylase Termamyl and glucoamylase Dextrozyme was higher than in the experiments carried out with amylase Liquozyme and Dextrozyme. Mathematical model of the complete reaction system was developed. Using the model, a possible explanation of the synergism between the amylase and glucoamylase was provided.

Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis: effect of calcium, pH and temperature.

The first step of starch hydrolysis, i.e. liquefaction has been studied in this work. Two commercial α-amylases from Bacilllus licheniformis, known as Termamyl and Liquozyme have been used for this purpose. Using starch as the substrate, kinetics of both enzymes has been determined at optimal pH and temperature (pH 7, T = 80 °C) and at 65 °C and pH 5.5. Michaelis-Menten model with uncompetitive product inhibition was used to describe enzyme kinetics. Mathematical models were developed and validated in the repetitive batch and fed-batch reactor. Enzyme inactivation was described by the two-step inactivation model. All experiments were performed with and without calcium ions. The activities of both tested amylases are approximately one hundred times higher at 80 °C than at 65 °C. Lower inactivation rates of enzymes were noticed in the experiments performed at 65 °C without the addition of calcium than in the experiments at 80 °C. Calcium ions in the reaction medium significantly enhance amylase stability at 80 °C and pH 7. At other process conditions (65 °C and pH 5.5) a weaker calcium stabilizing effect was detected.