Enzymes are biocatalysts that exhibit numerous advantages over chemical catalysts. Despite this, enzymes face limitations in industrial applications, which is why efforts are made to modify and improve them using enzyme engineering techniques. One of the key reactions for which enzymes are being modified is the synthesis of carbon-carbon bonds. Aldol condensation is the most common method for the synthesis of these bonds, and this reaction is catalyzed by enzymes called aldolases. N-acetylneuraminic acid synthase (NeuS) is a phosphoenolpyruvate-dependent aldolase that catalyzes the synthesis of N-acetylneuraminic acid (Neu5Ac). Neu5Ac belongs to the family of sialic acids, which have various functions in physiological processes. In this study, the activity of analogs of N-acetylneuraminic acid synthase was tested on sugars L-arabinose, D-galactose, D-lactose, D-lyxose, D-(+)-mannose, and D-(+)-xylose as substrates. The analogs of N-acetylneuraminic acid synthase used were the artificially obtained PNH3, PNH6, PNH11 and PNH17 enzymes, as well as the NmNeuS enzyme from the bacterium Neisseria meningitidis (Nm). The activity of these enzymes was tested using initial reaction rates by monitoring the concentrations of reactants and reaction products over time using high-performance liquid chromatography (HPLC), and the substrate conversions and reaction yields were calculated from the collected experimental data. Based on the results obtained, it was concluded that the PNH3 and PNH6 enzymes have greater activity towards hexoses (D-galactose, D-(+)-mannose) as substrates than towards pentoses (L-arabinose, D-lyxose). In the case of the NmNeuS cell extract, the reverse was true. NmNeuS showed the highest values of conversion and yield for all substrates. By comparing the yields of reactions catalyzed by the NmNeuS cell extract enzyme with its purified form, it was concluded that the purified enzyme is more specific towards substrates than the NmNeuS cell extract enzyme.