Amidohydrolases are a large class of enzymes catalyzing the hydrolysis of a wide range of substrates bearing amide or ester functional groups at the carbon and phosphorus centers. Due to their functionality, amidohydrolases are applicable in various fields, starting from research assaying their role in metabolic pathways, such as the metabolism of certain amino acids and nucleotides, and ending with the usage of these enzymes in chemical synthesis, food, and cosmetic industry. Nevertheless, a remaining conundrum regarding the application of amidohydrolases in biotherapy is the targeted delivery of these enzymes to malignant cells or affected tissues. Therefore, the aim of this study was to develop modified variants of YqfB amidohydrolase which possess alterations in the amino acid sequence not interfering with its catalytic activity, but providing improved biotherapeutic applications.
Firstly, targeted mutagenesis of the sequence encoding YqfB amidohydrolase was performed by PCR using synthetic primers. Next, the mutant sequences were transformed into an Escherichia coli strain (HMS174∆pyrF∆yqfB), in which the protein expression was later induced using IPTG at 20 °C for 22 hours. The bacterial biomass was collected and lysed. The final steps included the collection of the soluble protein extract via centrifugation and the verification of enzyme activity by thin-layer chromatography using the soluble protein extract and N\(^{4}\)-acetylcytosine as a substrate. The results showed that modifications that were tested in the amino acid sequence of YqfB did not interfere with its enzymatic activity towards N\(^{4}\)-acetylcytosine. This not only indicated sites in the protein’s amino acid sequence amenable to modification without compromising enzymatic activity, but also opened up the possibility of more efficient application of modified YqfB variants in biotherapy, which requires further research.