چكيده لاتين
Terminal deoxynucleotidyl transferase (TdT) is an unconventional and unique DNA polymerase that, unlike most polymerases, does not require a template strand for its polymerization reaction. This enzyme is capable of non-template-dependent addition of deoxynucleotides to the 3ʹ end of a DNA strand. This unique characteristic makes TdT a powerful and versatile tool in biotechnology, finding widespread use in aptamer design, DNA nanostructure construction, molecular labeling, and polynucleotide synthesis.
The structural stability and catalytic activity of the TdT enzyme are critical factors for its successful and optimal utilization in industrial and research applications. Maintaining its proper function is especially important when the enzyme is exposed to high temperatures, osmotic pressure, or chemical additives. In this regard, glycerol, a three-carbon compound with osmolytic properties, has long been used as a protein structure stabilizer in various laboratory environments, and its effects on enzyme activity have attracted researchersʹ attention.
In the present study, aiming to investigate the effect of glycerol on TdT enzyme activity, the enzyme was first expressed and purified under laboratory conditions. After assessing its purity and confirming its molecular weight, the Michaelis-Menten constant (Km) was determined for the enzyme with respect to its iDNA and dNTP substrates. Subsequently, the effect of three different glycerol concentrations (20%, 40%, and 60%) on TdT activity was investigated to reveal the potential relationship between the concentration of this additive and enzyme efficiency.
The results of this research indicated that a low concentration of glycerol (20%) led to a decrease in TdT enzyme activity, while an intermediate concentration (40%) showed a mild and limited positive effect on enzyme performance. Furthermore, a higher concentration of glycerol (60%) did not show a significant increase or decrease in enzyme activity, suggesting that its presence at this level has a neutral effect on TdT activity. These findings can contribute to optimizing the storage and usage conditions of the TdT enzyme in biological reaction environments and DNA synthesis, and can be effective in better designing molecular systems based on this enzyme.
