Abstract
Deoxynucleoside triphosphates (dNTP) are essential biological materials for synthesizing DNA. The compounds are also used in molecular biotechnology such as the polymerase chain reaction (PCR) and other DNA polymerase-based applications. Nowadays, dNTPs are commercially produced by chemical synthesis due to their low concentrations in living organisms. However, the chemical synthesis method utilizes toxic solvents and the yield is low. To solve these problems, this study attempt to establish an in vitro enzymatic method for complete biosynthesis of dNTP. The process involves two stages of enzymatic phosphorylation procedures. The deoxynucleoside monophosphates (dNMPs) are used in the first step. The products of the first and second stages are deoxynucleoside diphosphates (dNDP) and dNTP, respectively. Five genes from the Thermus thermophilus HB8, which encode four kinds of deoxynucleoside monophosphate kinases (NMKs) and one nucleoside diphosphate kinase (NDK), were cloned and expressed in high quality in E. coli BL21 (DE3) and NovaBlue (DE3). We have performed functional and kinetic property analysis of NMKs and NDK. The results showed that the five enzymes exhibited optimum activity at the pH value of 7.0 to 8.0 and at the temperature between 75OC to 80 OC. Furthermore, all of them displayed excellent thermal stability. Approximate 80% and 50% of enzyme activities were retained after incubation at 75 OC for 3 hr and 24hr, respectively. The kinetic property analysis showed that five enzymes have higher catalytic efficiency under optimum environment as compared to the mesophilic bacteria. Later, we combined these crude enzymes preliminarily to produce dNTP. The PCR performed using the enzymes-synthesized dNTP showed the same efficiency compared to the commercial one. Finally, this system provides a clean and better method of preparation that has potential for commercialization.