Enhancement of Activity of Thermophilic Inorganic Pyrophosphatase Ton1914 via Site-Directed Mutagenesis

Inorganic pyrophosphatase (PPase) is an enzyme that catalyzes the hydrolysis of pyrophosphate (PPi) into two phosphates. Ton1914, a thermophilic inorganic pyrophosphatase derived from <i>Thermococcus onnurineus</i> NA1, has good thermal stability and an extremely high optimum temperature and has been shown to reduce pyrophosphate inhibition. In this study, eight sites were selected based on sequence alignment and software calculations, and multiple single mutants were successfully constructed. After saturation and superposition mutations, six superior mutants were obtained. The enzyme activities of E97Y, D101K and L42F were increased 2.57-, 2.47- and 2.15-fold, respectively, while those of L42F/E97Y, L42F/D101K and E97Y/D101K were increased 2.60-, 2.63- and 1.88-fold, respectively, relative to the wild-type enzyme. Compared to Ton1914, all mutants more effectively increased PCR product quantity, reduced the number of qPCR cycles required to reach the threshold, and improved the efficiency of gene amplification. In the UDP-Galactose (UDP-Gal) synthesis reaction, the addition of mutants could further improve yield. When Ton1914 and mutants with the same activity were added, the yield of UDP-Gal was almost identical, effectively reducing the dosage of pyrophosphatase. Overall, the mutants showed greater prospects for industrial application.