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Article

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

1
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China
2
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130021, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Biomolecules 2025, 15(10), 1395; https://doi.org/10.3390/biom15101395
Submission received: 11 August 2025 / Revised: 28 September 2025 / Accepted: 29 September 2025 / Published: 30 September 2025

Abstract

Inorganic pyrophosphatase (PPase) is an enzyme that catalyzes the hydrolysis of pyrophosphate (PPi) into two phosphates. Ton1914, a thermophilic inorganic pyrophosphatase derived from Thermococcus onnurineus 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.
Keywords: inorganic pyrophosphorylase; thermophilic enzyme; site-directed mutagenesis; PCR enhancer; UDP-Gal synthesis inorganic pyrophosphorylase; thermophilic enzyme; site-directed mutagenesis; PCR enhancer; UDP-Gal synthesis

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MDPI and ACS Style

Liu, S.; Yang, X.; Gao, R.; Xie, G. Enhancement of Activity of Thermophilic Inorganic Pyrophosphatase Ton1914 via Site-Directed Mutagenesis. Biomolecules 2025, 15, 1395. https://doi.org/10.3390/biom15101395

AMA Style

Liu S, Yang X, Gao R, Xie G. Enhancement of Activity of Thermophilic Inorganic Pyrophosphatase Ton1914 via Site-Directed Mutagenesis. Biomolecules. 2025; 15(10):1395. https://doi.org/10.3390/biom15101395

Chicago/Turabian Style

Liu, Siyao, Xinrui Yang, Renjun Gao, and Guiqiu Xie. 2025. "Enhancement of Activity of Thermophilic Inorganic Pyrophosphatase Ton1914 via Site-Directed Mutagenesis" Biomolecules 15, no. 10: 1395. https://doi.org/10.3390/biom15101395

APA Style

Liu, S., Yang, X., Gao, R., & Xie, G. (2025). Enhancement of Activity of Thermophilic Inorganic Pyrophosphatase Ton1914 via Site-Directed Mutagenesis. Biomolecules, 15(10), 1395. https://doi.org/10.3390/biom15101395

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