Expression and enzymatic properties of pyrophosphatase in Escherichia coli

The pyrophosphatase (PPase) gene of Escherichia coli Rosetta (DE3) was amplified and cloned by polymerase chain reaction (PCR), the pyrophosphatase expression vector pET-28a-ppa was constructed by seamless cloning technique, transformed into competence E. coli Rosetta (DE3), and the recombinant strain E. coli PPase was constructed. The induced expression conditions were optimized by single factor tests, recombinant PPase was purified by nickel ion magnetic beads, and the enzymatic properties were studied. The results showed that the recombinant strain E. coli PPa was successfully constructed. The recombinant PPase activity reached the highest level (129.05 IU/ml) under the conditions: isopropyl-β-d-thiogalactoside (IPTG) induction concentration, induction temperature, and induction time were 20 ℃, 0.4 mmol/L, and 8 h, respectively, which was 3.14 times higher than that before optimization. The theoretical molecular mass of recombinant PPase was 19.7 kDa. The optimal reaction temperature and pH were 55 ℃ and 9 ℃, respectively, and stable in the range of temperature 10-30 ℃ and pH 7.0-9.0. Metal ions K⁺, Mg²⁺, and Na⁺ could significantly enhance the enzyme activity (P<0.01), while Mn²⁺ and Zn²⁺ significantly inhibited the enzyme activity (P<0.01). The kinetic parameters of the recombinant PPase were as follows: Michaelis constant (K_m value) 3.58 μmol/ml, maximum reaction rate (V_max value) 314.66 μmol/(mL·min), catalytic number (K_cat value) 87 300.56 S^-1, and half-inactivation time 10 min. This study provided a basis for the subsequent industrial production of PPase and rational design of the enzyme.