Improvement in the Thermal Stability of Pyrophosphatase by Conjugation to Poly(N‑isopropylacrylamide): Application to the Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a powerful method for nucleic acid amplification. However, the PCR is inhibited in its yield due to its byproduct, pyrophosphate (PPi), a byproduct of the reaction; the yield is thereby limited. The conventional method for hydrolysis of PPi by pyrophosphatase (PPase) is not well adapted for operation at elevated temperatures over long times as required during the PCR. In this work, we reported a strategy to improve the PCR yield using a conjugate of the enzyme with the thermally responsive polymer poly(N-isopropylacrylamide) (PNIPAM). Pyrophosphatase (PPase) was conjugated to PNIPAM site-specifically near the active center. As compared to the free enzyme, the optimum temperature of the conjugate was shown to increase from 45 to 60 °C. For the conjugate, about 77% enzyme activity was retained after incubation at 60 °C for 3 h, representing a 6.8-fold increase as compared to the unconjugated enzyme. For the PCR using the conjugate, the yield was 1.5-fold greater than using the unconjugated enzyme. As well as improving the yield of the PCR (and possibly other biological reactions) at elevated temperature, polymer conjugation may also provide a strategy to improve the heat resistance of proteins more generally.