Construction of artificial metalloenzymes by protein refolding

Abstract Hemin was packed into the inside of bovine serum albumin by protein refolding. The as-prepared artificial hemeprotein was characterized by UV-vis, CD, and MALDI-TOF-MS, which exhibited peroxidase-like activities with an optical pH at pH 7.0 and an optical temperature at 65°C. The Michaelis-Menten constant (Km) and maximum velocity (Vm) with guaiacol for the as-prepared hemeprotein was calculated to be 0.131 mM and 0.166 mM/s, which was significantly lower compared with the value of 39.7 mM and 44.4 mM/s of native horseradish peroxidase. The artificial hemeprotein had much more affinity to the guaiacol than HRP, and had lower guaiacol oxidation efficiency than HRP.