ИСТИНА

Horseradish peroxidase (HRP) is one of the most widely used enzyme in analytical biochemistry and medicine diagnostics. Chemiluminescent reaction of luminol oxidation catalysed by HRP provides very high sensitivity of analysis. Drawbacks of this detection system are fast decrease of chemiluminescent signal due to enzyme inactivation and use of special compounds (enchancers) to increase intensity of signal. Compared to HRP, tobacco peroxidase (TOP) provides higher signal in reaction of chemiluminescent luminol oxidation and it does not require use of enchancers, but there is still rather fast signal decay during reaction. Analysis of crystal structure of HRP and TOP showed resulted in selection of Phe142 and Phe179 residues in HRP and Phe140 and Thr151 residues in TOP. Amino acid changes Phe142Tyr, Phe142Trp and Phe1792Tyr, Phe179Trp were introduced into HRP amino acid sequence as well as single substitutions Phe140Tyr, Thr151Trp and double Phe140Tyr/Thr151Trp were done in TOP. Mutant enzymes were expressed in E.coli cell. After refolding procedure mutant HRP Phe142Tyr, HRP Phe142Trp and HRP, TOP Phe140Tyr, TOP Thr151Trp and TOP Phe140Tyr/Thr151Trp were obtained from inclusion bodies as active enzymes. HRP with mutation Phe179Trp could not be reactivated. Substrate specificity and thermal stability of mutant HRP and TOP were studied. It was found that all three mutant HRP Phe142Tyr, HRP Phe142Trp and HRP as well as two mutant TOP Phe140Tyr and TOP Phe140Tyr/Thr151Trp showed higher thermal stability compared to wild-type enzymes. Optimal conditions for chemiluminescent luminol oxidation reaction catalysed by mutant HRPs and TOPs were determined. In the case of one mutant HRP Phe142Tyr and two mutant TOP Phe140Tyr and TOP Phe140Tyr/Thr151Trp more stable signal was observed compared to wild-type enzymes.