Horseradish Peroxidase (hrp - Dl Properties And Its Application In Environmental Testing

Horseradish peroxidase (HRP) is widely used in the fields of biological engineering, wastewater treatment and enzyme linked immunosorbent assay (Elisa). The studies of the properties, stress resistance and resistance protective agent of HRP are important.In order to provide a excellent nature of HRP, and then provide protection method of thermal stability of HRP, horseradish peroxidase (HRP-DL) is extracted and purified from the horseradish of Dalian Pulandian in this article. The enzymatic properties of HRP-DL are investigated. The stabilities of HRP-DL in a variety of inverse environment are studied. While taking advantage of the intrinsic fluorescence technique to investigate the relationship between the thermal stability and structure of HRP-DL. The protective effect a class of osmotic compensation solute on the HRP-DL stable has been investigated. This paper also explored the protection role of osmotic compensation solute on the HRP stability in Elisa determination.Optimum temperature of the HRP-DL is30℃, optimum pH of the HRP-DL is5.5. Ca2+, Mn2+, Zn2+, Pb2+, Mg2+which the concentration are5mmol/L on HRP-DL show activation effect;0.01mmol/L Cu2+and Hg2+,0.001mmol/L Ag+,4mmol/L Fe2+have the inhibited effect on HRP-DL.When HRP-DL is processed at the temperature greater than or equal to35℃, the residual enzyme activity is reduced, When HRP-DL is treated at60℃for15min by heating and adding Hectoine(1,4,5,6-tetrahydro-2-methyl-5-hydroxy-4-pyrimdine carboxylic acid, Hydroxyectoine), its relative enzyme is increased by14.43%. The results of intrinsic fluorescence spectrum analysis show that, Heating treatment cause the intrinsic tryptophan of HRP-DL exposing, changes in the structure led to the reduction of the residual enzyme activity, and to add Hectoine can maintain structure stability of HRP-DL. The activity of HRP-DL is significantly inhibited by NaCl, with the increase of salinity in the reaction system, enzymatic activity is gradually decreased by43.84%.When applicating ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimdine carboxylic acid) to the actual detection of the Elisa kit. Detection at high temperature (40℃), adding the ectoine than no adding, the OD450is increased by28.3%, it can protect the thermal stability of the HRP-DL, the determination accuracy of the Elisa test kit the interferencein of the high-temperature is improved. In the Elisa kits for detecting the antigen-antibody binding process, adding3%NaCl as the confounding factors, the resulting OD450is decreased by51.58%, the detection accuracy is interfered; to add ectoine as the protective agent, the OD450is improved by26.19%than no adding, the role of antigen-antibody binding is promoted, the interference of the salt is reduced, improving the accuracy of detection of the Elisa.