Investigation of the thermal stability of plant Lepidium draba L. peroxidase at different temperatures

Peroxidases are enzymes found in various organisms such as animals, plants and microorganisms. These enzymes are oxidoreductases that can use various compounds as hydrogen donors in the presence of hydrogen peroxide and are widely used in the decomposition of pollutants, wastewater treatment and in biosensors. The main purpose of this study was to investigate the thermal stability of plant Lepidium draba L. peroxidase (EC ۱.۱۱.۱.۷) at different temperatures. In this study, after the expression of the recombinant enzyme in liquid LB medium in the presence of IPTG, its purification was performed using ۵- nickel- sepharose affinity chromatography column and a separating buffer containing imidazole. The Protein purity was determined using SDS-PAGE and its concentration was measured using Bradford method. In order to evaluate the thermal stability, the enzyme with the concentration of ۰.۵ mg/ml was incubated in the temperature range of ۳۰ to ۸۰ °C for ۱۰ minutes and then it was kept at room temperature for ۵ minutes. Thereafter, the activity of enzyme was measured using H۲O۲ and TMB as the enzyme substrates in potassium phosphate buffer (pH=۷) at ۶۵۳ nm using a spectrophotometer (carry۶۰). The results showed that the activity of the enzyme was higher in the temperature range of ۳۰ to ۴۰ °C and its activity decreased with increasing temperature. So that at the highest temperature (۸۰ °C) the enzyme maintained about ۵۳% of the initial activity. The application of mutagenesis methods is suggested to increase the thermal stability for using of this enzyme in higher temperatures.

کلیدواژه ها:

Activity ، H۲O۲ ، Purification