Purification and Characterization of Peroxidase from Thai Black Glutinous Rice (Oryza sativa L. cv. Luem Pua)

Abstract

Plant peroxidases are oxidoreductases that play important roles in several plant metabolic processes and are also useful in many applications. Peroxidase is involved in enzymatic browning, change of bioactive phytochemicals, and nutritional value which are the limiting factors for post-harvest process and shelf-life of various rice and cereals. Therefore, characteristics of peroxidase from Thai Black Glutinous Rice (Oryza sativa L. cv. Luem Pua) which is a popularly consumed variety in Northern Thailand were investigated. The rice seeds were germinated for 7 days, followed by crude enzyme extraction. The extract was further separated using two different processes including ammonium sulfate precipitation at 80% saturation and aqueous two-phase extraction. Toyopearl CM-650 cation exchange chromatography was selected for Luem Pua rice peroxidase purification. Elution chromatographic profiles of two purification procedures showed similar pattern that at least two cationic peroxidase isozymes were revealed. Peroxidase activity was assayed using guaiacol as a substrate, while protein content was determined using Bradford assay. Results showed specific activity of purified peroxidase obtained by ammonium sulfate precipitation and Toyopearl CM-650 column of 12.97 unit/mg protein with purification fold of 2.60 and enzyme recovery of 5.67%. While, purified peroxidase obtained by aqueous two-phase extraction and Toyopearl CM-650 column showed specific activity of 1.04 unit/mg protein with purification fold and enzyme recovery of 0.45 and 3.44%, respectively. The major isoform purified by aqueous two-phase partitioning and Toyopearl CM-650 chromatography was analyzed for its physical and catalytic characterization. The molecular weights of purified peroxidase estimated by size exclusion chromatography and SDS-PAGE were 37.5 and 37.6 kDa, respectively. Purified peroxidase showed maximum activity at pH 7.0 with optimum temperature of 30 °C. After incubation for 5 hours, the enzyme showed the highest percentage of residual activity at pH 11.0 with thermal stability between 30 °C and 50 °C. Activity of peroxidase rapidly decreased between pH 2.0 and pH 3.0, while temperature at 70 °C inactivated the enzyme within the first hour. Furthermore, the purified enzyme showed the highest affinity with o-phenylenediamine, followed by o-dianisidine and guaiacol, considering Km values of 10.21 mM, 15.65 mM, and 302.82 mM, respectively. Whereas, guaiacol provided the maximum velocity of purified enzyme with Vmax value of 0.4050 unit/ml. The major isoform of peroxidase purified from seedling of Oryza sativa L. cv. Luem Pua can be inactivated by acidic condition and temperature above 70 °C. This enzyme also has specificity for catalysis when phenolic compounds are used as substrates.