A Potent Peroxidase from Solid Cell Culture of Ocimum Basilicum with High Sensitivity for Blood Glucose Determination

Abstract

Extensive applications of peroxidase (POX) have raised the global market demand at a considerable rate during the forecast period of 2020 - 2025. Nonetheless, the large-scale POX preparation still relies on the extraction from agricultural products, while there is an accumulative driving force toward employing biotechnological processes with agricultural hassle free identity. In pursuit of this trend, a novel heme peroxidase was purified to homogeneity (MW of 40 kD) from the callus culture of Ocimum basilicum L. in darkness on Murashige-Skoog medium supplemented by 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The highest activity of the purified peroxidase (ObPOX) was observed in Tris-base buffer at pH 7.5 and 80 °C. ObPOX showed high stability over pH(s) 5 to 7.5 and temperatures of 15 to 60 °C. ObPOX specific activity was 1245.142 AU mg-1 in the presence of phenol, 4 times higher than that of HRP. ObPOX showed moderate affinity for guaiacol (Km = 21.5 mM), but obtained an exceptionally high specificity constant (kcat/Km = 66743.7 s-1M-1) for GASA (4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid), the introduced substrate for determination of blood sugar. Applying ObPOX instead of HRP in glucose measurements of the real samples improved the regression constant of the correlation diagram between the tests and the lab results from 0.958 to 0.981. Physicochemical properties of ObPOX as well as the growth rate of basil callus (5.04 g L-1 per day) and the yield of ObPOX production (35 mg per 100 g dry biomass per subculture) designates O. basilicum cell culture for large-scale production of a robust peroxidase.