Analysis, purification, and characterization of peroxidases in rosemary using chromatography and spectrometry techniques

Rosemary (Salvia Rosmarinus) peroxidase was purified by chromatographic techniques, yielding an enzyme recovery of 35%. The estimated molecular mass was 47 kDa, determined by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and it has an isoelectric focusing point at 4.33. The optimal pH and temperature were 6.0 and 40ºC, respectively. Michaelis constant (Km) values were 3.7 mg mL-1, and the specific activity (SA) values were 28.10 U min-1 mL-1 towards guaiacol. The purified peroxidase activity was increased with calcium, ferric, magnesium, and manganese ions but decreased with copper, mercury, and zinc ions at all tested concentrations. The data indicate that this enzyme is a promising source of industrial peroxidase. Flame atomic absorption spectroscopy (FAAS) was used to detect metals in rosemary. To validate peroxidase levels in rosemary using high-performance liquid chromatography (HPLC), we would typically extract the enzyme, purify it (ammonium sulfate), and then separate and quantify it using an HPLC-UV method. The method usually involves using a C18 column and UV detection, with specific wavelengths chosen based on the peroxidase products. The mean LOD and LOQ methods, based on microgram injections, were determined to be 0.051 and 0.153 µg mL-1, respectively, using the standard deviation of blank peak areas from 10 blank samples (RSD% < 4%). The rosemary extract is injected into the HPLC, and the calibration curve identifies the peroxidase peak.