Isolation and characterization of peroxidase from potato leaves Solanum tuberosum: Application in glucose diagnostic kit

• Potato leaves peroxidase (PLPOD) was purified through two chromatographic steps. • PLPOD achieved 11.8-fold purification times with a specific activity of 705.7 U/mg. • PLPOD molecular weight was ranging from ∼ 40–60 kDa. • PLPOD exhibited optimal activity at pH 5.2, with Zn 2+ and Ni 2+ enhancing activity. • PLPOD was applied in constructing a glucose diagnostic kit. Peroxidases play a pivotal role in many medical applications such as diagnostic kits and ELISA assays. This study reports the purification and biochemical characterization of peroxidase from potato leaves (PLPOD) and its application in the formulation of a glucose diagnostic kit. PLPOD was purified through CM-cellulose ion-exchange and Sephacryl S-300 gel filtration chromatography, achieving an 11.8-fold purification with 48 % recovery and a final specific activity of 705.7 U/mg. Native PAGE and activity staining confirmed the enzyme’s purity and homogeneity. PLPOD molecular weight was estimated from gel filtration column as 64 kDa, but on SDS gel, there were three PLPOD isoforms of approximated molecular weights ranging from ∼ 40–60 kDa. PLPOD exhibited optimal activity at pH 5.2, with Zn 2+ and Ni 2+ enhancing activity, while Ca 2+ and Fe 2+ inhibited it. Inhibitor analysis confirmed the heme-dependent nature of the enzyme. The K m values for guaiacol and H 2 O 2 were 0.067 mM and 40 mM, respectively, consistent with typical plant peroxidases. A glucose diagnostic kit developed using PLPOD showed strong concordance with a commercial glucose kit when tested on normal and diabetic serum samples demonstrating its clinical applicability. These findings suggest that PLPOD is a viable cost-effective for use in diagnostic assays and kits.