International Science Community Association Research Journal of Recent Sciences 15 1 2026 01 2 Peroxidase-Mediated Thymoquinone Biosynthesis in Nigella sativa: A pH-Dependent Enzymatic Insight 5 10 EN Ingale Vaishnavi Anilrao 1 Department of Botany, Faculty of Mycology, Shri Shivaji Science College, Amravati, Maharashtra, India Hande Dilip Vinayakrao 2 Mahatma Fule Arts, Commerce & Sitaramji Chaudhari Science College, Warud, District Amravati, Maharashtra, India Gyaneshwari Upagya 1 Department of Biotechnology, School of Life Sciences, Mahatma Gandhi Central University, Motihari-845401, India Pandey Brijesh 2 Department of Biotechnology, School of Life Sciences, Mahatma Gandhi Central University, Motihari-845401, India > Research Paper 2025 11 30 2026 01 2 Nigella sativa (N. sativa) is widely recognized for its pharmacological potential, largely attributed to thymoquinone (TQ), a key bioactive compound. Although TQ biosynthesis has been reported in various plants and microorganisms, its enzymatic pathway in N. sativa remains inadequately characterized. This study investigates the role of peroxidase enzymes in TQ biosynthesis, using thymol-a known precursoras the substrate, hypothesizing that the antioxidant capacity of N. sativa arises from robust enzymatic defense mechanisms. Peroxidase activity was evaluated in the leaves, buds, and fruits of the Rajendra Shyama cultivar using 1% hydrogen peroxide (H₂O₂) in Tris buffer at different pH 6.5, 7.0 and 7.5. Absorbance analyses at 254 nm and 274 nm were used to monitor TQ and thymol formation, respectively. Results revealed plant part-specific variations in enzymatic activity, with leaves unveiling the highest and fruits the lowest peroxidase activity. Optimal activity was observed at pH 6.5, which suggests a preference for slightly acidic to neutral conditions for effective TQ biosynthesis. Notably, differential absorbance patterns and correlation studies indicate the possible involvement of metabolites other than thymol in the biosynthetic pathway. These findings provide new insights into the enzymatic dynamics of N. sativa, with significant implications for therapeutic exploitation and metabolic engineering. Copyright@ International Science Community Association