Biochemical and toxicological characteristics of polyphenol oxidase from red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract

Red palm weevil (RPW) Rhynchophorus ferrugineus is the most destructive insect pests of numerous palm species in the world. The introduction of botanical extract(s) as integral part of an integrated pest management (IPM) programs against RPW will reduce the use of chemical insecticides. Polyphenol oxidase (PPO) is one of the RPW innate immune mechanisms and inhibition of such enzyme could result in a disorder of the insect's immune system. A one single PO isoenzyme has been purified from the hemolymph of the 12th instar larvae of RPW. Using L-DOPA as substrate, R. ferrugineus PPO exhibited specific activity 428 Units/mg proteins with 8.3-fold purification, optimum pH and temperature for activity at 7.5 and 40 °C, respectively and is enhanced by Cu²⁺ with 1.76-fold. The rank order for oxidizing R. ferrugineus PPO different substrates is catechol > pyrogallol > L-DOPA > pyrocatechuic acid and not tyrosine. The kinetic parameters Km, Vmax and Vmax/Km for L-DOPA are 3.3 mM, 1.3 μmol/ml/min, and 0.39, respectively. The catalytic efficiency of the enzyme towards catechol is 5.3-fold higher than that for L-DOPA. The enzyme completely inhibited by thiourea, ascorbic acid, dithiothreitol, and SDS. R. ferrugineus PPO is a catechol oxidase di-phenol: O₂ oxidoreductase. Based on the toxicological studies of various botanical extracts, the IC₅₀ ranged from 20 to 90 mg/ml. The enzyme completely inhibited by 50 mg/ml Cinnamomum camphora. Gallic acid, the major phenolic compound, has IC₅₀ 0.8 mM and competitively inhibited the enzyme with Ki 0.54 mM. C. camphora could be a useful natural RPW-controlling agent and used as integral part in IPM programs. This interpretation can be validated in future through an in vivo investigation.