Cuticular Polyphenoloxidase From The Silkworm, Bombyx Mori; Purification And Characterization

Insects and hardening of the insect cuticle, pigment formation, wound healing, as well as resist infection, such as pathogenic micro-organisms play an important role in the process. And is catalyzed by a series of phenoloxidase phenols were generated quinones redox material, after the non-quinone oxidation-reduction reaction to generate the final response to the process of melanin pigment, phenoloxidase is the key enzyme in this process (phenoloxidase, PO) . Phenoloxidase including tyrosinase-type (EC1.14.18.1) and laccase-type phenoloxidase (EC1.10.3.2). In this study, silkworm (Bombyx mori) cuticle for material phenoloxidase isolation, identification and characterization studies, can be generated for the study of insect cuticle, immunization and the target enzyme for inhibitors of new pests and provide a theoretical basis. The major findings of this study are as follows:1. Bombyx cuticle isolation and identification of phenoloxidase. The results showed that: tyrosinase-type phenoloxidase enzyme activity at 20-30% saturation ammonium sulfate, the salting-out precipitation of proteins has been the highest enzyme activity; Laccase-type phenoloxidase enzyme activity at 40-70% saturation ammonium sulfate, the salting-out precipitation of proteins has been the highest enzyme activity. And again SephadexG-200 gel filtration chromatography of the silkworm cuticle purified phenoloxidase. Purified by two steps, tyrosinase-type phenoloxidase enzyme activity was 39.70-fold; laccase-type phenoloxidase enzyme activity was 4.94-fold.2. Phenoloxidase study the basic properties. The results showed that: the optimum pH was 7.0 and the best temperature was 30℃for the tested silkworm cuticle tyrosinase-type phenoloxidase. Thermal stability and strong; The optimum pH was 5.0 and the best temperature was 60℃for the tested silkworm cuticle laccase-type phenoloxidase. Poor thermal stability; Tyrosinase-type phenoloxidase-catalyzed catechol Km and Vm were 2.73mmol·L^-1 and 0.91μmol·L^-1·min^-1. Laccase-type phenoloxidase-catalyzed ABTS Km and Vm were 0.327mmol·L^-1 and 0.376μmol·L^-1·min^-1. Low concentration of metal ions Ag+, Cu2+, Mn2+, Zn2+ on the silkworm cuticle tyrosinase and laccase-type phenoloxidase activation must have, and must have a high concentration of its inhibitory effect.3. Kinetics of ascorbic acid inhibition. Ascorbic acid can inhibit the tyrosine silkworm cuticle and laccase-type phenol oxidase activity, which I50 were 175μM and 190μM. Catechol as a substrate, ascorbic acid tyrosine on the silkworm cuticle-type phenoloxidase showed irreversible inhibition; ABTS as a substrate, ascorbic acid on the silkworm cuticle laccase-type phenoloxidase showed irreversible inhibition.4. Inhibition Kinetics of citric acid. Citric acid tyrosine can inhibit epidermal silkworm and laccase-type phenol oxidase activity, which I50 were 400μM and 720μM. Catechol as a substrate, citric acid tyrosine are bombycis epidermal-type phenoloxidase competitive inhibitor; ABTS as a substrate, citric acid is a laccase-type silkworm cuticle phenoloxidase competitive inhibitors.