A trypsin-solubilized laccase from pharate pupal integument of the tobacco hornworm, Manduca sexta

Abstract

A laccase-like enzyme was solubilized from Manduca sexta pharate pupal integument by trypsin treatment and partially purified by ammonium sulfate precipitation, gel filtration, ultracentrifugation and anion exchange high performance liquid chromatography (HPLC). The molecular weight of the laccase as determined by gel filtration HPLC was 250 ± 30 kDa. The most purified preparation contained a single oxidase with typical laccase activity as determined by substrate and inhibitor specificities, enzyme kinetics, and a single band of enzyme activity on nondenaturing polyacrylamide gels with syringaldazine (SYR) and 3,4-dihydroxyphenylalanine (DOPA) substrates. The specific activity of the most purified enzyme preparation was 18.0 μmol of quinone and 1.1 μmol of ϱ-quinoneimine formed min −1 mg −1 from 2-methylhydroquinone (MHQ) and DOPA, respectively, at pH 6. The pH optimum of the laccase was 6 for MHQ and about 7 for N-acetyldopamine. Incubation of the M. sexta laccase with selective laccase and tyrosinase inhibitors demonstrated that the enzyme is generally inhibited like fungal laccase with p-diphenolic substrates and like mushroom tyrosinase with o-diphenolic substrates. Manduca laccase affinity for MHQ was 400-fold greater than that for DOPA and the catalytic efficiency ( V max K m ) for oxidation of the p-diphenol was 2000-fold higher than that for the o-diphenol. Topical application of SYR to pharate pupal forewing cuticle revealed laccase activity in situ. The natural tanning precursor, N- β-alanyldopamine (NBAD), was the best o-diphenolic substrate tested. N- β-Alanylnorepinephrine was also formed at a high rate when NBAD was incubated with M. sexta laccase, but at a low rate with fungal laccase, suggesting specificity for side chain oxidation through a p-quinone methide intermediate. The results indicate that M. sexta laccase is important for oxidizing cuticular o-diphenols to o-quinone and p-quinone methide tanning agents for sclerotization of the M. sexta pupal exoskeleton.