Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore

Aphids are among the most serious insect pests of agricultural crops in the world. They often have specific hosts, and the cabbage aphid (Brevicoryne brassicae) is a specialist on Cruciferae. It has previously been described that certain insects contain the enzyme myrosinase (EC 3.2.3.1), which is c onsidered an important defence enzyme of crucifers. Myrosinase was purified to homogeneity from cabbage aphid soluble extracts using anion-exchange an d phenyl-Sepharose chromatography. The protein has an apparent subunit mole cular moss of 57-58 kDa and is a dimer. The isoelectric point is 4.9 and th e enzyme has a temperature optimum around 40 degreesC. The enzyme was activ e towards the glucosinolates tested, sinigrin and glucotropaeolin, but was inhibited by ascorbate at concentrations that normally activate plant myros inases. Using sinigrin as the substrate K-m was determined as 0.41 mM, and the k(cat) as 36 s(-1). With glucotropaeolin the K-m and k(cat) values were determined as 0.52 mM and 22.8 s(-1), respectively. The enzyme was stable upon storage at 4 degreesC for many months, but lost some activity upon fre ezing. The insect myrosinase did not cross-react with antibodies raised to plant myrosinase. Peptide sequencing of a tryptic digest of the protein sho wed homology to beta -glucosidases. The presence of myrosinase in an insect pest specialist may be an example of a coevolution process that facilitate s host specialization.