CHARACTERIZATION OF A THERMOSTABLE LYSINE-SPECIFIC METALLOENDOPEPTIDASE FROM THE FRUITING BODIES OF A BASIDIOMYCETE, GRIFOLA-FRONDOSA

A zinc-metalloendopeptidase, MEP, capable of catalyzing specific cleavage of acyl-lysine bonds (-X-Lys-) in polypeptides has been purified 212-fold in a yield of 24.7% from the fruiting bodies of Grifola frondosa, which is a popular edible mushroom called ''MAITAKE'' in Japan, The purified enzyme consists of a single polypeptide chain with an apparent molecular mass of 20 kDa and a pi value of 7.46, contains 1 atom of zinc/molecule and can be inactivated with EDTA or 1,10-phenanthroline, Treatment of MFP with EDTA affords an apoenzyme, whose activity can be fully restored by the addition of Mn2+, Zn2+, Ca2+, or Co2+, Prominent features of MEP are its remarkable heat stability and its high affinity for beta-D-glucans and chitin. It hydrolyzes proteins maximally at pH 9-10, liberating only lysyl-peptides, Polylysine and lysine copolymers with alanine, phenylalanine, or glutamic acid can serve as good substrates, Lysylalanine was liberated from bovine insulin and its oxidized B chain by the action of MEP, Mass spectrometric analysis by Frit-FAB RIS of the fragments generated from horse heart cytochrome c presented unambiguous evidence to corroborate the specificity of MEP for acyl-lysine bonds.