Purification and Properties ofβ-N-Acetyl-D-glucosaminidase from Alimentary Canal of the Silkworm,Bombyx mori

Abstract

β-N-Acetyl-d-glucosaminidase (EC 3.2.1.30) was purified from the alimentary canal of the silkworm, Bombyx mori, by ammonium sulfate fractionation and chromatography with hydroxylapatite, DEAE Bio-Gel A, chromatofocusing, and Sephacryl S-200. The purified enzyme was a single band on disc-PAGE. The molecular weight was 119,000 by gel filtration and 125,000 by SDS-PAGE. The enzyme was separated into two peptides whose apparent molecular weights were 67,500 and 57,500 by SDS-PAGE. The pi was 4.86 by chromatofocusing. The optimum pH was 5.5 to 6.0 and the optimum temperature, 45°C, using pNp-β-GlcNAc as the substrate. The enzyme was stable from pH 5.5 to 8.5 and below 30°C. It was strongly inhibited by HgCl2. Small N-acetylchitooligomers were as good substrates as pNp-β-GlcNAc, and the enzyme cleaved colloidal chitin to GlcNAc, even though the relative velocity was slow. Smaller N-acetylchitooligomers were preferred substrates with Km 0.787 to 0.056mm, A:cat 1013 to 52sec–1, and kcat/Km 1690 to 754 mm–1 sec–1. The enzyme precipitated in as band with moulting fluid chitobiase antiserum, but not with haemolymph exo-β-N-acetylglucosaminidase antiserum. The results suggest that this enzyme is an exo-type enzyme which is involved in the degradation of chitin to GlcNAc.