Abstract
β- N-Acetylglucosaminidase secreted by Entamoeba histolytica was extracted from the growth medium by affinity chromatography on CH-Sepharose 4 B coupled to p-aminophenyl-1-thio-β-2-acetamido-2-deoxyglucopyranoside. The enzyme was further purified by isoelectric focusing, by sequential chromatography on DEAE-cellulose and Sephadex G-150, and by preparative disc gel electrophoresis. Chitobiose (βGlcNAcl-4GlcNAc) derived from chitin as well as the oligosaccharides βGlcNAc1-4βGlcUA1-3GlcNAc, βGlcNAc1-4βGlcUA1-3βGlcNAc1-4GlcUA, and βGlcNAc1-4βGlcUA1-3βGlcNAc1-4βGlcUA1-3βGlcNAc1-4GlcUA derived from hyaluronic acid were tested as potential physiological substrates. All these oligosaccharides are susceptible to action of β- N-acetylglucos-aminidase from E. histolytica. Under identical conditions chitobiose is cleaved 38–48 times faster than hyaluronate oligosaccharides. No release of N-acetylglucosamine was observed when glycopeptides from ovalbumin were used as substrate. The pH optimum of hydrolase activity was 4.5 when chitobiose was used as substrate. Optimal hydrolysis of hyaluronate oligosaccharides was observed at pH 3.0 for trisaccharide and pH 2.0 for tetra- and hexasaccharide, respectively. Estimation of molecular weight by means of gel filtration gave values of 75 000. The isoelectric point was 5.02. β- N-Acetylglucosaminidase from E. histolytica does not act on macromolecular chitin and hyaluronic acid.
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