The occurrence of lysozyme activity in calf rennet and abomasal fluid has recently been reported. In addition, ß-N-acetyl-D-hexosaminidase activity (Mr ca. 80'000) was also detected in the course of the purification procedure used for this lysozyme. We now report the isolation of four multiple molecular forms exhibiting ß-N-acetyl-D-glucosaminidase activity (EC 3.2.1.30). In addition to their in vivo physiological role, such enzymes could be useful for the structural analysis of various glycoproteins.
Crude calf rennet was used as the starting material, and the purification steps included precipitation in the presence of ammonium sulfate, gel filtration, cation and anion exchange chromatography. The ß-N-acetyl-D-hexosaminidase activity was tested at the various purification steps with the following substrates: 4-nitrophenyl-2-acetamido-2-deoxy-ß-D-glucopyranoside, 4-nitrophenyl-2-acetamido-2-deoxy-ß-D-galactopyranoside, colloidal chitin and chitobiose.
The four isolated enzyme forms were most active when hydrolyzing the synthetic glucosaminide substrate, and three of them also hydrolyzed the galactosaminide substrate (relative reaction rate: ca. 13%). By contrast, no significant endo-chitinase activity could be detected, whereas chitobiose was susceptible to enzymatic hydrolysis. The isolated enzyme forms thus appear to act preferentially on low Mr substrates.