Abstract
Zymolyase is a commercially available endo-(1→3)-β-D-glucanase preparation, and is widely used to lyse fungal cell walls. In this paper, the active center of this enzyme preparation was examined by using chemical modification of functional groups of amino acid residues, ultraviolet (UV) spectroscopy, optical rotation measurement ant analysis of the enzymic products. First of all, chemical modifications of the amino acid residues of Zymolyase, by using 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide-HCl or 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide metho-p-toluenesulforate for carboxyl, and N-bromosuccinimide (NBS) for tryptophan residues, caused the loss of (1→3)-β-D-glucanase activity. These inactivations were prevented by the addition of substrate to the reaction mixture. In the presence of the substrate analogues, e.g. laminarabiose or laminaratriitol, a UV difference spectrum attributed to tryptophan residues was observed. This difference spectrum disappeared after NBS oxidation. Secondly, when the specific rotation of carboxymethylated curdlan, a linear (1→3)-β-D-glucan obtained from Alcaligenes faecalis, was measured during the enzymic reaction, the value was more negative than that after mutarotation. These results suggest that carboxyl and tryptophan residues are essential to the enzyme activity and the anomeric specificity of the reducing end of the product is β.
