DETECTION AND QUANTIFICATION OF N-ACETYL-BETA-D-GLUCOSAMINIDASE, CHITOBIOSIDASE, AND ENDOCHITINASE IN SOLUTIONS AND ON GELS

Procedures are described for the direct assay of N-acetyl-β-glucosaminidase (EC 3.2.1.30), chitobiosidase, and endochitinase (EC 3.2.1.14) after separation on starch or polyacrylamide electrophoresis gels. The enzymes were visualized as fluorescent bands by using an agarose overlay containing 4-methylumbelliferyl derivatives of N-acetyl-β-D-glucosaminide, β-D-N, N'-diacetylchitobioside, or β-D-N, N', N''-triacetylchitotriose for N-acetyl-β-glucosaminidase, chitobiosidase, or endochitinase, respectively. For quantitative assay of N-acetyl-β-glucosaminidase and chitobiosidase in solutions, a rapid technique using nitrophenyl-N-acetyl-β-D-glucosaminide and nitrophenyl-β-D-N, N'-diacetylchitobiose, respectively, was used. Endochitinase activity was quantitatively measured by determining the percentage reduction in turbidity of a reaction mixture that contained purified colloidal chitin. Trichoderma harzianum strain P1 was shown to produce three kinds of chitinolytic enzymes, and there were multiple forms of some of these. © 1994 Academic Press, Inc. All rights reserved.