Detection and quantification of N-acetyl-beta-D-glucosaminidase, chitobiosidase, and endochitinase in solutions and on gels

Abstract

Procedures are described for the direct assay of N-acetyl-beta-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-beta-D-glucosaminide, beta-D-N,N'-di-acetylchitobioside, or beta-D-N,N',N"-triacetylchitotriose for N-acetyl-beta-glucosaminidase, chitobiosidase, or endochitinase, respectively. For quantitative assay of N-acetyl-beta-glucosaminidase and chitobiosidase in solutions, a rapid technique using nitrophenyl-N-acetyl-beta-D-glucosaminide and nitrophenyl-beta-D-N,N'-diacetyl-chitobiose, 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.