A microtitre plate assay for measuring glycosidase activity

Figures & data

Table I.  Assay conditions and kinetic parameters investigated for enzymes used in this study. Product was quantified by reference to a standard curve (see experimental).

	Enzyme and organism	Buffer/pH	Substrate^1	Km (mM)	Vmax (μmol/min/mg)
1	β-Galactosidase from A. oryzae	Citric acid-NaOH, pH 4.5	3	1.5	29.1
2	β-Galactosidase from E. coli	HEPES-NaOH, pH 7.3	3	0.16	70
3	α-glucosidase from S. cerevisiae	MOPS-NaOH, pH 6.8	2	1.3	16.8
4	β-glucosidase from A. dulces	Citric acid-NaOH, pH 5.0	1	1.8	110
5	β-Glucuronidase from bovine liver	Citric acid-NaOH, pH 5.0	4	0.94	0.216
6	β-glucuronidase from E. coli	MOPS-NaOH, pH 6.8	4	0.23	280
7	β-glucuronidase from P. vulgate	Citric acid-NaOH, pH 3.8	4	0.052	0.295

¹Substrates used in kinetic assays, numbers refer to structures shown in Scheme 1.

Scheme 1 (A). Reactions catalysed by glycosidases as exemplified by β-glucosidase. Under the alkali quenching conditions 4-nitrophenol (pKa = 7.2) ionises to 4-nitrophenolate to give a yellow colour (ε_401nm = 18.5 mM^{− 1} cm^{− 1} [11]); (B). Structures of substrates used in this study: 1, 4-nitrophenyl-β-D-glucopyranoside; 2, 4-nitrophenyl-α-D-glucopyranoside; 3, 4-nitrophenyl-β-D-galactopyranoside; 4, 4-nitrophenyl-β-D-glucuronide; 5, phenolphthalein-β-D-glucuronide.

Ferreira C, Torres BB, Terra WR. Substrate specificities of midgut β-glycosidases from insects of different orders. Comp Biochem Physiol B-Biochem Mol Biol 1998; 119: 219–225

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