COMPARISON OF SEVERAL NEW CHROMOGENIC GALACTOSIDES AS SUBSTRATES FOR VARIOUS BETA-D-GALACTOSIDASES

The kinetic characteristics of beta-galactosidases from bovine liver a nd testes, Escherichia coli, Aspergillus niger and Jack bean were stud ied using five newly-developed colorimetric substrates. All the chromo phores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate in hibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant probl em if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli beta-D-galactosid ase resulting in low K(m) values. The data suggest that the chemical p roperties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the sub strate inhibition can be attributed to interactions not involving the catalytic site. When the product of the maximum observed velocity (v(m )) and the molar absorption coefficient is calculated for each substra te, the relative merits of the substrates for the assay of each enzyme can be assessed. The beta-D-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that th ey may be of particular value in areas of molecular biology and biotec hnology.