beta-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalactan side chains of the complex pectin network in plant cell walls. Two fungal beta-1,4-galactanases from Aspergillus aculeatus, Meripileus giganteus and one bacterial enzyme from Bacillus licheniformis have been kinetically characterized using the chromogenic substrate analog 4-nitrophenyl beta-1,4-d-thiogalactobioside synthesized by the thioglycoligase approach. Values of k(cat)/K(m) for this substrate with A. aculeatus beta-1,4-galactanase at pH 4.4 and for M. giganteus beta-1,4-galactanase at pH 5.5 are 333M(-1)s(-1) and 62M(-1)s(-1), respectively. By contrast the B. licheniformis beta-1,4-galactanase did not hydrolyze 4-nitrophenyl beta-1,4-d-thiogalactobioside. The different kinetic behavior observed between the two fungal and the bacterial beta-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the structure of B. licheniformis beta-1,4-galactanase. This loop contains substrate binding subsites -3 and -4, which presumably cause B. licheniformis beta-1,4-galactanase to bind 4-nitrophenyl -1,4-beta-d-thiogalactobioside non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-beta-d-thiogalactobioside, the two fungal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-beta-d-galactopyranoside, but kinetic parameters could not be determined because of transglycosylation at substrate concentrations above 4mM.