Activity of three β-1,4-galactanases on small chromogenic substrates.

Abstract β-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 β-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 β-1,4- d -thiogalactobioside synthesized by the thioglycoligase approach. Values of k cat / K m for this substrate with A. aculeatus β-1,4-galactanase at pH 4.4 and for M. giganteus β-1,4-galactanase at pH 5.5 are 333 M −1  s −1 and 62 M −1  s −1 , respectively. By contrast the B. licheniformis β-1,4-galactanase did not hydrolyze 4-nitrophenyl β-1,4- d -thiogalactobioside. The different kinetic behavior observed between the two fungal and the bacterial β-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the structure of B. licheniformis β-1,4-galactanase. This loop contains substrate binding subsites −3 and −4, which presumably cause B. licheniformis β-1,4-galactanase to bind 4-nitrophenyl -1,4-β- d -thiogalactobioside non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-β- d -thiogalactobioside, the two fungal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-β- d -galactopyranoside, but kinetic parameters could not be determined because of transglycosylation at substrate concentrations above 4 mM.