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

TY - JOUR

T1 - Activity of three ß-1,4-galactanases on small chromogenic substrates

AU - Torpenholt, Søs Katja

AU - Le Nours, Jerome

AU - Christensen, Ulla

AU - Jahn, Michael

AU - Withers, Stephen

AU - Østergaard, Peter

AU - Borchert, Torben V.

AU - Poulsen, Jens-Christian Navarro

AU - Lo Leggio, Leila

PY - 2011/9

Y1 - 2011/9

N2 - 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 kcat/Km 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.

AB - 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 kcat/Km 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.

M3 - Journal article

SN - 0008-6215

VL - 346

SP - 2028

EP - 2033

JO - Carbohydrate Research

JF - Carbohydrate Research

ER -