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

Sos Torpenholt, Jerome Le Nours, Ulla Christensen, Michael Jahn, Stephen Withers, Peter Ostergaard, Torben Borchert, Jens-Christian Poulsen, Leila Lo Leggio

Research output: Contribution to journalArticleResearchpeer-review

13 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)2028 - 2033
Number of pages6
JournalCarbohydrate Research
Volume346
Issue number13
DOIs
Publication statusPublished - 2011

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