An Exo-β-1,3-galactanase Having a Novel β-1,3-Galactan-binding Module from Phanerochaete chrysosporium*

By H. Ichinose, Makoto Yoshida, Toshihisa Kotake, A. Kuno, K. Igarashi, Y. Tsumuraya, M. Samejima, J. Hirabayashi, Hideyuki Kobayashi, S. Kaneko. Journal of Biological Chemistry, 2005

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An exo-β-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-β-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward β-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze β-1,4-linked galacto-oligosaccharides, β-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze β-1,3-galactosyl galactosaminide, β-1,3-galactosyl glucosaminide, or β-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal β-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from β-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl β-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass β-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and β-1,3-glucan, although it bound β-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a β-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.

Subfields: Biomedical Engineering, Nutrition and Dietetics, Plant Science

View Paper: https://www.semanticscholar.org/paper/e0ac7ce3a82db57f74f313f6160b531f22ccaa9f

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