An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium

An exo-beta- 1,3- galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta- 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 carbohydratebinding 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 beta- 1,3- linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta- 1,4- linked galacto- oligosaccharides, beta- 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 beta- 1,3- galactosyl galactosaminide, beta- 1,3- galactosyl glucosaminide, or beta- 1,3- galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta- 1,3- linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta- 1,3- galactan in an exo- acting manner. However, no activity toward p- nitrophenyl beta- galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta- 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 beta- 1,3- glucan, although it bound beta- 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 beta- 1,3- galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.