An Exo-β-1,3-galactanase Having a Novel β-1,3-Galactan-bindingModule from Phanerochaetechrysosporium

Abstract

An exo-␤-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-␤-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 ␤-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze ␤-1,4-linked galacto-oligosaccharides, ␤-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 ␤-1,3-galactosyl galactosaminide, ␤-1,3-galactosyl glucosaminide, or ␤-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal ␤-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from ␤-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl ␤-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass ␤-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 ␤-1,3-glucan, although it bound ␤-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 ␤-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.Arabinogalactan proteins (AGPs) 1 are a family of complex proteoglycans widely distributed in plants. They are found in the extracellular matrix associated with the plasma membrane and cell wall (1, 2). Although their precise functions are unknown, AGPs have been implicated in diverse developmental roles, including differentiation, cell-cell recognition, and embryogenesis (3-5). AGPs are characterized by large amounts of carbohydrate components rich in galactose (sugars in the present study are D series unless otherwise designated) and Larabinose, and protein components rich in hydroxyproline, serine, threonine, alanine, and glycine (1). The carbohydrate moieties of AGPs have a common structure consisting of a ␤-1,3-galactosyl backbone to which side chains of ␤-1,6-galactosyl residues are attached through O-6. L-Arabinose and lesser amounts of other auxiliary sugars, such as glucuronic acid, L-rhamnose, and L-fucose, are attached to t...

Characterization of an Exo-β-1,3-D-galactanase fromStreptomyces avermitilisNBRC14893 Acting on Arabinogalactan-Proteins

“…-1,3-Galactan, -1,3--1,6-galactan from Prototheca zopfii, native and -L-arabinofuranosidasetreated AGPs from radish, -1,3-and -1,6-linked galactooligosaccharides, and arabinan were prepared as described previously. [4][5][6][7][8] Methyl -glycoside of -1,3-linked galactotetraose and -pentaose, and -1,6-linked galactopentaose and -hexaose were a kind gift from Dr. P. Kováč of the NIDDK (National Institutes of Health, Bethesda, MD). -1,3-Galactosyl galactosaminide and -1,3-galactobiose were purchased from Dextra Laboratories (Reading, UK).…”

“…The activity of the recombinant enzyme was determined by the method described previously. 6,7) The reaction mixture consisted of 20 ml of McIlvaine buffer (0.2 M Na 2 HPO 4 -0.1 M citric acid), pH 5.0, and 25 ml of 1% (w/v) -1,3-galactan. After 5 min of preincubation at 37 C, 5 ml of enzyme was added to the solution, and the mixture was incubated at 37 C for 10 min.…”

“…Hence, we focused on enzymes capable of degradation of AGPs. [4][5][6][7][8][9] Exo--1,3-galactanase is an especially powerful tool to analyze AGP since it is able to accommodate branched side chains when the enzyme hydrolyzes the main-chain -1,3-linkage of the carbohydrate moiety of AGPs. In spite of its usefulness, so far, exo--1,3-galactanase has been purified from only two organisms, Irpex lacteus (Il1,3Gal) and Aspergillus niger (An1,3Gal).…”

“…Recently, we succeeded for the first time in cloning an exo--1,3-galactanase gene from Phanerochaete chrysosporium (Pc1,3Gal43A). 6) Very recently, y To whom correspondence should be addressed. Fax: +81-298-38-7996; E-mail: sakaneko@affrc.go.jp Abbreviations: AGP, arabinogalactan-protein; Sa1,3Gal43A, Streptomyces avermitilis exo--1,3-D-galactanase; Il1,3Gal, Irpex lacteus exo--1,3-D-galactanase; An1,3Gal, Aspergillus niger exo--1,3-D-galactanase; Pc1,3Gal43A, Phanerochaete chrysosporium exo--1,3-D-galactanase; Ct1,3Gal43A, Clostridium thermocellum exo--1,3-D-galactanase; GH, glycoside hydrolase family; CBM, carbohydrate-binding module; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; McIlvaine buffer, 0.2 M Na 2 HPO 4 -0.1 M citric acid; HPAEC-PAD, highperformance anion-exchange chromatography with a pulsed amperometric detection system; dp, degree of polymerization; CM-curdlan, carboxymethyl-curdlan; Gal--1,3-GalNAc, -1,3-D-galactosyl galactosaminide; PCR, polymerase chain reaction; 4-O-MeGlcA, 4-O-methylglucuronic acid using the amino acid sequence of Pc1,3Gal43A, we found a first bacterial exo--1,3-galactanase from Clostridium thermocellum (Ct1,3Gal43A), and suggested that the enzyme appears to be widely distributed in nature.…”

“…Fax: +81-298-38-7996; E-mail: sakaneko@affrc.go.jp Abbreviations: AGP, arabinogalactan-protein; Sa1,3Gal43A, Streptomyces avermitilis exo--1,3-D-galactanase; Il1,3Gal, Irpex lacteus exo--1,3-D-galactanase; An1,3Gal, Aspergillus niger exo--1,3-D-galactanase; Pc1,3Gal43A, Phanerochaete chrysosporium exo--1,3-D-galactanase; Ct1,3Gal43A, Clostridium thermocellum exo--1,3-D-galactanase; GH, glycoside hydrolase family; CBM, carbohydrate-binding module; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; McIlvaine buffer, 0.2 M Na 2 HPO 4 -0.1 M citric acid; HPAEC-PAD, highperformance anion-exchange chromatography with a pulsed amperometric detection system; dp, degree of polymerization; CM-curdlan, carboxymethyl-curdlan; Gal--1,3-GalNAc, -1,3-D-galactosyl galactosaminide; PCR, polymerase chain reaction; 4-O-MeGlcA, 4-O-methylglucuronic acid using the amino acid sequence of Pc1,3Gal43A, we found a first bacterial exo--1,3-galactanase from Clostridium thermocellum (Ct1,3Gal43A), and suggested that the enzyme appears to be widely distributed in nature. 7) Four kinds of exo--1,3-galactanases have been characterized, [6][7][8]10) but limited information is available on the enzymes to characterize AGPs, because, so many kinds of AGP exist and it is known that the structure of AGPs from different organs of the same plant are quite different. 1,2,11,12) Larger numbers of exo--1,3-galactanases that have various substrate specificities are necessary for analysis of AGPs.…”