title: An exo-β-(1→3)-D-galactanase from Streptomyces sp. provides insights into type II arabinogalactan structure.
abstract: An exo-β-(1→3)-D-galactanase (SGalase1) that specifically cleaves the β-(1→3)-D-galactan backbone of arabinogalactan-proteins (AGPs) was isolated from culture filtrates of a soil Streptomyces sp. Internal peptide sequence information was used to clone and recombinantly express the gene in E. coli. The molecular mass of the isolated enzyme was ~45 kDa, similar to the 48.2 kDa mass predicted from the amino acid sequence. The pI, pH and temperature optima for the enzyme were ~7.45, 3.8 and 48 °C, respectively. The native and recombinant enzymes specifically hydrolysed β-(1→3)-D-galacto-oligo- or poly-saccharides from the upstream (non-reducing) end, typical of an exo-acting enzyme. A second homologous Streptomyces gene (SGalase2) was also cloned and expressed. SGalase2 was similar in size (47.9 kDa) and enzyme activity to SGalase1 but differed in its pH optimum (pH 5). Both SGalase1 and SGalase2 are predicted to belong to the CAZy glycosyl hydrolase family GH 43 based on activity, sequence homology and phylogenetic analysis. The K(m) and V(max) of the native exo-β-(1→3)-D-galactanase for de-arabinosylated gum arabic (dGA) were 19 mg/ml and 9.7 μmol D-Gal/min/mg protein, respectively. The activity of these enzymes is well suited for the study of type II galactan structures and provides an important tool for the investigation of the biological role of AGPs in plants. De-arabinosylated gum arabic (dGA) was used as a model to investigate the use of these enzymes in defining type II galactan structure. Exhaustive hydrolysis of dGA resulted in a limited number of oligosaccharide products with a trisaccharide of Gal(2)GlcA(1) predominating.
authors.affiliation: Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3010, Australia.
authors.forename: Naomi X-Y
authors.initials: NX
authors.lastname: Ling
authors.forename: Joanne
authors.initials: J
authors.lastname: Lee
authors.forename: Miriam
authors.initials: M
authors.lastname: Ellis
authors.forename: Ming-Long
authors.initials: ML
authors.lastname: Liao
authors.forename: Shaio-Lim
authors.initials: SL
authors.lastname: Mau
authors.forename: David
authors.initials: D
authors.lastname: Guest
authors.forename: Peter H
authors.initials: PH
authors.lastname: Janssen
authors.forename: Pavol
authors.initials: P
authors.lastname: Kováč
authors.forename: Antony
authors.initials: A
authors.lastname: Bacic
authors.forename: Filomena A
authors.initials: FA
authors.lastname: Pettolino
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005685:Galactans / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012988:Soil Microbiology; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D005685:Galactans; D006026:Glycoside Hydrolases; C063901:exo-beta-(1--3)-D-galactanase; C005653:arabinogalactan