title: Molecular cloning and expression in Pichia pastoris of a Irpex lacteus exo-beta-(1-->3)-galactanase gene.
abstract: A gene encoding exo-beta-(1-->3)-galactanase from Irpex lacteus was cloned by reverse transcriptase-PCR. The deduced amino acid sequence showed high similarity with exo-beta-(1-->3)-galactanases from other sources. The molecular mass of the mature form was calculated to be 45,520 Da. The gene product expressed in Pichia pastoris specifically hydrolyzed beta-(1-->3)-galactooligosaccharides, as did other exo-beta-(1-->3)-galactanases. The recombinant enzyme showed high activity toward arabinogalactan-proteins (AGPs) from radish as well as beta-(1-->3)-galactan. Product analysis revealed that the enzyme released beta-(1-->6)-galactobiose, beta-(1-->6)-galactotriose, and alpha-L-arabinofuranosyl-(1-->3)-beta-galactosyl-(1-->6)-galactose together with Gal from beta-(1-->3)-galactans attached with and without beta-(1-->6)-galactosyl branches prepared from acacia gum. These results indicate that the exo-beta-(1-->3)-galactanase from I. lacteus efficiently hydrolyzes beta-(1-->3)-galactan main chains of AGPs by bypassing beta-(1-->6)-galactosyl side chains.
authors.affiliation: Division of Life Science, Graduate School of Science and Engineering, Saitama University, Japan.
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Kotake
authors.forename: Kiminari
authors.initials: K
authors.lastname: Kitazawa
authors.forename: Ryohei
authors.initials: R
authors.lastname: Takata
authors.forename: Kohei
authors.initials: K
authors.lastname: Okabe
authors.forename: Hitomi
authors.initials: H
authors.lastname: Ichinose
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kaneko
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Tsumuraya
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005685:Galactans / Q000378:metabolism; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D020072:Polyporales / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D005685:Galactans; D009088:Mucoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C006619:arabinogalactan proteins; D006026:Glycoside Hydrolases; C063901:exo-beta-(1--3)-D-galactanase