title: Biochemical characterization of a GH53 endo-β-1,4-galactanase and a GH35 exo-β-1,4-galactanase from Penicillium chrysogenum.
abstract: An endo-β-1,4-galactanase (PcGAL1) and an exo-β-1,4-galactanase (PcGALX35C) were purified from the culture filtrate of Penicillium chrysogenum 31B. Pcgal1 and Pcgalx35C cDNAs encoding PcGAL1 and PcGALX35C were isolated by in vitro cloning. The deduced amino acid sequences of PcGAL1 and PcGALX35C are highly similar to a putative endo-β-1,4-galactanase of Aspergillus terreus (70% amino acid identity) and a putative β-galactosidase of Neosartorya fischeri (72%), respectively. Pfam analysis revealed a "Glyco_hydro_53" domain in PcGAL1. PcGALX35C is composed of five distinct domains including "Glyco_hydro_35," "BetaGal_dom2," "BetaGal_dom3," and two "BetaGal_dom4_5" domains. Recombinant enzymes (rPcGAL1 and rPcGALX35C) expressed in Escherichia coli and Pichia pastoris, respectively, were active against lupin galactan. The reaction products of lupin galactan revealed that rPcGAL1 cleaved the substrate in an endo manner. The enzyme accumulated galactose and galactobiose as the main products. The smallest substrate for rPcGAL1 was β-1,4-galactotriose. On the other hand, rPcGALX35C released only galactose from lupin galactan throughout the reaction, indicating that it is an exo-β-1,4-galactanase. rPcGALX35C was active on both β-1,4-galactobiose and triose, but not on lactose, β-1,3- or β-1,6-galactooligosaccharides even after 24 h of incubation. To our knowledge, this is the first report of a gene encoding a microbial exo-β-1,4-galactanase. rPcGAL1 and rPcGALX35C acted synergistically in the degradation of lupin galactan and soybean arabinogalactan. Lupin galactan was almost completely degraded to galactose by the combined actions of rPcGAL1 and rPcGALX35C. Surprisingly, neither rPcGAL1 nor rPcGALX35C released any galactose from sugar beet pectin.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan. sakamoto@biochem.osakafu-u.ac.jp
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Makino
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Sunagawa
authors.forename: Naoki
authors.initials: N
authors.lastname: Harada
mesh_terms: D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004187:Disaccharides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005685:Galactans / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D006868:Hydrolysis; D010408:Penicillium chrysogenum / Q000201:enzymology*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D004187:Disaccharides; D005685:Galactans; D011994:Recombinant Proteins; C056853:6-O-galactopyranosylgalactose; D006026:Glycoside Hydrolases; C066350:exo-1,4-beta-galactanase; C094192:arabinogalactan endo-1,4-beta-galactosidase; D005690:Galactose