title: Characterization of an alginate lyase, FlAlyA, from Flavobacterium sp. strain UMI-01 and its expression in Escherichia coli.
abstract: A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I-Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. inouea21@fish.hokudai.ac.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Inoue
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. k.takadono@gmail.com.
authors.forename: Kohei
authors.initials: K
authors.lastname: Takadono
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. nsym2480@ec.hokudai.ac.jp.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Nishiyama
authors.affiliation: Laboratory of Molecular Materials Chemistry, Faculty of Engineering, Hokkaido University, Sapporo, Hokkaido 060-8626, Japan. ktajima@eng.hokudai.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Tajima
authors.affiliation: Hokkaido Industrial Technology Center, Kikyou, Hakodate, Hokkaido 041-0801, Japan. kobayashi@techakodate.or.jp.
authors.forename: Takanori
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan. ojima@fish.hokudai.ac.jp.
authors.forename: Takao
authors.initials: T
authors.lastname: Ojima
mesh_terms: D000464:Alginates; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005417:Flavobacterium / Q000235:genetics*; D020723:Glucuronic Acid / Q000235:genetics; D006603:Hexuronic Acids; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D021382:Protein Sorting Signals; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase