title: Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone.
abstract: A metagenomic fosmid library was constructed using a genomic DNA mixture extracted from the gut microflora of abalone. The library gave an alginate lyase positive clone (AlyDW) harboring a 31.7-kbp insert. The AlyDW insert consisted of 22 open reading frames (ORFs). The deduced amino acid sequences of ORFs 11-13 were similar to those of known alginate lyase genes, which are found adjacent in the genome of Klebsiella pneumoniae subsp. aerogenes, Vibrio splendidus, and Vibrio sp. belonging to the phylum Gammaproteobacteria. Among the three recombinant proteins expressed from the three ORFs, alginate lyase activity was only observed in the recombinant protein (AlyDW11) coded by ORF 11. The expressed protein (AlyDW11) had the highest alginate lyase activity at pH 7.0 and 45°C in the presence of 1 mM AgNO(3). The alginate lyase activity of ORF 11 was confirmed to be endolytic by thin-layer chromatography. AlyDW11 preferred poly(β-D: -mannuronate) as a substrate over poly(α-L: -guluronate). AlyDW11 contained three highly conserved regions, RSEL, QIH, and YFKAGVYNQ, which may act to stabilize the three-dimensional conformation and function of the alginate lyase.
authors.affiliation: Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea.
authors.forename: Su-Jung
authors.initials: SJ
authors.lastname: Sim
authors.forename: Keun Sik
authors.initials: KS
authors.lastname: Baik
authors.forename: Seong Chan
authors.initials: SC
authors.lastname: Park
authors.forename: Han Na
authors.initials: HN
authors.lastname: Choe
authors.forename: Chi Nam
authors.initials: CN
authors.lastname: Seong
authors.forename: Tai-Sun
authors.initials: TS
authors.lastname: Shin
authors.forename: Hee Chul
authors.initials: HC
authors.lastname: Woo
authors.forename: Jeong-Yong
authors.initials: JY
authors.lastname: Cho
authors.forename: Duwoon
authors.initials: D
authors.lastname: Kim
mesh_terms: D000464:Alginates / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020563:Gammaproteobacteria / Q000201:enzymology / Q000235:genetics; D049851:Gastropoda / Q000382:microbiology*; D015723:Gene Library; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D007422:Intestines / Q000382:microbiology; D056186:Metagenomics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D011994:Recombinant Proteins; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase
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