title: Cloning and Characterization of a Novel Agarase from a Newly Isolated Bacterium Simiduia sp. Strain TM-2 Able to Degrade Various Seaweeds.
abstract: A new bacterial strain capable of reducing thalli of various seaweeds (red, green, and brown algae) was isolated from marine sediments of Uozu in Toyama Prefecture, Japan. We designated the strain Simiduia sp. TM-2 based on analyses of the 16S rRNA gene and gyrB gene sequences and its biochemical and morphological characteristics. Zymography methods revealed numerous active bands of alginate lyases, cellulases, and agarases in the cells and culture supernatants of TM-2, showing that the strain possessed multiple polysaccharide lyases. A novel agarase gene (agaTM2) was cloned from TM-2 and expressed in Escherichia coli. The resulting DNA sequence contained an open reading frame of 1764 bp that encoded a protein of 587 amino acids with an estimated molecular mass of 64 kDa and pI of 4.62. The deduced amino acid sequence, AgaTM2, had a typical signal peptide followed by a glycoside hydrolase family 16 catalytic domain and two carbohydrate-binding modules 6. A BLAST search indicated that AgaTM2 shared 75.5 % amino acid sequence identity with agarase from Simiduia agarivorans SA1. The cloned and purified AgaTM2 protein showed optimal activity at 35 °C and pH 8.0, and its thermostability increased in the presence of calcium ions. AgaTM2 degraded agarose to tetraose and hexaose.
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Tawara
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan. sakatoku@sci.u-toyama.ac.jp.|Graduate School of Science and Engineering, Department of Environmental and Energy Science, Faculty of Earth and Environmental Systems, University of Toyama, Toyama, 930-8555, Japan. sakatoku@sci.u-toyama.ac.jp.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Sakatoku
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan.
authors.forename: Rosine E
authors.initials: RE
authors.lastname: Tiodjio
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tanaka
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Nakamura
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics; D003001:Cloning, Molecular; D027081:DNA Gyrase / Q000235:genetics; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D019015:Geologic Sediments / Q000382:microbiology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012624:Seaweed / Q000378:metabolism*; D017421:Sequence Analysis
chemical_list: D012336:RNA, Ribosomal, 16S; D006026:Glycoside Hydrolases; D002480:Cellulase; C019624:agarase; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase; D027081:DNA Gyrase
keywords: Agarase; Degradation; Seaweeds; Simiduia