title: Biochemical characterization of a novel iron-dependent GH16 β-agarase, AgaH92, from an agarolytic bacterium Pseudoalteromonas sp. H9.
abstract: A putative agarase gene (agaH92) encoding a primary translation product (50.1 kDa) of 445 amino acids with a 19-amino-acid signal peptide and glycoside hydrolase 16 and RICIN superfamily domains was identified in an agarolytic marine bacterium, Pseudoalteromonas sp. H9 ( = KCTC23887). The heterologously expressed protein rAgaH92 in Escherichia coli had an apparent molecular weight of 51 kDa on SDS-PAGE, consistent with the calculated molecular weight. Agarase activity of rAgaH92 was confirmed by a zymogram assay. rAgaH92 hydrolyzed p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for rAgaH92 were 6.0 and 45°C, respectively. It was thermostable and retained more than 85% of its initial activity after heat treatment at 50°C for 1 h. rAgaH92 required Fe(2+) for agarase activity and inhibition by EDTA was compensated by Fe(2+). TLC analysis, mass spectrometry and NMR spectrometry of the GST-AgaH71 hydrolysis products revealed that rAgaH92 is an endo-type β-agarase, hydrolyzing agarose into neoagarotetraose and neoagarohexaose.
authors.affiliation: Biological and Genetic Resource Assessment Division, National Institute of Biological Resource, Hwangyeong-ro 42, Seo-gu, Incheon 404-170, Korea.
authors.forename: Won-Jae
authors.initials: WJ
authors.lastname: Chi
authors.affiliation: Department of Biological Science and Bioinformatics, Myongji University, Nam-dong, Cheoin-gu, Yongin, Gyeonggido 449-728, Korea.
authors.forename: Chang-Ro
authors.initials: CR
authors.lastname: Lee
authors.affiliation: Department of Biological Science and Bioinformatics, Myongji University, Nam-dong, Cheoin-gu, Yongin, Gyeonggido 449-728, Korea.
authors.forename: Saruul
authors.initials: S
authors.lastname: Dugerjonjuu
authors.affiliation: Department of Biological Science and Bioinformatics, Myongji University, Nam-dong, Cheoin-gu, Yongin, Gyeonggido 449-728, Korea.
authors.forename: Jae-Seon
authors.initials: JS
authors.lastname: Park
authors.affiliation: Department of Animal Resources Science, Dankook University, Dandae-ro 119, Cheonan 330-714, Korea.
authors.forename: Dae-Kyung
authors.initials: DK
authors.lastname: Kang
authors.affiliation: Department of Biological Science and Bioinformatics, Myongji University, Nam-dong, Cheoin-gu, Yongin, Gyeonggido 449-728, Korea skhong@mju.ac.kr.
authors.forename: Soon-Kwang
authors.initials: SK
authors.lastname: Hong
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005697:Galactosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008970:Molecular Weight; D009598:Nitrophenylgalactosides / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D021382:Protein Sorting Signals; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics*; D012685:Sepharose / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005697:Galactosides; D009844:Oligosaccharides; D021382:Protein Sorting Signals; C081798:neoagarotetraose; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D012685:Sepharose; D007501:Iron; D006026:Glycoside Hydrolases; C019624:agarase
keywords: Pseudoalteromonas sp. H9; neoagarohexaose; neoagarotetraose; β-1,4-agarase