title: Cloning, expression, and biochemical characterization of a GH16 β-agarase AgaH71 from Pseudoalteromonas hodoensis H7.
abstract: An agarase gene (agaH71) was identified from Pseudoalteromonas hodoensis, an agar utilizing marine bacterium. The nucleotide sequence revealed that AgaH71 had significant homology to glycosyl hydrolase (GH) 16 agarases. agaH71 encodes a primary translation product (32.7 kDa) of 290 amino acids, including a 21-amino-acid signal peptide. The entire AgaH71 was expressed in a fused protein with glutathione-S-transferase (GST) at its N-terminal (GST-AgaH71) in Escherichia coli. Purified GST-AgaH71 had an apparent molecular weight of 59 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was consistent with the calculated molecular weight (58.7 kDa). Agarase activity of the purified protein was confirmed by zymogram assay. GST-AgaH71 could hydrolyze p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for GST-AgaH71 were 6.0 and 45 °C, respectively. GST-AgaH71 retained more than 95 and 90 % of its initial activity at 40 and 45 °C after heat treatment for 60 min, respectively. The K m and V max for agarose were 28.33 mg/ml and 88.25 U/mg, respectively. GST-AgaH71 did not require metal ions for its activity, but severe inhibition by divalent metal ions was observed. Thin-layer chromatography (TLC) analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectrometry of the GST-AgaH71 hydrolysis products revealed that GST-AgaH71 is an endo-type β-agarase that hydrolyzes agarose into predominantly neoagarotetraose and small proportions of neoagarobiose and neoagarohexaose.
authors.affiliation: Division of Bioscience and Bioinformatics, Myongji University, San 38-2, Namdong, Yongin, Gyeonggi-do, 449-728, South Korea.
authors.forename: Da Yeon
authors.initials: DY
authors.lastname: Park
authors.forename: Won-Jae
authors.initials: WJ
authors.lastname: Chi
authors.forename: Jae-Seon
authors.initials: JS
authors.lastname: Park
authors.forename: Yong-Keun
authors.initials: YK
authors.lastname: Chang
authors.forename: Soon-Kwang
authors.initials: SK
authors.lastname: Hong
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004187:Disaccharides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005697:Galactosides / Q000737:chemistry; D015870:Gene Expression; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D016366:Open Reading Frames; D021382:Protein Sorting Signals; D031161:Pseudoalteromonas / Q000737:chemistry* / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012685:Sepharose / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D005697:Galactosides; D009844:Oligosaccharides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C081798:neoagarotetraose; C068544:neoagarobiose; D012685:Sepharose; D005982:Glutathione Transferase; D006026:Glycoside Hydrolases; C019624:agarase