title: Extracellular production of a novel endo-β-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose.
abstract: The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type β-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products.
authors.affiliation: Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 202, Taiwan. phsu@mail.ntou.edu.tw.
authors.forename: Pang-Hung
authors.initials: PH
authors.lastname: Hsu
authors.affiliation: Department of Food Science, National Taiwan Ocean University, Keelung 202, Taiwan. hank199021@hotmail.com.
authors.forename: Chien-Han
authors.initials: CH
authors.lastname: Wei
authors.affiliation: Department of Food Science, National Taiwan Ocean University, Keelung 202, Taiwan. miss350100@yahoo.com.tw.
authors.forename: Wen-Jung
authors.initials: WJ
authors.lastname: Lu
authors.affiliation: Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 202, Taiwan. leo3220@livemail.tw.
authors.forename: Fen
authors.initials: F
authors.lastname: Shen
authors.affiliation: Department of Food Science, National Taiwan Ocean University, Keelung 202, Taiwan. b0037@ntou.edu.tw.
authors.forename: Chorng-Liang
authors.initials: CL
authors.lastname: Pan
authors.affiliation: Department of Food Science, National Taiwan Ocean University, Keelung 202, Taiwan. HL358@ntou.edu.tw.|Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung 202, Taiwan. HL358@ntou.edu.tw.
authors.forename: Hong-Ting Victor
authors.initials: HT
authors.lastname: Lin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D005697:Galactosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D021382:Protein Sorting Signals; D021381:Protein Transport; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D012685:Sepharose / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005697:Galactosides; D009844:Oligosaccharides; D021382:Protein Sorting Signals; C081798:neoagarotetraose; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase