title: Cloning, expression, and characterization of a glycoside hydrolase family 118 beta-agarase from Agarivorans sp. JA-1.
abstract: We report a glycoside hydrolase (GH)-118 beta-agarase from a strain of Agarivorans, in which we previously reported recombinant expression and characterization of the GH- 50 beta-agarase. The GH comprised an open reading frame of 1,437 base pairs, which encoded a protein of 52,580 daltons consisting of 478 amino acid residues. Assessment of the entire sequence showed that the enzyme had 97% nucleotide and 99% amino acid sequence similarities to those of GH-118 beta-agarase from Pseudoalteromonas sp. CY24, which belongs to a different order within the same class. The gene corresponding to a mature protein of 440 amino acids was inserted, recombinantly expressed in Escherichia coli, and purified to homogeneity with affinity chromatography. It had maximal activity at 35 degrees C and pH 7.0 and had 208.1 units/mg in the presence of 300 mM NaCl and 1 mM CaCl2. More than 80% activity was maintained after 2 h exposure to 35 degrees C; however, < 40% activity remained at 45 degrees C. The enzyme hydrolyzed agarose to yield neoagarooctaose as the main product. This enzyme could be useful for industrial production of functional neoagarooligosaccharides.
authors.affiliation: Bioscience and Biotechnology, Graduate School, Silla University, Busan, Korea.
authors.forename: Dong-Geun
authors.initials: DG
authors.lastname: Lee
authors.forename: Myong Je
authors.initials: MJ
authors.lastname: Jeon
authors.forename: Sang-Hyeon
authors.initials: SH
authors.lastname: Lee
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012685:Sepharose / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase