Purification and characterization of a novel beta-agarase, AgaA34, from Agarivorans albus YKW-34.
An extracellular beta-agarase (AgaA34) was purified from a newly isolated marine bacterium, Agarivorans albus YKW-34 from the gut of a turban shell. AgaA34 was purified to homogeneity by ion exchange and gel filtration chromatographies with a recovery of 30% and a fold of ten. AgaA34 was composed of a single polypeptide chain with the molecular mass of 50 kDa. N-terminal amino acid sequencing revealed a sequence of ASLVTSFEEA, which exhibited a high similarity (90%) with those of agarases from glycoside hydrolase family 50. The pH and temperature optima of AgaA34 were pH 8.0 and 40 degrees C, respectively. It was stable over pH 6.0-11.0 and at temperature up to 50 degrees C. Hydrolysis of agarose by AgaA34 produced neoagarobiose (75 mol%) and neoagarotetraose (25 mol%), whose structures were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy and (13)C NMR. AgaA34 cleaved both neoagarohexaose and neoagarotetraose into neoagarobiose. The k (cat)/K (m) values for hydrolysis agarose and neoagarotetraose were 4.04 x 10(3) and 8.1 x 10(2) s(-1) M(-1), respectively. AgaA34 was resistant to denaturing reagents (sodium dodecyl sulfate and urea). Metal ions were not required for its activity, while reducing reagents (beta-Me and dithiothreitol, DTT) increased its activity by 30%.
Fu
Xiao Ting
XT
College of Food Science & Engineering, Ocean University of China, Qingdao, 266003, China.
Lin
Hong
H
Kim
Sang Moo
SM
D034241:Alteromonadaceae / Q000201:enzymology*; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005697:Galactosides / Q000378:metabolism; D049851:Gastropoda / Q000382:microbiology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D004269:DNA, Bacterial; D004187:Disaccharides; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005697:Galactosides; D009844:Oligosaccharides; C081798:neoagarotetraose; D012967:Sodium Dodecyl Sulfate; C068544:neoagarobiose; D008623:Mercaptoethanol; D014508:Urea; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase; D004229:Dithiothreitol