title: beta-Agarase from Pseudomonas sp. W7: purification of the recombinant enzyme from Escherichia coli and the effects of salt on its activity.
abstract: The recombinant plasmid (pJAI), harbouring the agarase gene (pjaA) of Pseudomonas sp. W7, was introduced and expressed in Escherichia coli JM83. The agarase was purified using a combination of acetone precipitation and anion-exchange, gel-filtration and affinity chromatographies, with overall yield of 10% from the culture supernatant of E. coli JM83 (pJAI). The purified agarase migrated as a single band (molecular mass 59 kDa) on SDS/PAGE and was found to be beta-agarase, which could hydrolyse the beta-1,4 linkage of agarose to yield neoagarotetraose as the main product. Optimal enzyme activity was at pH 7.8 and the temperature optimum spanned the broad range 20-40 degrees C. The recombinant agarase was halophilic, maximum activity being exhibited at 0.9 M NaCl. This halophilic property could improve the production of neoagaro-oligosaccharides available in a marine environment.
authors.affiliation: Department of Biotechnology and Bioengineering, Pukyong National University, Pusan, South Korea.
authors.forename: J C
authors.initials: JC
authors.lastname: Ha
authors.forename: G T
authors.initials: GT
authors.lastname: Kim
authors.forename: S K
authors.initials: SK
authors.lastname: Kim
authors.forename: T K
authors.initials: TK
authors.lastname: Oh
authors.forename: J H
authors.initials: JH
authors.lastname: Yu
authors.forename: I S
authors.initials: IS
authors.lastname: Kong
mesh_terms: D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D012965:Sodium Chloride; D006026:Glycoside Hydrolases; C019624:agarase