beta-Agarase from Pseudomonas sp. W7: purification of the recombinant enzyme from Escherichia coli and the effects of salt on its activity.
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.
Ha
J C
JC
Department of Biotechnology and Bioengineering, Pukyong National University, Pusan, South Korea.
Kim
G T
GT
Kim
S K
SK
Oh
T K
TK
Yu
J H
JH
Kong
I S
IS
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
D011994:Recombinant Proteins; D012965:Sodium Chloride; D006026:Glycoside Hydrolases; C019624:agarase