Purification and characterization of arylsulfatase from Sphingomonas sp. AS6330.
Arylsulfatase was purified from Sphingomonas sp. AS6330 through ionic exchange, hydrophobic- and gel-chromatographies. The purity increased 12,800-fold with approximately 19.1% yield against cell homogenate. The enzyme was a monomeric protein with apparent molecular weight of 62 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 41 kDa as determined by gel filtration. The enzyme had optimum reaction conditions for hydrolysis of sulfate ester bonds in agar and p-nitrophenyl sulfate (NPS) at pH 7.0 and 45 degrees C, with a specific activity of 3.93 and 97.2 U, respectively. The enzyme showed higher activity towards agar than other sulfated marine polysaccharides such as porphyran, fucoidan and carrageenan. The K(m) and V(max) of the enzyme for hydrolysis of NPS were 54.9 microM and 113 mM/min, respectively. With reaction of 200 g agar with 100 U arylsulfatase for 8 h at 45 degrees C, gel strength increased 2.44-fold, and 97.7% of the sulfate in the agar was hydrolyzed.
Department of Biological Science, Pusan National University, 609-735 Busan, South Korea.
J-H
JH
Kim
D-S
DS
Byun
J S
JS
Godber
J-S
JS
Choi
W-C
WC
Choi
H-R
HR
Kim
D000362:Agar / Q000378:metabolism; D001192:Arylsulfatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001711:Biotransformation; D002351:Carrageenan / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods; D008970:Molecular Weight; D009578:Nitrobenzenes / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives* / Q000378:metabolism; D020578:Sphingomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D009578:Nitrobenzenes; D011134:Polysaccharides; C023379:4-nitrophenyl sulfate; C038549:porphyran; D002351:Carrageenan; D000362:Agar; D012685:Sepharose; C007789:fucoidan; D001192:Arylsulfatases