Purification and characterization of the recombinant arylsulfatase cloned from Pseudoalteromonas carrageenovora.
Arylsulfatase cloned from a marine aerobic Gram-negative bacterium, Pseudoalteromonas carrageenovora, was overexpressed in Escherichia coli with 10 microM IPTG induction. The expressed recombinant arylsulfatase was purified to homogeneity from the harvested cells through osmotic disruption and column chromatography methods, such as DEAE-cellulose anion exchange chromatography and Heparin-Sepharose affinity chromatography. The purified arylsulfatase was kinetically characterized using the synthetic substrate of phenolic ester, p-nitrophenyl sulfate (pNPS). One unit of arylsulfatase catalyzes the liberation of 1.0 micromol p-nitrophenol from pNPS per minute. The purified enzyme has a specific activity of 468 U/mg with a purification yield of 27% from the cell lysate, and exhibited an estimated molecular mass of 33 kDa in SDS-PAGE analysis. The precursor polypeptide of 36 kDa was processed by releasing a putative signal peptide, and the mature arylsulfatase of 33.1 kDa with a N-terminal sequence of S-E-T-K-N was trafficked to periplasmic space. The enzyme had optimum reaction conditions for activity at pH 7.0 and at a temperature range of 40-45 degrees C. The apparent K(M) and k(cat) of the enzyme for hydrolysis of pNPS at pH 7.0 and at 45 degrees C were determined to be 1.15 mM and 1000 s-1, respectively. Based on inhibitor studies along with optimal pH values and preferential periplasmic location of the enzyme, we suggest that the recombinant arylsulfatase from P. carrageenovora is probably similar to the Klebsiella sulfatase with serine residue in the active site.
Kim
Dong-Eun
DE
Department of Biotechnology and Bioengineering, Dong-Eui University, Busan 614-714, Republic of Korea.
Kim
Kyung-Hwa
KH
Bae
Yu-Jin
YJ
Lee
Jung-Hee
JH
Jang
Yhon-Hwa
YH
Nam
Soo-Wan
SW
D001192:Arylsulfatases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D031161:Pseudoalteromonas / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors
D001192:Arylsulfatases