Purification and characterization of methyl parathion hydrolase from Burkholderia cepacia capable of degrading organophosphate insecticides.
Methyl parathion hydrolase (MPH) from a methyl parathion-degrading Burkholderia cepacia indigenous to Thailand was purified to apparent homogeneity by three steps of column chromatography using Resource S, Sephadex G100, and Octyl Sepharose 4FF columns. Its molecular mass was determined to be 35 kDa, and the pI to be 8.5. The recombinant plasmid pGT1, containing the MPH-encoding gene, mpdB, cloned into pGEX-4T-2 was over-expressed in Escherichia coli as GST-MPH fusion protein. The recombinant MPH was purified to homogeneity by a single step, using GSTPrep FF affinity column, with the molecular mass identical to that of the native enzyme. The purified enzyme had the specific activity of about 1,600 unit mg(-1) protein and the yield of about 75%, a 39-fold increase in recovery compared to that of the native enzyme. The optimal temperature and pH were 25°C and 9.0, respectively. The MPH was stable, with its activity unchanged for 48 h at 4°C, and reduced to 50% after 5 h and to 45% after 48 h at 25°C. The enzyme activity remained 80-90% after 8-15 h at pH 6-7. Cd(2+), Co(2+), and Zn(2+) ions at the concentration of 1 mM enhanced the activity; while sodium dodecyl sulfate (SDS), dithiothreitol (DTT) and ethylenediaminetetraacetate (EDTA) reduced it. The enzyme also showed cross reactivity with other insecticides within the organophosphate group, and the kinetic parameters for individual substrates were investigated. Since MPH from B. cepacia has wide potential applications in detoxification and detection of organophosphate compounds, this study provides important basis for its future use.
Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Sanam-Chandra Palace Campus, Nakhon Pathom, 73000, Thailand.
Anirut
A
Ekkhunnatham
Boonsri
B
Jongsareejit
Wanphen
W
Yamkunthong
Jesdawan
J
Wichitwechkarn
D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000378:metabolism*; D007526:Isoelectric Point; D008743:Methyl Parathion / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010755:Organophosphates / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D013785:Thailand
D004269:DNA, Bacterial; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007306:Insecticides; D010755:Organophosphates; D011994:Recombinant Proteins; D008743:Methyl Parathion; D010744:Phosphoric Monoester Hydrolases