Purification and characterization of methyl parathion hydrolase from Burkholderia cepacia capable of degrading organophosphate insecticides

Abstract

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.

In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

“…Soluble OpdA has been shown to have an apparent melting temperature of 62°C; however, activity rapidly decreased after 55°C (10). No structural or stability data are currently available for OpdA from A. radiobacter; however, other similar phosphotriesterases have been shown to be stable up to 50°C and between pH 6.0 and 10.0 (31,32). The isoelectric points of BLA and NanA are 6.9 and 4.5, respectively (27,29), while the isoelectric point of a well-characterized homologous phosphotriesterase from Brevundimonas diminuta is 8.3 (33).…”

Enhancing methyl parathion degradation by the immobilization of Burkholderia sp. isolated from agricultural soils

“…There is a report in B. cepacia of the mpd gene coding for methyl parathion hydrolase, which is responsible for the hydrolysis of MP (Ekkhunnatham, Jongsareejit, Yamkunthong, & Wichitwechkarn, ). Vikram et al.…”

“…There is a report in B. cepacia of the mpd gene coding for methyl parathion hydrolase, which is responsible for the hydrolysis of MP (Ekkhunnatham, Jongsareejit, Yamkunthong, & Wichitwechkarn, 2012). Vikram et al (2012) found in the Burkholderia strain SJ98 genes encoding monooxygenases responsible for the degradation of aromatic compounds.…”

Organophosphate-degrading metallohydrolases: Structure and function of potent catalysts for applications in bioremediation

“…and Burkholderia sp. [107][108][109][110][111][112]. MPH does not share any sequence similarity to OPHs such as PTE or OpdA.…”