Department of Biology, Karadeniz Technical University, 61080 Trabzon, Turkey
PROCESS BIOCHEMISTRY (Impact Factor: 2.52). 12/2009; 44(12):1366-1373. DOI: 10.1016/j.procbio.2009.07.014
Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase. Phosphotriesterase is a hydrolytic, bacterial enzyme with unusual substrate specificity for synthetic organophosphate triesters, common constituents of chemical warfare agents and agricultural pesticides. PHP may belong to the family of proteins from which phosphotriesterase evolved. The PHP gene from the thermophilic bacterium Geobacillus caldoxylosilyticus TK4 was cloned and overexpressed in Escherichia coli with 6×His tag in the N-terminal. The recombinant protein was purified with nickel affinity chromatography and characterized in detail. The enzyme did not have any activity against paraoxon. The highest activities were observed with p-nitrophenyl acetate (pNPA) and p-nitrophenyl butyrate. pH and temperature optima were determined as 8.0 and 50 °C, respectively, with pNPA. The enzyme activity was not largely affected by the incubation of the enzyme at 50 °C in the different buffer solutions (pHs between 3.0 and 9.0) for 7 days. After the incubation at 90 °C for 7 days, G. caldoxylosilyticus TK4 PHP retained 62% of its original activity. The enzyme was also resistant to some metal ions and organic solvents. These results suggest that this is the first reported PHP having an extremely pH- and thermo-stable esterase activity.
") parathion hydrolase (P0A433), Flavobacterium sp. MTCC 2495 organophosphorus hydrolase (Q5UB52), Homo sapiens phosphotriesterase-related protein (Q96BW5), Rattus norvegicus phosphotriesterase-related protein (Q63530), Mus musculus phosphotriesterase-related protein (A2AUR4), E. coli PHP (P45548) and M. tuberculosis PHP (P96413) with the maximum parsimony method (Yildirim et al., 2009) "
[Show abstract][Hide abstract] ABSTRACT: Organophosphorus (OP) pesticides evolved in the 1940s as chemical warfare agents and subsequently manufactured into formulations to be used as insecticides both in the agriculture and domestic sectors. The user of OPs are limited up to 1960 since the organochlorines were considered more potent killer of insect pests and the persistence of these chemicals were unknown. After the discovery of environmental effects and recalcitrance of DDT in 1960, attention was given to the chemicals which are more effective and less persistent (sort half life) in the environment. The OP chemicals satisfied both the criteria and gradually replaced OC chemicals as pest control agents. Since then OP insecticides have been used in large quantities throughout the world. Problem of contamination resulting from surplus pesticides and the wastewater from pesticide manufacturing units have become obvious. The transformation of pesticides in the environment results from physicochemical reactions as well as the activities from cellular extracellular components of biota (microorganisms, plants and animals), but principal biodegradation pathway is microbial degradation.
Biodegradation of insecticides plays an important role in determining the efficiency and application of the pest control operations. Biodegradation of OP chemicals by different organisms has been reported much before the exact mechanism of the process was developed. The current research evidences suggest that OP chemicals are degraded by three important pathways viz- 1) Reduced glutathione (GSH) dependent decarboxylation or dearylation, 2) P450 enzymes mediated microsomal oxidation and 3) Phosphotriesterase mediated deesterification.
Applenten and Nakatsugawa (1972) observed the cleavage of ester linkage as an effective pathway of degradation of OP insecticides and such hydrolytic breakdown was found to be catalyzed by an enzyme which does not have any natural substrate. The enzymes are now popularly called as Phosphotriesterases (PTEs). Since its discovery a lot of informations have been gathered on the origin, structure, catalytic activity, evolution, specificity and application of the enzymes but there is hardly any literature to give an in-depth information about this novel enzyme. This review provides an exhaustive information on the PTEs and its role in human welfare.
"In contrast to our results, esterase from G. thermoleovorans YN and G. caldoxylosilyticus TK4 was strongly inhibited in the presence of Zn 2? (Yildirim et al. 2009; Soliman et al. 2007). The most effective increase of esterase activity (*25%) was observed in the presence of NaCl. "
[Show abstract][Hide abstract] ABSTRACT: Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E.coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27.5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC(2)) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5-10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.
[Show abstract][Hide abstract] ABSTRACT: Bombyx mori cytoplasmic polyhedrosis virus is one of the major viral pathogens for the silkworm. The immune response of silkworm to the virus infection is obscure. A phosphotriesterase-re-lated protein gene of silkworm, Bombyx mori (BmPTERP) was found in our previous mi-croarry analysis of the midgut infected with the virus. In the present study, we cloned and ana-lyzed the full-length cDNA of BmPTERP gene by means of rapid amplification of complementary DNA ends (RACE) and bioinformatic analysis for exploring its functions in interaction between the silkworm and the virus. The nucleotide se-quence of the gene is 1349-bp and contains a 131 bp 5'UTR and a 165 bp 3'UTR. The 1053 bp open reading frame encodes a 350 amino acid protein. The deduced protein contains specific hits of phosphotriesterase-related proteins and belongs to the amidohydrolase superfamily. RT-PCR analysis revealed that BmPTERP gene was expressed in all the tissues tested, including midgut, hemocyte, gonad, fat body and silk gland. Real-time quantitative polymerase chain reac-tion analysis indicated that the relative tran-script of BmPTERP gene in the infected midgut was 19.32 fold lower than that in normal midgut at 72 hours post inoculation.
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