Properties of hexahistidine-tagged organophosphate hydrolase

Votchitseva, Yu. A.; Ефременко, Елена; Алиев, Т. К.; Varfolomeyev, S.D.

doi:10.1134/s0006297906020088

Cited by 43 publications

(31 citation statements)

References 24 publications

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“…Because most biological based assays have the influence of temperature, pH and substrate concentration on the enzyme activity. Free enzyme was showing maximum activity at pH.7.5 and temperature 35 o C. Our results are correlated well with the previously reported studies 24,26,27 . Most biological-based assays are influenced by the assay temperature.…”

Section: Discussionsupporting

confidence: 82%

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Extraction and Purification of Organophosphorus hydrolase Enzyme from Soil Microorganism Pseudomonas diminuta

2017

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Synthetic organophosphorus compounds are highly toxic hence, widely used as pesticides, insecticides and chemical warfare agents. Organophosphorus hydrolase (OPH) is an organophosphotriester hydrolysing enzyme; effectively hydrolyse a range of organophosphate esters. The objective of the present study was extraction and purification of OPH enzyme from Pseudomonas diminuta bacteria (soil microorganism) and to study kinetic properties of the purified enzyme. Enzyme was extracted and purified from bacteria by ammonium sulphate precipitation and ion exchange chromatography. Purity of an enzyme was determined by sodium dodecyl sulphate-polyacryamide gel electrophoresis (SDS-PAGE). Purified OPH enzyme specific activity was found to be 27.7 fold of 32.8 U/mg protein, molecular weight of 72 Kda and it is a homodimer since it has shown a single band in SDS-PAGE separation. Maximum activity of the free OPH enzyme was found at Optimum pH 7.5 and temperature 35 o C with the incubation time of 10 min. Michaelis constant (K m ) and maximum velocity (V max ) values of free OPH enzyme for methyl parathion as substrate was found to be 286.2 µM and 2.5 µM/min respectively.

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Section: Discussionsupporting

confidence: 82%

“…Under these conditions, there was no free enzyme present and the concentration of enzyme-substrate complex was the total enzyme concentration present. Michaelis constant (K m ) and maximal velocity (V max ) values of free OPH enzyme for methyl parathion as substrate which were compared with previously reported methods 26,27 .…”

Section: Discussionmentioning

confidence: 99%

Extraction and Purification of Organophosphorus hydrolase Enzyme from Soil Microorganism Pseudomonas diminuta

2017

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“…The K m values indicate a high affinity of urate oxidase for uric acid as substrate and are comparable with C. utilis (33.7 μ M ) and Bacillus fastidiosus (180 μ M ) [Bongaerts et al, 1978;Liu et al, 2011]. The reason for differences in pH, temperature profiles and kinetic parameters of Auox6hp may be caused by the C-terminal HisTag which can have influence on the protein structure and protein charge [Votchitseva et al, 2006]. The enzymatic activity of both recombinant and endogenous urate oxidase was stable up to 50 ° C for at least 1 h. This physicochemical property will be useful in using urate oxidase to treat heated food products.…”

Section: Discussionmentioning

confidence: 88%

<b><i>Arxula adeninivorans</i></b> Recombinant Urate Oxidase and Its Application in the Production of Food with Low Uric Acid Content

Trautwein-Schult

Jankowska

Cordes³

et al. 2013

Microb Physiol

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Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food.

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“…The purified preparation of His 6 -OPH was characterized by enzymatic activity as described earlier [17]. The concentration of protein was determined by Bradford assay, and protein purity was analyzed by SDS-PAGE in 12% polyacrylamide gel using Mini-PROTEAN II cell (Bio-Rad, Hercules, CA, USA) followed by Coomassie Blue (R-250) staining.…”

Section: Preparation Of Enzyme Samplesmentioning

confidence: 99%

“…The organophosphorus hydrolase activity was determined as described earlier [17] with 7.7 mM aqueous Paraoxon stock solution at 405 nm using the Agilent 8453 UV-visible spectroscopy system (Agilent Technology, Waldbronn, Germany) equipped with a thermostatted analytical cell. The reaction was carried out in a 0.1 M carbonate buffer (pH 10.5).…”

Section: Measurement Of Enzyme Activitymentioning

confidence: 99%

Catalytic Characteristics of New Antibacterials Based on Hexahistidine-Containing Organophosphorus Hydrolase

et al. 2017

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Catalytic characteristics of hexahistidine-containing organophosphorus hydrolase (His 6 -OPH) and its enzyme-polyelectrolyte complexes with poly-L-glutamic acid or poly-L-aspartic acid (His 6 -OPH/PLD 50 ), hydrolyzing organophosphorous compounds, and N-acyl homoserine lactones were studied in the presence of various antibiotics (ampicillin, gentamicin, kanamycin, and rifampicin). The antibiotics at concentrations below 1 g·L −1 had a negligible inhibiting effect on the His 6 -OPH activity. Mixed inhibition of His 6 -OPH was established for higher antibiotic concentrations, and rifampicin was the most potent inhibitor. Stabilization of the His 6 -OPH activity was observed in the presence of antibiotics at a concentration of 0.2 g·L −1 during exposure at 25-41 • C. Molecular docking of antibiotics to the surface of His 6 -OPH dimer revealed the antibiotics binding both to the area near active centers of the enzyme subunits and to the region of contact between subunits of the dimer. Such interactions between antibiotics and His 6 -OPH were verified with Fourier-transform infrared (FTIR) spectroscopy. Considering all the results of the study, the combination of His 6 -OPH/PLD 50 with β-lactam antibiotic ampicillin was established as the optimal one in terms of exhibition and persistence of maximal lactonase activity of the enzyme.

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Properties of hexahistidine-tagged organophosphate hydrolase

Cited by 43 publications

References 24 publications

Extraction and Purification of Organophosphorus hydrolase Enzyme from Soil Microorganism Pseudomonas diminuta

Extraction and Purification of Organophosphorus hydrolase Enzyme from Soil Microorganism Pseudomonas diminuta

<b><i>Arxula adeninivorans</i></b> Recombinant Urate Oxidase and Its Application in the Production of Food with Low Uric Acid Content

Catalytic Characteristics of New Antibacterials Based on Hexahistidine-Containing Organophosphorus Hydrolase

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