Purification of His6–organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography

Ефременко, Елена; Votchitseva, Yu. A.; Plieva, Fatima M.; Galaev, Igor Yu.; Mattìasson, Bo

doi:10.1007/s00253-005-0103-x

Cited by 65 publications

(31 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In the first case, the cryopolymerization proceeded in the non-frozen microphase remaining after freezing of the reaction feed and resulted in the formation of a macroporous structure with highly interconnected pores. It should be noted that such structures have been successfully used as chromatographic monolithic sorbents for chromatography of particulate-containing fluids [18,26,33,60], or microbial cells [28,30,61,62], or as cell culture reactors [41].…”

Section: Control Of Porous Propertiesmentioning

confidence: 99%

Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate‐containing fluids and cell culture applications

Plieva

Galaev

Mattìasson³

2007

J of Separation Science

180

134

View full text Add to dashboard Cite

Macroporous gels (MGs) with a broad variety of morphologies are prepared using the cryotropic gelation technique, i. e. gelation at subzero temperatures. These highly elastic hydrophilic materials can be produced from practically any gel-forming system with a broad range of porosity extending from elastic and porous gels with pore sizes up to 1.0 microm to elastic and sponge-like gels with pore sizes up to 100 microm. The versatility of the cryogelation technique is demonstrated by use of different chemical reactions (hydrogen bond formation, chemical cross-linking of polymers, free radical polymerization) mainly in an aqueous medium. Appropriate control over solvent crystallization (formation of solvent crystals) and rate of chemical reaction during the cryogelation allows the reproducible preparation of cryogels with tailored properties. Different approaches, such as chemical modification of reactive groups, grafting of the pore surface with an appropriate polymer, or direct copolymerization with functional monomers are used for control of the surface chemistry of MGs. Typically, MGs with pore sizes up to 1.0 microm are produced in the shape of beads and MGs with pore size up to 100 microm are prepared as monoliths, discs, and sheets. The difference in porous structure of MGs defines the main applications of these porous materials. Elastic beaded MGs are mostly used as carriers for cell and enzyme immobilization or for capture of low-molecular weight targets from particulate-containing fluids in expanded-bed mode. However, the elastic and sponge-like MG monoliths with interconnected pores measuring hundreds of mum have been successfully used as monolithic columns for chromatography of particulate-containing fluids (crude cell homogenates, viruses, whole cells, wastewater effluents) and as three-dimensional scaffolds for mammalian cell culture applications.

show abstract

Section: Control Of Porous Propertiesmentioning

confidence: 99%

Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate‐containing fluids and cell culture applications

Plieva

Galaev

Mattìasson³

2007

J of Separation Science

180

134

View full text Add to dashboard Cite

show abstract

“…Recombinant cells of Escherichia coli SG13009[pREP4] were cultivated, and the enzyme was isolated and purified by methods published previously [31]. The purified preparation of His 6 -OPH was characterized by enzymatic activity as described earlier [17].…”

Section: Preparation Of Enzyme Samplesmentioning

confidence: 99%

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

et al. 2017

View full text Add to dashboard Cite

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.

show abstract

“…Catalytic activity of His 6 -OPH was evaluated, as described earlier [28]. The rates of enzymatic hydrolysis of Paraoxon used as substrate was determined with UV-visible spectroscopy by the formation of p-nitrophenol (405 nm, ε405 = 18,000 M −1 cm −1 ) at 25 • C in 50 mM carbonate buffer (pH 10.5).…”

Section: Analysis Methodsmentioning

confidence: 99%

“…Cell biomass was suspended in 0.05 M K-phosphate buffer (pH 7.5), containing 0.3 M NaCl in ratio 1:5 and then homogenized by sonication on ice [28]. Cell debris containing PFH was removed by centrifugation for 20 min at 12,000 g and used again in the experiments.…”

Section: General Experimental Proceduresmentioning

confidence: 99%

Intensification of Organophosphorus Hydrolase Synthesis by Using Substances with Gas-Transport Function

et al. 2017

View full text Add to dashboard Cite

Abstract:We have performed studies and comparative analysis of the biosynthesis characteristics of intracellular recombinant enzyme, such as hexahistidine-containing organophosphorus hydrolase (His 6 -OPH) in Escherichia coli SG13009[pREP4] cells when various perfluorocarbon compounds (PFC) were introduced into the medium for cell cultivation. The PFC were found to facilitate the biosynthesis of His 6 -OPH: increased levels of the total OPH-activity (up to 37%) were measured upon introduction of 1,1,1,2,2,3,3,4,4,5,5,6,6,6-tetradecafluorohexane (PFH) and 4,7,10,13,16,19,22,25,28,31-decaoxaperfluoro-5,8,11,14,17,18,21,24,27,30-decamethyl tetratriacontane (Polyether II) into culture medium. We have demonstrated the possibility of effective and multiple (at least five-fold) use of PFH for biosynthesis of intracellular recombinant protein His 6 -OPH, which catalyzes the hydrolysis of organophosphorus pesticides (OP), is widely used in agriculture and can be applied as new antidote for OP-detoxification in vivo. The multiple use of PFH was achieved through recycling of this substance: sediment of Escherichia coli SG13009[pREP4] cell biomass was collected at the end of each culture growing step and disintegrated with ultrasound, and obtained residue containing almost all of the initially introduced PFC was then added to the medium at the start of the following culture growing step.

show abstract

Purification of His6–organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography

Cited by 65 publications

References 15 publications

Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate‐containing fluids and cell culture applications

Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate‐containing fluids and cell culture applications

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

Intensification of Organophosphorus Hydrolase Synthesis by Using Substances with Gas-Transport Function

Contact Info

Product

Resources

About