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Results for “Enzyme Stability”

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At least 865 records · Page 48Linked to original sources
PubMed2005

Encapsulation of glucose oxidase and an oxygen-quenched fluorophore in polyelectrolyte-coated calcium alginate microspheres as optical glucose sensor systems.

Microspheres coated with polyelectrolyte multilayers (PEM's) are being investigated for potential use as implantable biosensors-so-called "smart tattoos." In this work, the feasibility of this approach for glucose sensors was demonstrated by glucose oxidase encapsulated within calcium alginate microspheres, followed by entrapment of an oxygen-quenched ruthenium compound in the same microstructure. A novel feature of these microdevices is the formation of multilayer nanofilms on the surface of the microspheres, used to stabilize enzyme entrapment and control substrate diffusion. Confocal microscopy was used to confirm the stable encapsulation of sensor chemistry. The reversible response of sensors to step changes in glucose was observed, and preliminary experimental data were compared to theoretical predictions produced by a computational model. These findings demonstrate the promise of the described nanoengineering approach for production of functional implantable glucose sensor materials.

Alginates↗
PubMed2004

Use of chemically modified PMMA microspheres for enzyme immobilization.

Modified poly(methyl methacrylate) (PMMA) microspheres, about 7microm in diameter, carrying aldehyde groups on their surfaces were synthesized and used as the support for enzyme immobilization. The immobilizing behavior as well as the properties of immobilized enzyme was studied. The amount of bound enzyme can be extended to 76.8mg g(-1) support, which is relatively much higher than other supports. The kinetic investigation derived from three typical models shows that the practical process is more complicated than the ideal condition, with one or more interactions being involved in the immobilization process. The K(m) value is actually larger and V(max) is smaller in the immobilized form than those in the free form. The increased resistance of the immobilized enzyme against the changes of temperature indicates that immobilizing enzyme onto the modified microspheres is useful for enzyme immobilization.

Adsorption↗
PubMed2005

Dissipation and maintenance of stable states in an enzymatic system: analysis and simulation.

The constraint-based analysis has emerged as a useful tool for analysis of biochemical networks. An essential assumption for constraint-based analysis is the formation of a stable steady state. This work investigates dissipation and maintenance of stable states in a simple reversible enzymatic reaction with substrate inhibition. Under mass-action kinetics, the conditions under which the reaction maintains a stable steady state are analytically derived and numerically confirmed. It is shown that, in order to maintain a steady state in the regulated reaction, maximal enzyme activity must be much higher than input rate. Moreover, it is revealed that requirements for large enzyme activity are due to substrate inhibition. It is suggested that high activities of enzymes may play a vital role in protecting a stable state from its catastrophic collapse, giving an additional explanation to an intriguing problem--why the activities of some enzymes greatly exceed the flux capacity of a pathway. In addition, dissipation of the enzymatic reaction is analysed. It is shown that the collapse of stable states is always associated with a point at which dissipation is the highest. Therefore, in order to maintain a stable state, dissipation of the reaction must be less than a critical value. Moreover, although external forcing may not change net mass flow, it may lead to collapse of stable states. Furthermore, when stable states collapse at a critical forcing amplitude and period, dissipation also reaches a highest value. It is concluded that collapse of stable steady state in the enzyme system with substrate inhibition always corresponds to critical points at which dissipation is highest, regardless if the reaction is forced or not. Therefore, for the substrate inhibited reaction, maintenance of stable states is intrinsically related to level of dissipation.

Computer Simulation↗
PubMed2004

Recent progress in biocatalyst discovery and optimization.

The use of enzymes in industrial catalysis continues to grow because of the considerable advantages of natural catalytic systems. The need for enantiomerically pure fine chemicals and the movement away from chemically burdened technologies will drive the acceptance of enzyme-assisted processes. New technologies for enzyme discovery and optimization have enabled the application of enzymes in harsh industrial conditions and in processes demanding stringent selectivity. These discovery and laboratory evolution methods entail genomic approaches that by their nature engender screening of extremely large numbers of gene types and variants. By extension, the fitness of an individual high-throughput screen requires an intelligent, process-targeted assay amenable to a chosen screening platform.

Biotechnology↗
PubMed2005

Recent progress towards the application of hyperthermophiles and their enzymes.

The discovery of extremophiles has drastically changed our understanding towards the diversity of life itself and the conditions under which it can be sustained. Extremophiles have evolved to withstand and multiply under the extremes of temperature, pressure, pH and salinity. Hyperthermophiles are the group that have adapted to high temperature; many have been found to grow at temperatures above the boiling point of water. This review focuses on recent advances in application-based research on hyperthermophiles and their thermostable enzymes.

Archaea↗
PubMed2005

Immobilised enzymes: science or art?

Enzyme immobilisation is experiencing an important transition. Combinatorial approaches are increasingly applied in the design of robust immobilised enzymes by rational combination of fundamental immobilisation techniques (i.e. non-covalent adsorption, covalent binding, entrapment and encapsulation) or with other relevant technologies. The objective is to solve specific problems that cannot be solved by one of these basic immobilisation techniques.

Combinatorial Chemistry Techniques↗
PubMed2005

Isolation, characterization, and cDNA-derived amino acid sequence of glycocyamine kinase from the tropical marine worm Namalycastis sp.

We isolated cytoplasmic glycocyamine kinase (GK) and creatine kinase (CK) from the tropical marine worm Namalycastis sp. by ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-5PW chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the isolated GK is highly purified and appears to be a heterodimer of two distinct subunits, alpha and beta, with molecular masses of approximately 40 kDa. The complete nucleotide sequences of the cDNAs for Namalycastis GKalpha and GKbeta were 1527 (encoding 374 amino acids) and 1579 bp (encoding 390 amino acids), respectively. The predicted amino acid sequences differ only in the N-terminal 50 residues. This is consistent with the characteristics of Neanthes GKalpha and GKbeta chains, which we have previously shown to be generated by alternative splicing. The recombinant enzymes GKalpha, GKbeta, and CK from Namalycastis were successfully expressed in Escherichia coli as maltose-binding protein fusion proteins. In contrast to the stable GKbeta enzyme, GKalpha was quite unstable, and its activity decreased remarkably with time. Thus, the N-terminal 50 residues appear to play a key role in enzyme stability. The kinetic parameters for the native GK heterodimer were similar to GKbeta, suggesting that GKalpha would have an activity similar to GKbeta if part of a heterodimer. This is the first report of precise kinetic parameters for GK. Finally, based on our results, we present a model for pluriphosphagen function in Namalycastis wherein cytoplasmic GK and CK and mitochondrial CK function together with phosphocreatine and phosphoglycocyamine to enable cells to respond quickly to a sudden large energy requirement.

Amino Acid Sequence↗
PubMed2004

Absence of telomerase and shortened telomeres have minimal effects on skin and pancreatic carcinogenesis elicited by viral oncogenes.

The telomere-stabilizing enzyme telomerase is induced in tumors and functionally associated with unlimited replicative potential. To further explore its necessity, transgenic mice expressing SV40 or HPV16 oncogenes, which elicit carcinomas in pancreas and skin, respectively, were rendered telomerase-deficient. Absence of telomerase had minimal impact on tumorigenesis, even in terc(-/-) generations (G5-7) exhibiting shortened telomeres and phenotypic abnormalities in multiple organs. Analyses of chromosomal aberrations were not indicative of telomere dysfunction or increased genomic instability in tumors. Quantitative image analysis of telomere repeat intensities comparing biopsies of skin hyperplasia, dysplasia, and carcinoma revealed that telomere numbers and relative lengths were maintained during progression, implicating a means for preserving telomere repeats and functionality in the absence of telomerase.

Anaphase↗
PubMed2005

RGS2 interacts with Gs and adenylyl cyclase in living cells.

Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Protein-tagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gsalpha, or the Gs-coupled beta2-adrenergic receptor (beta2AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gsalpha-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACVI fused to Renilla luciferase. Additionally, RGS2 was found to interact with the beta2AR. Purified RGS2 selectively bound to the third intracellular loop of the beta2AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and beta2AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production.

Adenylyl Cyclases↗
PubMed2005

Cyanide detoxification by recombinant bacterial rhodanese.

Cyanide is a major environmental pollutant of the chemical and metallurgical industries. Although extremely toxic, cyanide can enzymatically be converted to the less toxic thiocyanate by rhodaneses (thiosulfate:cyanide sulfurtransferases, EC 2.8.1.1). We engineered a genetic system to express high levels of recombinant Pseudomonas aeruginosa rhodanese (r-RhdA) in Escherichia coli, and used this organism to test the role of r-RhdA in cyanide detoxification. Inducible expression of the rhdA gene under the control of the hybrid T7-lacO promoter yielded active r-RhdA over a 4-h period, though r-RhdA-expressing E. coli showed decreased viability starting from 1 h post-induction. At this time, Western blot analysis and enzymatic assay showed r-RhdA partition between the cytoplasm (95%) and the periplasm (5%). The accessibility of thiosulfate to r-RhdA was a limiting step for the sulfur transfer reaction in the cellular system, but cyanide conversion to thiocyanate could be increased upon permeabilization of the bacterial membrane. Specific r-RhdA activity was higher in the whole-cell assay than in the in vitro assay with pure enzyme (2154 vs. 816 micromol min-1 mg-1 r-RhdA, respectively), likely reflecting enzyme stability. The r-RhdA-dependent cyanide detoxification resulted in increased resistance of r-RhdA overexpressing E. coli to 5 mM cyanide. Bacterial survival was paralleled by release of thiocyanate into the medium. Our results indicate that cyanide detoxification by engineered E. coli cells is feasible under laboratory conditions, and suggest that microbial rhodaneses may contribute to cyanide transformation in natural environments.

Biodegradation, Environmental↗
PubMed2005

Comparative activity of doripenem and three other carbapenems tested against Gram-negative bacilli with various beta-lactamase resistance mechanisms.

Doripenem (formerly S-4661), a novel carbapenem antimicrobial, was compared with ertapenem, imipenem, and meropenem using reference broth microdilution test methods against wild-type and various resistant microbial subsets (380 strains). Doripenem and meropenem were consistently more potent than ertapenem or imipenem when tested against Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. Ertapenem exhibited minimum inhibitory concentration (MIC) elevations for some isolates producing AmpC and extended-spectrum beta-lactamases, in contrast to greater enzyme stability for doripenem and other carbapenems tested. Multiple beta-lactamase (TEM, SHV, CTX-M, OXA, CMY types)-producing Escherichia coli had doripenem MIC values at </=0.016 mug/mL (with higher values for ertapenem). Doripenem appears to be a promising, potent carbapenem for parenteral use against contemporary Gram-negative bacilli producing various beta-lactamases.

Anti-Bacterial Agents↗
PubMed2004

Chemical and enzymatic stability evaluation of lipoamino acid esters of idebenone.

Lipophilic conjugates of idebenone (IDE) with short-chain alkylamino acids were previously synthesized and evaluated in vitro for their antioxidant properties. In this study, their susceptibility to chemical and enzymatic hydrolysis was evaluated. Results indicated that these derivatives release the parent drug quantitatively via enzymatic hydrolysis by serum and liver esterases, with a cleavage rate related to the length of the alkyl side chain. Consequently, the present lipoamino acid conjugates of IDE are prodrugs and their in vivo effects are mediated through the parent compound released in the body.

Amino Acids↗
PubMed2006

Extended release of high pI proteins from alginate microspheres via a novel encapsulation technique.

Alginate has potential as a matrix for controlled delivery of protein-based drugs that require site-specific long-term delivery. In the current work albumin, lysozyme and chymotrypsin were encapsulated into alginate microspheres using a novel method that involved soaking the microspheres in a protein-containing NaCl solution. This was followed by recrosslinking with calcium chloride. High pI proteins also appeared to physically crosslink the sodium alginate which resulted in more sustained release. Release was affected by the nature of the releasate solution. In TRIS buffered saline, the high pI proteins chymotrypsin and lysozyme showed sustained release lasting over 150 h. Release into 0.15% NaCl led to relatively constant release of lysozyme and chymotrypsin over more than 2000 h; reduction of the releasate volume lengthened the lysozyme release to greater than 8 months. Released lysozyme was shown to remain active for at least 16 days, in some cases with activity greater than 100% of the active control. This encapsulation technique can therefore be used to rapidly load alginate microspheres with proteins, with high isoelectric point proteins showing particular promise. Furthermore, the interactions between the high pI proteins and the alginate gel could potentially be exploited to generate new protein delivery systems.

Alginates↗
PubMed2005

Application of TZERO calibrated modulated temperature differential scanning calorimetry to characterize model protein formulations.

The objective of this study was to evaluate the feasibility of using T(ZERO) modulated temperature differential scanning calorimetry (MDSC) as a novel technique to characterize protein solutions using lysozyme as a model protein and IgG as a model monoclonal antibody. MDSC involves the application of modulated heating program, along with the standard heating program that enables the separation of overlapping thermal transitions. Although characterization of unfolding transitions for protein solutions requires the application of high sensitive DSC, separation of overlapping transitions like aggregation and other exothermic events may be possible only by use of MDSC. A newer T(ZERO) calibrated MDSC model from TA instruments that has improved sensitivity than previous models was used. MDSC analysis showed total, reversing and non-reversing heat flow signals. Total heat flow signals showed a combination of melting endotherms and overlapping exothermic events. Under the operating conditions used, the melting endotherms were seen in reversing heat flow signal while the exothermic events were seen in non-reversing heat flow signal. This enabled the separation of overlapping thermal transitions, improved data analysis and decreased baseline noise. MDSC was used here for characterization of lysozyme solutions, but its feasibility for characterizing therapeutic protein solutions needs further assessment.

Buffers↗
PubMed2006

Periodically interrupted amperometry. A way of improving analytical performance of membrane coated electrodes.

Amperometry is a powerful voltammetric measuring method. Its application is specially advantageous when used in combination with a separation step or with some other sample treatment method providing selectivity. The selectivity is often achieved by coating the amperometric working electrode surface with a membrane of special character. Size exclusion membrane, immobilized enzyme containing reaction layer, protecting dialysis membrane, perm selective ion exchange film etc can be mentioned here. In conventional amperometry the measuring potential is continuously applied, therefore in case of membrane coated electrodes the electrode process depletes the diffusion layer. In this work the performance of a new periodically interrupted amperometric (PIA) measuring program has been investigated in case of glucose enzyme sensor. The measuring program allowing time for reloading the diffusion layer provided higher current and therefore improved sensitivity and lower limit of detection.

Biosensing Techniques↗
PubMed2004

Purification and partial characterization of an acidic polygalacturonase from Aspergillus kawachii.

An endo-polygalacturonase, named PGI, was purified to homogeneity from the culture filtrate of Aspergillus kawachii IFO 4033 grown in a glucose-tryptone medium. The molecular mass of PGI was estimated to be 60 kDa by SDS-PAGE and 40 kDa by gel filtration on Sephacryl S-100. The isoelectric point was 3.55 as determined by isoelectic focusing. PGI exhibited binding properties to ConA-Sepharose suggesting that the protein is glycosylated. The N-terminal amino acid sequence was also determined as S-T-C-T-F-T-D-A-A-T-A-S-E-S-K. The remarkable property of PGI was its high activity in the pH range 2.0-3.0 towards soluble and insoluble substrates, while being inactive at pH 5.0. Enzyme stability at low pHs was markedly enhanced by different compounds, such as proteins, polysaccharides, simple sugars and the substrate pectin. PGI was very efficient to extract pectin from lemmon protopectin and to macerate carrot tissues at pH 2.0. These properties make PGI an interesting biocatalyst for industrial applications under highly acidic conditions.

Amino Acid Sequence↗
PubMed2005

Investigations of the transfructosylation reaction by fructosyltransferase from B. subtilis NCIMB 11871 for the synthesis of the sucrose analogue galactosyl-fructoside.

The exo-fructosyltransferase produced from B. subtilis NCIMB 11871 strain transfers the fructose moiety from donor alpha12 linked saccharides such as sucrose, raffinose and stachyose to the acceptor d-galactose, leading to the sucrose analogue, galactosyl-fructoside. Here, we report detailed kinetic studies. The enzyme showed a remarkably high optimal temperature at 50 degrees C and was effectively immobilised on Eupergit C 250 L and Trisopor-Amino. This is also the first report about the equilibrium of the transfructosylation reaction, its activation energy determination, the structure of the product and its preparative scale isolation.

Bacillus subtilis↗