Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Enzyme Stability”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,099 records · Page 61Linked to original sources
PubMed1998

Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction.

We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 microgram = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25 degrees C) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

Animals↗
PubMedDate not supplied

p-Nitrophenetole deethylase activity of rat liver microsomes entrapped in polyelectrolyte capsules.

Liver microsomes from phenobarbital induced rats are entrapped in capsules prepared from polyelectrolytes. A comparative analysis of the deethylase activity against p-nitrophenetole by encapsulated and freely suspended microsomes is carried out. The pH optimum occurs at about 7.2 for encapsulated as well as free microsomes. The pH activity profile of encapsulated microsomes, however, is strongly flattened. The maximal velocity of entrapped microsomes is about a quarter of that of free microsomes. The Michaelis-Menten constants are virtually equal. Despite of the lowered activity of encapsulated microsomes this kind of immobilization of enzymes may be useful in biotechnology and medicine because mild immobilization conditions like room temperatures and aqueous solutions are realized.

Animals↗
PubMed1995

Viability and enzymatic activity of bifidobacteria in milk.

Bifidobacterium breve NCFB 2258, Bifidobacterium bifidum NCFB 2715, Bifidobacterium longum ATCC 15707, Bifidobacterium angulatum ATCC 27535, and Lactobacillus acidophilus N2 were evaluated for viability and beta-galactosidase and alpha-galactosidase activities under conditions of refrigerated and frozen storage in reconstituted NDM. beta- and alpha-Galactosidase activities were variable. Bifidobacterium angulatum 27535 exhibited much greater enzymatic activities than those of the other strains. All strains demonstrated culture and enzyme stability upon refrigerated storage in fermented and unfermented reconstituted NDM. Bifidobacteria were significantly less tolerant of low temperature storage than was L. acidophilus N2.

Animals↗
PubMed1998

Heat stability of virulence-associated enzymes from Listeria monocytogenes SLCC 5764.

The enzyme activities of listeriolysin O (LLO), phosphatidylinositol-specific phospholipase C (PI-PLC), catalase (CA), and superoxide dismutase (SOD) from Listeria monocytogenes SLCC 5764 were examined after heat treatment, growth at elevated temperatures, and anaerobic growth. Growth at elevated temperatures may influence virulence as expressed by virulence enzyme activity. The enzymes were heated for 0 and 10 min at temperatures ranging from 40 to 100 degrees C. The production of LLO was examined when the bacterium was grown at elevated temperatures, and the production of LLO, CA, and SOD were examined after anaerobic growth. LLO was the most heat-labile factor, losing almost all activity when heated above 50 degrees C. CA was more heat stable, showing no decrease in activity until it was heated at 55 degrees C. The PI-PLC enzyme was most heat resistant: the activity decreased between 40 and 50 degrees C and continued to decrease when heated between 65 and 100 degrees C. When L. monocytogenes was grown at elevated temperatures, it produced less LLO than when grown at the optimum growth temperature of 37 degrees C. When the organism was grown under anaerobic conditions, the levels of CA and LLO decreased, while the SOD level remained unchanged.

Bacterial Toxins↗
PubMed2002

Disrupting Escherichia coli: a comparison of methods.

The often-encountered problem of disrupting bacteria for the purpose of extracting soluble protein has generated various methods. Many require specialized equipment. Very often, especially during preliminary studies, investigators need a simple, fast, and inexpensive method for cell disruption that preserves biological activity. This paper compares some simple and inexpensive methods for cell disruption, such as bead-vortexing, freezing-thawing, French pressing, and sonication. It also provides some tips to increase protein yield and preserve biological activity. If performed under optimal conditions, bead-vortexing gives protein yields that are comparable to French pressing and sonication. It also preserves the activities of labile enzymes and releases periplasmic enzymes. Vortexing with glass beads appears to be the simplest method for cell disruption.

Bacteriological Techniques↗
PubMed1975

A spectrophotometric assay for gentamicin.

A rapid and accurate spectrophotometric assay has been developed for the determination of blood werum levels of gentamicin and related antibiotics. The assay uses a purified enzyme from Escherichia coli JR88/C600 that acetylates gentamicin with the production of coenzyme A, linked to a chemical reaction with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore, monitored in the visible spectrum. The system provides advantages of speed, cost, convenience, accuracy, and enzyme stability to the desirable characteristics encountered with previous enzymatic methods.

Acetyltransferases↗
PubMed1998

Bilayer permeability-based substrate selectivity of an enzyme in liposomes.

Liposomes were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which contained the water soluble proteinase alpha-chymotrypsin. This liposome entrapped enzyme showed selectivity for externally added substrates in that only small substrates (benzoyl-l-Tyr-p-nitroanilide or acetyl-l-Phe-p-nitro-anilide)-for which the liposome bilayer was permeable-were transformed into products. Large substrates (succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide or casein) could not penetrate from the external aqueous phase into the liposomes, and were not hydrolyzed. This substrate selectivity is entirely based on the compartimentation and permeability properties of the liposome microreactor.

Biotechnology↗
PubMed1998

Site-directed and random immobilization of subtilisin on functionalized membranes: activity determination in aqueous and organic media.

Kinetic comparisons have been made between a randomly immobilized and a site-specifically immobilized subtilisin BPN' on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site-directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild-type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica-containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site-directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site-directed immobilized systems using nearly anhydrous hexane as the solvent.

1-Butanol↗
PubMed1999

pH gradients in immobilized amidases and their influence on rates and yields of beta-lactam hydrolysis.

The pH gradients developing within immobilized biocatalysts during hydrolysis of penicillin G and glutaryl-7-aminocephalosporanic acid have been estimated both theoretically and experimentally. For the latter a fluorimetric method for the direct measurement of the average pH value within the carrier during reaction has been developed using the pH-dependent fluorescence intensity of an enzyme-bound fluorophore determined with a fiber bundle. The theoretical calculations were based on a model for the hydrolysis with immobilized enzymes using a kinetic expression with five pH-dependent, measurable kinetic and equilibrium constants. The transport reaction differential equation which considers the laminar boundary layer has been solved numerically for the key component. The calculated values agreed well with the experimental data. Under the typical reaction conditions of penicillin G hydrolysis the average pH value in the carrier was 1 and 2.5 pH units below the bulk pH (=8) with and without buffer, respectively. The corresponding changes for the hydrolysis of glutaryl-7-aminocephalosporanic acid at bulk pH 8 in the presence of buffer was 0.5. This demonstrates the existence of considerable pH gradients in carriers during hydrolytic reactions, even in buffered systems with negligible mass transfer resistance. The low pH value causes suboptimal reaction rates, reduced equilibrium conversion, and reduced enzyme stability. These pH gradients can be minimised by using buffers with pK values approximately equal to the bulk pH used for the hydrolysis. The prediction quality of the model has been tested applying it to fixed bed reactor design. The reduction in rate and yield due to concentration and pH gradients can be overcome with simple measures such as high initial pH value and pH adjustments in segmented or recycling fixed bed reactors. Thus, enzymatic conversions with high yield and high operational effectiveness are achieved.

Amidohydrolases↗
PubMed1999

A novel efficient enzyme-immobilization reaction on NH2 polymers by means of L-ascorbic acid.

A new enzyme-immobilization reaction by means of L-ascorbic acid (ASA) is described using NH(2) polymers based on cellulose or poly(vinyl alcohol) with the example of oxidoreductase enzymes. In this way, enzyme proteins such as glucose oxidase (GOD), glutamate oxidase, lactate oxidase, urate oxidase and peroxidase can be covalently fixed with a high surface loading to ultrathin and transparent NH(2)-polymer films if their surfaces are previously treated with an ASA solution, in, for example, N,N-dimethyl acetamide, DMSO or methanol. ASA then obviously reacts like a diketo compound with amino groups of the NH(2)-polymer film and enzyme protein, forming dehydroascorbic acid derivatives with neighbouring Schiff's-base structures. In a subsequent fragmentation reaction, the latter presumably form stable oxalic acid diamide derivatives as coupling structures between enzyme protein and NH(2)-polymer film, as suggested by results from investigations of the ASA reaction with n-butylamine. The immobilized enzymes can be stored at 4 degrees C in bidistilled water for at least 1 month without becoming detached from the NH(2)-polymer film and without diminished enzyme activity. The apparent K(m) values of the immobilized enzymes are in part clearly smaller than those of the dissolved enzymes or those found in other immobilization processes such as the diazo coupling or the bifunctional glutardialdehyde reaction. For example, the K(m) value of the immobilized GOD with different NH(2) polymers as the matrix structure is smaller by a factor of approx. 20 than that of the dissolved enzyme.

Acetamides↗
PubMed2000

Preparation, morphological characterization, and activity of thin films of horseradish peroxidase.

Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/SiO(2) or glass supports previously activated by silanization and succinylation. Labeling by fluorescent or by Electron Spin Resonance (ESR) probes was used to quantify the surface density of active groups and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non-uniform protein adsorption due to physical interactions was present when the supports were not activated for covalent binding and was, in large part, removed by washing. The enzyme deposited by covalent binding formed homogeneous layers with a height in the range 60-90 A. By using a fluorescent label, we calculated a protein density of 3.6 x 10(12) molecules cm(-2) on Si/SiO(2), corresponding to an estimated area per molecule of 2800 A(2) which is in agreement with the value expected on the basis of the crystallographic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 x10(12) molecules cm(-2)). The enzyme immobilized on both supports showed a k(cat)/K(M) being of the order of 3-5x10(5) M(-1)s(-1) that is 1/20th of free HRP. The half-life time of the activity of the enzyme immobilized by covalent binding was longer than 40 days at 6 degrees C.

Bisphenol A-Glycidyl Methacrylate↗
PubMed2001

Optimization of production and downstream processing of the almond beta-glucosidase-mediated glucosylation of glycerol.

This article describes the synthesis of glyceryl glucoside from glycerol and glucose with almond beta-glucosidase as the catalyst. A yield of 54% (0.45 mmol/g) was obtained. The influence of the enzyme stability, the water concentration, and the water activity on the glucoside yield were determined. A molar fraction-based equilibrium constant of 2.4 +/- 0.6 was found, with which the glucoside yield could be calculated for all possible combinations of initial substrate and water fractions in the reaction mixture. A model was used to optimize the glucoside yield while minimizing one of the substrate concentrations at equilibrium. This straightforward model gives a good prediction of the measured glucoside yield, according to a parity plot.

Glucose↗
PubMed1975

Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties.

Kidney alkaline phosphatase was purified to homogeneity. It is a glycoprotein of about 172,000 molecular weight. Analyses of the subunit structure by sedimentation equilibrium in 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate indicate that the alkaline phosphatase is a dimer comprising two very similar or identical subunits of about 87,000 molecular weight. The native enzyme contains 4.5 +/- 0.2 g atoms of zinc per mol of protein. Reconstitution experiments from the apophosphatase show that binding of 4 Zn2+ per mol of dimer is essential for full activity. The kinetic data of Zn2+ binding to the apoprotein require at least a two-step mechanism, in which one of the steps corresponds to a conformational change within the enzyme. This paper also presents data concerning amino acid composition, sugar content, enzyme stability, absorbance index, and sedimentation velocity.

Alkaline Phosphatase↗
PubMed1975

Effect of methotrexate on thymidylate synthetase in cultured parenchymal cells isolated from regenerating rat liver.

The effect of methotrexate (MTX) on thymidylate synthetase activity during liver regeneration was examined with parenchymal cells isolated 22 and 44 hr after partial hepatectomy and cultured as a monolayer. The synthetase activity in these cells decreased with a half-life of 18 to 24 hr, but if MTX (1.5 X 10(-6) to 1.5 x 10(-5) M) was present in the culture media, this decline could be delayed for at least 48 hr. In contrast, thymidine kinase activity decreased at a rate which we unaffected by MTX. Dihydrofolate reductase was inhibited at all concentrations of MTX used to block the decrease in synthetase activity. Folic acid at 10(-4) M, although less effective than MTX, also delayed the decrease in synthetase activity. The addition of cycloheximide, puromycin, or antinomycin D to the culture media did not alter the response of the synthetase to MTX. The latter studies, coupled with those indicating that the rapid loss of synthetase activity in crude extracts could be prevented by MTX or, more effectively, by MTX plus deoxyuridine 5'-monophosphate, suggest that the primary effect of MTX on thymidylate synthetase in vivo is that of enzyme stabilization. Similar stabilizing effects were obtained in liver cell extracts with 10(-5) M deoxyuridine 5'-monophosphate in combination with 10(-4) M folate or 10(-4) M dihydrofolate.

Animals↗
PubMedDate not supplied

[Purification and characteristics of alkaline proteinase from alkalophylic Bacillus sp].

Alkaline protease was purified from Bacillus sp. isolated from soil. The pH optimum was 11.5 at 37 degrees C. Calcium divalent cation was effective to stabilize the enzyme especially at higher temperatures. The proteolytic activity was inhibited by active site inhibitors of PMSF (Phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, Hg. The enzyme was stable in the presence of some detergents, such as Triton-X-100, Tween-80, SDS (sodium dodecyl sulfate) and EDTA (ethylendiaminetetraacetic acid), pH 11.5 and 37 degrees C for 30 min. The optimum pH was 11.5 at 37 degrees C and the optimum temperature was 62 degrees C at pH 11.5.

Bacillus↗
PubMedDate not supplied

[Effect of boron-containing mineral waters on the lipid peroxidation status and antioxidant defense factors in experimental gastroduodenitis].

We made experiments to study effects of effervescent hydrocarbonate mangesium-calcium-sodium boronic mineral water on lipid peroxidation and factors of antioxidant defense in gastric tissue in gastroduodenitis. A course treatment with mineral water improves histomorphology of the gastric mucosa, stabilizes enzymes of the antioxidant defense system and down-regulates intensity of lipid peroxidation.

Animals↗
PubMed2001

[Studies on the immobilized penicillin acylase on polymer beads].

The extracellular penicillin acylase from Bacillus megaterium was immobilized on oxirane group of Eupergit C beads. The apparent activity of the immobilized enzyme was about 1400 u.g-1 (dry weight). The optimal pH and temperature were 8.0 and 55 degrees C for hydrolytic reaction of penicillin G, respectively. The immobilized enzyme was stable in the pH range of 6.0-8.5 and at temperature below 45 degrees C. The apparent Michaelis constant for penicillin G was inhibition constant of phenylacetic acid as competitive 2 x 10(-2) mol.L-1 and Vm was 1.33 mmol.g-1 min-1 (dry weight) at 37 degrees C and PH8.0. The inhibitor and 6-APA as non-competitive inhibitor were 2.8 x 10(-2) mol.L-1 and 0.125 mol.L-1 for the immobilized enzyme at pH 8.0 and 37 degrees C, respectively. The remained activity of the immobilized enzyme was about 80% after operating 200 times for hydrolysis of penicillin G to 6-APA, and the average yield of 6-APA was 89.48%.

Bacillus megaterium↗
PubMed1998

[Studies on some properties of the immobilized penicillin acylase by polyacrylonitrile fibres].

The extracellular penicillin acylase from Bacillus megaterium was immobilized by coupling to derivatives of polyacrylonitrile fibres. The apparent activity of the immobilized penicillin acylase was about 2,000 u/g (dry weight). The optimal pH and temperature were 9.0 and 50 degrees C for hydrolysis of penicillin G, respectively. The immobilized enzyme was stable in the pH range of 5.5-10.3, and at the temperature below 50 degrees C. The apparent Michaelis constant Ka and Vm of the immobilization enzyme were 1.33 x 10(-2) mol/L and 2.564 mmol.min-1, respectively. The inhibition constant of phenylacetic acid acted as a competitive inhibitor for The immobilization enzyme was 0.16 mol/L. The remained activity was about 80% after operating 20 times.

Acrylic Resins↗