Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Enzyme stabilization”

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 271 records · Page 15Linked to original sources
PubMed2001

Stabilization of Penicillium occitanis cellulases by spray drying in presence of Maltodextrin.

The stabilization of fungal cellulases by spray drying, the thermal stability of Penicillium occitanis cellulases and the effect of some additives were studied. We observed that CMCase activity presents a good stability at 50 degrees C, even after 60 h of incubation. On the other hand, beta-glucosidase activity was more sensitive (loss of 50%) and reacts on total cellulases activities (Filter paper activities). The addition of hydrophilic agents such as ethylene glycol, polyethylene glycol (PEG6000) enhanced enzyme activity. The effect of PEG and Maltodextrin, another water activity decreasing agent, were then tested during the spray drying of Pol6 cellulases. The presence of 1% PEG allowed the best recovery but had a negative effect on enzyme stability while 1% Maltodextrin showed a negative effect on enzyme recovery but a very positive effect on enzyme stabilization.

Journal Article↗
PubMed1999

Stabilization of the enzyme--substrate complex of the mutant Asp-67Asn inorganic pyrophosphatase from Escherichia coli by fluoride ions.

Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly. This enzyme--substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration.

Aspartic Acid↗
PubMed2007

Selective modification of surface-exposed thiol groups in Trigonopsis variabilis D-amino acid oxidase using poly(ethylene glycol) maleimide and its effect on activity and stability of the enzyme.

Covalent modification of purified Trigonopsis variabilis D-amino acid oxidase using maleimide-activated poly(ethylene glycol) 5000 yielded a stable bioconjugate in which three surface-exposed cysteine side chains were selectively derivatized. Compared with the native enzyme, the PEGylated variant displayed substantially (approximately 3.3-fold) slowed dissociation rate of FAD cofactor at 50 degrees C, and this caused a twofold thermostabilization of the enzyme activity. The stability under reaction conditions at 30 degrees C was also markedly enhanced in the PEG-oxidase conjugate. PEGylation did not affect steady-state kinetic parameters for oxidative deamination of D-methionine when 2,6-dichloroindophenol replaced dioxygen as the cosubstrate while it caused a ninefold decrease in substrate catalytic efficiency for the dioxygen-dependent reaction.

D-Amino-Acid Oxidase↗
PubMed1996

Site-directed mutagenesis of a highly conserved aspartate in the putative 10-formyl-tetrahydrofolate binding site of yeast C1-tetrahydrofolate synthase.

C1-tetrahydrofolate (THF) synthase is a eukaryotic trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. Although the 10-formyl-THF synthetase reaction (a reversible ATP-dependent formylation of THF) has been studied extensively, little is known about specific residues involved in the catalytic mechanism. In this study, we have examined the role of a highly conserved aspartate residue, Asp449 of yeast cytoplasmic C1-THF synthase. Asp449 is part of a putative folate binding site found in many proteins that bind 10-formyl-THF. The corresponding aspartate has been identified as a critical catalytic residue in Escherichia coli and human GAR transformylase, which catalyzes a 10-formyl-THF-dependent formyl transfer. In order to determine if Asp449 has a similar catalytic role in the 10-formyl-THF synthetase reaction, three mutant proteins were produced by site-directed mutagenesis in which Asp449 of yeast cytoplasmic C1-THF synthase was changed to Asn, Glu, or Ala. The mutant proteins were expressed in yeast, purified, and characterized with respect to kinetic properties and enzyme stability. All three of the mutant enzymes retained substantial 10-formyl-THF synthetase activity, indicating that Asp449 is not a critical catalytic residue. However, our data suggest that it does play a role in folate binding, probably by contributing to the proper conformation of the active site. Thus, these results suggest that the 10-formyl-THF binding site differs significantly between the GAR transformylase and 10-formyl-THF synthetase families, and that the conserved aspartate plays different roles in the two enzymes.

Acyltransferases↗
PubMed1991

Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues.

Transglutaminases catalyze the posttranslational modification of proteins by transamidation of available glutamine residues. This action results primarily in the formation of epsilon-(gamma-glutamyl)lysine cross-links but includes the incorporation of polyamines into suitable protein substrates as well. The covalent isopeptide crosslink is stable and resistant to proteolysis, thereby increasing the resistance of tissue to chemical, enzymatic, and mechanical disruption. The plasma transglutaminase, factor XIIIa, is formed at sites of blood coagulation and impedes blood loss by stabilizing the fibrin clot. The squamous epithelium constituting the protective callus layer of skin is formed by the action of keratinocyte transglutaminase (TGK) and epidermal transglutaminase (TGE). The tissue transglutaminase (TGC) is a cytoplasmic enzyme present in many cells including those in the blood vessel wall. TGC function is unknown, although it could function to stabilize intra- and extra-cellular molecules in a wide variety of physiologic or pathologic processes. The amino acid sequences of factor XIII, TGC, and TGK establish them as a homologous gene family and also reveal a striking homology to the erythrocyte membrane protein, band 4.2. This review summarizes the current information on structures, functions, and evolution of the most prominent members of this gene family.

Amino Acid Sequence↗
PubMed1994

Immobilization of beta-galactosidase on metal-chelate-substituted gels.

The use of copper, zinc, iron, nickel and calcium in three different chelating gels was investigated for preparing immobilized beta-galactosidase. The chelated ligands [Cu(2+)-iminodiacetate (IDA), Cu(2+)-Tris(carboxymethyl)ethylenediamine (TED), Ni(2+)-IDA and Fe(3+)-IDA] absorbed the protein so strongly that it can be considered a true immobilization. The obtained enzyme derivatives were investigated with regard to activity and stability. Enzymic activity was highly preserved in general for the TED derivates (90% when compared with that for Cu(2+)-TED). The immobilized Ni2+ derivatives were more stable to high temperature and to storage than the Cu2+ derivatives. Temperature-stability of the immobilized enzyme was very much improved by adding a strong metal-chelating gel such as carboxymethylated tetraethylenepentamine-agarose. The gel could be re-used and reloaded after elution with chelator. beta-Galactosidase from Escherichia coli was purified using immobilized-metal-ion-chelate chromatography (i.m.a.c.). The potential use of beta-galactosidase immobilized on i.m.a.c. gels for technical purposes is discussed.

Chelating Agents↗
PubMed1998

Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit.

DNA polymerase alpha-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg2+ and Mn2+ but not Zn2+. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase alpha-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.

Animals↗
PubMed1980

Chemical cross-linking stabilizes the enzymic activity and quaternary structure of formyltetrahydrofolate synthetase.

Clostridium cylindrosporum formyltetrahydrofolate synthetase tetramers cross-linked with dimethyl suberimidate remained active in the absence of the monovalent cations normally required for enzymic activity and the tetrameric conformation. The modified enzyme was analyzed by sodium dodecyl sulfate electrophoresis, sedimentation velocity, and gel permeation chromatography. Under the experimental conditions used, the enzyme was only partially cross-linked; 74% of the enzyme was cross-linked dimer or monomer. Nonetheless, the modified enzyme is able to retain enzymic activity and the tetrameric structure under conditions where native enzyme would be completely dissociated and inactivated. The result suggests that cross-linked dimers strongly associate with each other and with monomers. Flame emission spectroscopy indicates that cross-linked enzyme contains two monovalent cations per tetramer.

Ammonia↗
PubMed1995

Isepamicin (SCH 21420, 1-N-HAPA gentamicin B): microbiological characteristics including antimicrobial potency of spectrum of activity.

Isepamicin (formerly SCH 21420 or 1-N-HAPA-gentamicin B) is a novel broad-spectrum aminoglycoside which possesses a high level of stability to aminoglycoside inactivating enzymes and low levels of toxicity to the kidney and inner ear. The only modifying enzymes capable of inactivating isepamicin are ANT(4')-I (staphylococci), ANT(4')-II and APH(3')-VI, in addition to resistance mediated by permeability mutations. The spectrum of isepamicin is most similar to that of amikacin, another aminoglycoside with high enzyme stability. Reviews of isepamicin activity demonstrate MIC90s ranging from 1.1 to 8.5 mg/L for members of the Enterobacteriaceae, slightly more potent than amikacin. Pseudomonas aeruginosa, Acinetobacter spp. and other pseudomonads had isepamicin consensus MIC90s of 7.8, 7.2 and 6.8 mg/ml, respectively. Staphylococci were generally very susceptible to isepamicin (MIC90s 0.5-6.9 mg/L), but enterococci and Streptococcus spp. were resistant (MIC90s > or = 64 mg/L), as were anaerobes, Xanthomonas (Stenotrophomonas) maltophilia, pathogenic Neisseria spp., Flavobacterium spp., Pseudomonas (Burkholderia) cepacia, Alcaligenes spp. and Vibrio spp. Additional studies of isepamicin microbiology revealed: 1) MICs were adversely influenced by elevated divalent cation content of the medium; 2) minimum inoculum effects were observed by using elevated concentrations; 3) bactericidal action and concentration dependent killing was the rule; 4) excellent stability in the presence of high beta-lactam co-drug concentrations was documented in several studies; 5) predictable synergistic or additive interactions with broad spectrum antimicrobial agents such as cephalosporins, penicillins, carbapenems and fluoroquinolones was observed by numerous investigators; and 6) in vitro susceptibility testing criteria (National Committee for Clinical Laboratory Standards) and quality control guidelines are established for routine clinical use. Isepamicin's antimicrobial qualities position it as a potential alternative aminoglycoside in hospitals or in geographical areas where resistance to existing aminoglycosides has emerged. The wider stability of isepamicin to contemporary aminoglycoside inactivating enzymes, its predictable pharmacokinetics, lower toxicity risks and enhanced activity (synergy) with other broad spectrum antimicrobial agents, will make isepamicin a valuable addition to the antimicrobial armamentarium in areas where ACC(6') enzymes are prevalent (Europe, Latin America, Western Pacific) and amikacin has become less efficacious.

Anti-Bacterial Agents↗
PubMed1987

In vitro evaluations of U-76,252 (CS-807): antimicrobial spectrum, beta-lactamase stability, and enzyme inhibition.

Compound U-76,252 (Upjohn) is a cephalosporin ester that enhances oral absorption of the active free acid cephem, U-76,253. The active form structurally resembles parenteral aminothiazolyl-methoxyimino cephalosporins such as cefotaxime and its desacetyl metabolite. The g-negative antimicrobial activity of U-76,253 A (sodium salt of U-76,253) was most similar to that of cefixime and more potent than that of cefaclor or cefuroxime among the orally administered cephalosporins. Against g-positive bacteria, U-76,253 A was more active than cefixime. U-76,253 A was relatively stable to hydrolysis by five beta-lactamases (Type Ia, TEM-1, K1, CARB-2, and OXA-1), a stability most similar to cefotaxime and superior to that of cefaclor. Only the Type Ia (P99) enzyme was significantly inhibited by U-76,253 (IC 50 = 2.0 microM).

Anti-Bacterial Agents↗
PubMed2003

High stability of enzyme immunoassay for hepatitis C virus core antigen-evaluation before and after incubation at room temperature.

Hepatitis C virus (HCV) RNA is thought to be less stable than HCV core antigen (HCV-Ag), however there have been few studies on comparing the stability of HCV-Ag with that of HCV-RNA in vitro. The aim of this study is to evaluate serial levels of HCV-Ag and HCV-RNA in serum before and after incubation at 4 or 25 degrees C for 7 days to estimate an assay suitable for general laboratory use. In this study, we demonstrate that HCV-Ag levels are highly reproducible (coefficients of variation (CVs); 0.89-6.92%) and stable (84.8% of the initial level) with incubation of even 25 degrees C for 7 days, whereas HCV-RNA levels are much less reproducible (CVs; 9.13-29.66%) and decrease dramatically (15.1% of the initial level) after incubation, particularly at 25 degrees C. The measurement of the HCV-Ag level was found to be suitable for HCV quantification with serum samples stored either at 4 degrees C or under unknown conditions. Additionally, it successfully eliminated inhibitors such as heparin from plasma and could be applied to a variety of clinical specimens. Our data suggest the significance of measuring the HCV-Ag level during clinical management independently of the HCV-RNA level, particularly because of its high stability.

Journal Article↗
PubMedDate not supplied

[Study of immobilization and properties of urease for creation of a biosensor based on semiconductor structures].

Many-sided investigations of urease immobilization methods were carried out to create the biosensor devices on the base of semiconductor structures. Special attention was concentrated on the biomembrane formation by means of urease and bovine serum albumin (BSA) cross-linking by gaseous glutaraldehyde. Optimal conditions for the formation process were selected which preserve about 20% of total urease activity after the cross-linking. The properties of enzyme immobilized by the above-mentioned method have been comprehensively studied. They included the urease activity dependence on pH, ionic strength, incubation buffer capacity as well as the enzyme stability during its functioning, storing and thermoinactivation. As was shown, for immobilized ureas Km value for urea at pH 7.0 and 20 degrees C is 1.65 time less than for free enzyme. In the presence of EDTA (1 mM) the enzyme activity in the biomembrane is practically unchanged under a month storing. Biomembrane possesses good adhesion to silicon surface and its swelling level under different conditions does not exceed 35%. The conclusion is made about the prospects of the used method of biomembrane formation for biosensor technology based on semiconductor structures.

Enzyme Stability↗
PubMed1999

An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus.

A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a delta pyrE strain by a two step method. First, the leuB gene was replaced with the pyrE gene. Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid. A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host. When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme. These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes.

3-Isopropylmalate Dehydrogenase↗
PubMed2001

Enhancement of enzyme activity through three-phase partitioning: crystal structure of a modified serine proteinase at 1.5 A resolution.

Three-phase partitioning is fast developing as a novel bioseparation strategy with a wide range of applications including enzyme stability and enhancement of its catalytic activity. Despite all this, the enzyme behaviour in this process still remains unknown. A serine proteinase, proteinase K, was subjected to three-phase partitioning (TPP). A 3 ml volume of proteinase K solution (3 mg/ml in 0.05 M acetate buffer, pH 6.0) was brought to 30% (w/v) ammonium sulphate saturation by addition of saturated ammonium sulphate. tert-Butanol (6 ml) was added to this solution and the mixture was incubated at 25 degrees C for 1 h. The precipitated protein in the mid-layer was dissolved in 3 ml of 0.05 M acetate buffer, pH 6.0. The specific activity of the processed enzyme was estimated and was found to be 210% of the original enzyme activity. In order to understand the basis of this remarkable enhancement of the enzyme activity, the structure of the TPP-treated enzyme was determined by X-ray diffraction at 1.5 A resolution. The overall structure of the TPP-treated enzyme is similar to the original structure in an aqueous environment. The hydrogen bonding system of the catalytic triad is intact. However, the water structure in the substrate binding site has undergone a rearrangement as some of the water molecules are either displaced or completely absent. Two acetate ions were identified in the structure. One is located in the active site and seems to mimic the role of water in the enzyme activity and stability. The other is located at the surface of the molecule and is involved in stabilizing the local structure of the enzyme. The most striking observation in respect of the present structure pertains to a relatively higher overall temperature factor (B = 19.7 A(2)) than the value of 9.3 A(2) in the original enzyme. As a result of a higher B-factor, a number of residues, particularly their side chains, were found to adopt more than one conformation. It appears that the protein exists in an excited state which might be helping the enzyme to function more rapidly than the original enzyme in aqueous media. Summarily, the basis of increased enzymatic activity could be attributed to (i) the presence of an acetate ion at the active site and (ii) its excited state as reflected by an overall higher B-factor.

Crystallography, X-Ray↗