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PubMed1995

Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii.

The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens. Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D. J. Hei and D. S. Clark, Appl. Environ. Microbiol. 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.

Adenylate Kinase↗
PubMed2002

The WD40-repeat protein CreC interacts with and stabilizes the deubiquitinating enzyme CreB in vivo in Aspergillus nidulans.

Genetic dissection of carbon catabolite repression in Aspergillus nidulans has identified two genes, creB and creC, which, when mutated, affect expression of many genes in both carbon catabolite repressing and derepressing conditions. The creB gene encodes a functional deubiquitinating enzyme and the creC gene encodes a protein that contains five WD40 repeat motifs, and a proline-rich region. These findings have allowed the in vivo molecular analysis of a cellular switch involving deubiquitination. We demonstrate that overexpression of the CreB deubiquitinating enzyme can partially compensate for a lack of the CreC WD40-repeat protein in the cell, but not vice versa and, thus, the CreB deubiquitinating enzyme acts downstream of the CreC WD40-repeat protein. We demonstrate using co-immunoprecipitation experiments that the CreB deubiquitinating enzyme and the CreC WD40-repeat protein interact in vivo in both carbon catabolite repressing and carbon catabolite derepressing conditions. Further, we show that the CreC WD40-repeat protein is required to prevent the proteolysis of the CreB deubiquitinating enzyme in the absence of carbon catabolite repression. This is the first case in which a regulatory deubiquitinating enzyme has been shown to interact with another protein that is required for the stability of the deubiquitinating enzyme.

Aspergillus nidulans↗
PubMed2003

Improvement in enzyme activity and stability by addition of low molecular weight polyethylene glycol to sodium bis(2-ethyl-L-hexyl)sulfosuccinate/isooctane reverse micellar system.

The activity and stability of Chromobacterium viscosum lipase (glycerolester hydrolase, EC 3.1.1.3)-catalyzed olive oil hydrolysis in sodium bis (2-ethyl-l-hexyl)sulfosuccinate (AOT)/isooctane reverse micelles is increased appreciably when low molecular weight polyethylene glycol (PEG 400) is added to the reverse micelles. To understand the effect of PEG 400 on the phase behavior of the reverse micellar system, the phase diagram of AOT/ PEG 400/water/isooctane system was studied. The influences of relevant parameters on the catalytic activity in AOT/PEG 400 reverse micelles were investigated and compared with the results in the simple AOT reverse micelles. In the presence of PEG 400, the linear decreasing trend of the lipase activity with AOT concentration, which is observed in the simple AOT reverse micelles, disappeared. Enzyme entrapped in AOT/PEG reverse micelles was very stable, retaining >75% of its initial activity after 60 d, whereas the half-life in simple AOT reverse micelles was 38 d. The kinetics parameter maximum velocity (Vmax) exhibiting the temperature dependence and the activation energy obtained by Arrhenius plot was suppressed significantly by the addition of PEG 400.

Chromobacterium↗
PubMed2005

Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea.

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

Animals↗
PubMed1996

Pressure-induced inactivation of E. coli beta-galactosidase: influence of pH and temperature.

In order to assess the feasibility of a high-pressure immunodesorption process using a beta-galactosidase-anti-beta-galactosidase complex as a model, the influence of high hydrostatic pressure on the activation of E. coli beta-galactosidase has been investigated. The irreversible activity loss of beta-galactosidase was studied as a function of pH and temperature for pressures comprised between atmospheric pressure and 500 megapascal (MPa; 1 MPa = 10 bar). This enabled us to establish a practical pressure-temperature diagram of stability for this enzyme. The stability domains determined thus appeared to be strongly dependent on the pH under atmospheric pressure of phosphate buffer employed for pressurisation. Therefore, to interpret meaningfully this result, the influence of pressure on the pH-activity curve of beta-galactosidase was investigated by using a high-pressure stopped-flow device. It appeared that the pH-activity curve of this enzyme was also reversibly affected by pressures lower than 150 MPa. An interpretation of these results in relation to the high-pressure induced changes of ionisation constants is proposed. For our practical purpose, the implications for the elaboration of a high-pressure immunodesorption process using beta-galactosidase as a tag, are discussed.

Adsorption↗
PubMed2004

Substrate ground state binding energy concentration is realized as transition state stabilization in physiological enzyme catalysis.

Previously published kinetic data on the interactions of seventeen different enzymes with their physiological substrates are re-examined in order to understand the connection between ground state binding energy and transition state stabilization of the enzyme-catalyzed reactions. When the substrate ground state binding energies are normalized by the substrate molar volumes, binding of the substrate to the enzyme active site may be thought of as an energy concentration interaction; that is, binding of the substrate ground state brings in a certain concentration of energy. When kinetic data of the enzyme/substrate interactions are analyzed from this point of view, the following relationships are discovered: 1) smaller substrates possess more binding energy concentrations than do larger substrates with the effect dropping off exponentially, 2) larger enzymes (relative to substrate size) bind both the ground and transition states more tightly than smaller enzymes, and 3) high substrate ground state binding energy concentration is associated with greater reaction transition state stabilization. It is proposed that these observations are inconsistent with the conventional (Haldane) view of enzyme catalysis and are better reconciled with the shifting specificity model for enzyme catalysis.

Catalysis↗
PubMed1994

Glutamic acid 86 is important for positioning the 80's loop and arginine 54 at the active site of Escherichia coli aspartate transcarbamoylase and for the structural stabilization of the C1-C2 interface.

Glu-86, which interacts with the side chain of Arg-54 across the C1-C2 interface of Escherichia coli aspartate transcarbamoylase, tethers the end of the flexible 80's loop, which moves into the active site during the T to R transition. In order to determine whether this interaction is important for the correct positioning of the 80's loop and Arg-54 at the active site and also for the structural stabilization of the enzyme, a mutant version was created in which Glu-86 was replaced by Gln (Glu-86-->Gln). Although the mutant holoenzyme exhibits almost normal homotropic cooperativity, both the holoenzyme and catalytic subunit exhibit substantial reductions in activity and affinity for aspartate and carbamyl phosphate. Furthermore, the mutant holoenzyme shows a marked decrease in the activation by ATP and by the bisubstrate analog N-(phosphonoacetyl)-L-aspartate, reduced inhibition by CTP, as well as reduced affinities for these ligands. Results from molecular dynamics simulations of the Glu-86-->Gln and Glu-86-->Ala enzymes suggest that the positions of the 80's loop and Arg-54 are significantly perturbed by the introduction of these mutations. Taken together, these results indicate that the interaction between Glu-86 and Arg-54 is important for the formation of the high affinity, high activity form of the enzyme by stabilizing the correct position of the 80's loop and Arg-54 at the active site. Heat inactivation experiments also demonstrated that Glu-86 plays a significant role in the structural stabilization of the C1-C2 interface, since the temperature required for loss of half of the activity of the Glu-86-->Gln catalytic subunit is reduced by 5 degrees C relative to the wild-type catalytic subunit.

Aspartate Carbamoyltransferase↗
PubMed1992

Chemical crosslinking and the stabilization of proteins and enzymes.

The technique of chemical crosslinking has been used to enhance the stability of proteins and enzymes. In this procedure, the molecule is braced with chemical crosslinks either intramolecularly or intermolecularly to another species to reinforce its active structure. Various chemicals have been used for this purpose. The bifunctional reagents are the most prominent. These compounds are derived from group-specific reagents and may be classified into homobifunctional, heterobifunctional, and zero-length crosslinkers. Different physical and chemical characteristics have been incorporated into these chemicals. Their versatility holds great potential in preparing chemically, thermally, and mechanically stable proteins and enzymes for industrial applications.

Cross-Linking Reagents↗
PubMed1990

Purification and properties of glutamyl aminopeptidase from chicken egg-white.

Hydrolytic activities characteristic for different aminopeptidases were detected in the egg-white of unfertilized chicken eggs, and one aminopeptidase was isolated in an electrophoretically homogeneous form. The isolated aminopeptidase preferentially hydrolyzed bonds of alpha-glutamyl residue at the NH(2)-end of synthetic substrates and peptides. The enzyme is a dimer with an M(r) of 320,000 and pI of 4.2. Its optimal pH and temperature are 7.6 and 60 degrees C, respectively. EDTA, amastatin, and N-bromosuccinimide are inhibitors, while Ca2++ and Mn2+ are activators of the enzyme Ca2+ also stabilizes the enzyme. According to the observed properties, the isolated chicken egg-white aminopeptidase belongs to the glutamyl aminopeptidases.

Aminopeptidases↗
PubMedDate not supplied

A smart bioconjugate of alginate and pectinase with unusual biological activity toward chitosan.

The commercial preparation of pectinase (Pectinex Ultra SP-L) was conjugated to alginate by noncovalent interactions by employing 1% alginate during the conjugation protocol. The optimum "immobilization efficiency" was 0.76. The pH optimum and the thermal stability of the enzyme remained unchanged upon conjugation with alginate. The soluble bioconjugate showed a 3-fold increase in V(max)/K(m) as compared to the free enzyme when the smart biocatalyst was used for chitosan hydrolysis. Time course hydrolysis of chitosan thus showed higher conversion of chitosan into reducing oligosaccharides/sugars. The smart bioconjugate could be reused five times without any detectable loss of chitosanase activity.

Alginates↗
PubMed2003

Immobilized laccase for decolourization of Reactive Black 5 dyeing effluent.

Reactive Black 5 industrial dyeing effluent was decolourized by free and immobilized laccase. The stability of the enzyme (194 h free and 79 h immobilized) depended on the dyeing liquor composition and the chemical structure of the dye. In the decolourization experiments with immobilized laccase, two phenomenons were observed--decolourization due to adsorption on the support (79%) and dye degradation due to the enzyme action (4%). Dyeing in the enzymatically recycled effluent provided consistency of the colour with both bright and dark dyes.

Biodegradation, Environmental↗
PubMed1982

C3 convertase of human complement: enhanced formation and stability of the enzyme generated with nickel instead of magnesium.

We demonstrate that nickel++ (Ni) can replace Mg in the formation of the C3b,Bb enzyme, and that Ni is more efficient in enzyme formation than Mg. C3b-bearing sheep erythrocytes (EC3b) were used to measure radiolabeled Factor B uptake and C3 convertase activity. Up to nine times more Factor B was specifically bound to EC3b in the presence of Ni than in the presence of Mg under identical conditions. To form one effective hemolytic site (1 Z) per EC3b cell with Ni, three times less Factor B, 12 times less Factor D, and 66 times less metal ion were required than when using Mg. The C3b,Bb formed with Ni (C3b,Bb(Ni)) had a 5 to 10 times longer half-life at different temperatures than the enzyme formed with Mg (C3b,Bb(Mg)). Native properdin stabilized C3b,Bb(Ni) to the same extent as C3b,Bb(Mg) (four to five times) and Factor H accelerated similarly the decay of both enzymes. Ni could also replace Mg in the formation of the C4b,2a enzyme. C4b,2a formation was measured by using C1 and C4b-bearing sheep erythrocytes (EAC1,4b) and native C2. To form 1 Z per EAC1,4b cell with Ni, two times less C2 and 60 times less metal ion were required than when using Mg. The half-life of the C4b,2a formed with Ni was two times longer than that formed with Mg. Decay of both C3b,Bb and C4b,2a enzymes formed either with Mg or with Ni was unaffected by EDTA. These results show that Ni is more efficient than Mg in the generation of both enzymes and suggest that Ni binds to the Mg-binding sites of the enzymes with a higher affinity than Mg.

Complement Activating Enzymes↗
PubMed1986

Structural implications of the location and stability to proteolytic enzymes of immunodominant determinants of the human leukocyte common molecule.

In this report, we use 8 different mouse monoclonal antibodies to define 3 immunodominant determinants of the human leukocyte common (LC) antigen, and by defining the relative location and the stability to proteolytic enzymes of these determinants we make some structural predictions about the LC family of molecules. One lineage-restricted determinant is expressed predominantly on B lymphocytes and is located on the two higher (210 and 195) kDa bands of LC, with possibly some expression on the 180-kDa band. The two other determinants, one of which is a complex of 3 partially overlapping sites, are expressed on all leukocytes and found on all four (210, 195, 180, 160 kDa) LC bands. Pronase and trypsin treatment of peripheral blood lymphocytes and of Daudi cells gave complete cleavage of the lineage-restricted determinant from the cells, but the two common determinants were, surprisingly, left intact on the cell surface. By contrast, pronase and trypsin treatment of pure, detergent-solubilized LC resulted in rapid degradation of the common determinants, while the restricted determinant was sensitive to pronase but not to trypsin. Purification of the LC molecule from pronase-treated Daudi cells yielded a 130-kDa lentil lectin-binding protein, while undegraded LC from Daudi cells has a molecular mass of 210 kDa. Our results demonstrate that both common and restricted determinants are at least partly protein in nature and that the restricted determinant is external to and therefore on the presumed amino terminal side of the common determinants. Moreover, because LC has been reported to have a large (approximately 100 kDa) intracellular component, our data suggest the presence of a relatively small (approximately 25 kDa) membrane proximal domain containing both of the common LC determinants and at least one N-linked oligosaccharide chain. This domain is potentially susceptible to proteolytic cleavage, but it is interesting that on the intact cell it is both protected from proteolytic degradation and unable to be cleaved from the cell surface.

Antibodies, Monoclonal↗
PubMed2000

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal beta-glycosidases.

S beta gly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of S beta gly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure-function relationships in these enzymes.

Cellulase↗
PubMedDate not supplied

Covalent-bonded immobilization of lipase on poly(phenylene sulfide) dendrimers and their hydrolysis ability.

Covalent-bonded immobilization of lipase from burkholderia cepacia onto two poly(phenylene sulfide) (PPS) dendrimers with different generations (two and three) was achieved using carbodiimide as a coupling reagent. The hydrolysis activity of olive oil to fatty acid was studied on enzyme-immobilized PPS dendrimers. Enzyme activity was proportional to the enzyme loading, and highest recovered activity was obtained at the medium enzyme loading for both G2 and G3 dendrimers. The immobilization improved the optimum pH and caused the temperature range to widen. Immobilization of enzyme has enhanced the thermal stability of enzyme activity in comparison with free enzyme. The immobilized enzyme as a biocatalyst for batch hydrolysis of olive oil retained 80 approximately 90% activity even after 20 times of recycling. This retention of activity after recycle is very valuable and powerful in enzyme technology. The present noteworthy and vital availability on enzyme reaction of the covalently bonded immobilized lipase on dendrimer came from the structure of dendrimer with a large number of functional terminal groups, which are easily available for immobilization of many lipases at the situation keeping reactive enzymes on the surface of dendrimer.

Biocompatible Materials↗
PubMed2002

Purification and characterization of two polymorphic variants of short chain acyl-CoA dehydrogenase reveal reduction of catalytic activity and stability of the Gly185Ser enzyme.

Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the beta-oxidation of fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of ethylmalonic acid excretion in urine, a clinical characteristic of SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant proteins. Both variant proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial chaperonin GroEL/ES. The k(cat)/K(m) values of the green wild-type, R147W, and G185S SCAD enzymes coexpressed with GroEL/ES were 33, 30, and 10 microM(-)(1) s(-)(1), respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type enzymes coexpressed with GroEL/ES, and the R147W variant when butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant enzyme, however, was reduced compared to that of the wild-type enzyme. The thermal and guanidine HCl stability of the purified enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function.

Acyl-CoA Dehydrogenase↗