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PubMedDate not supplied

Evaluation of enzyme stability during preparation of polylactide-co-glycolide microspheres.

This work was aimed at studying enzyme prolidase stability and its interactions with the reagents and the process conditions involved in preparation, by an emulsification process, of prolidase loaded poly(lactide-co-glycolide) (PLGA) microparticulate systems. Enzyme stability was tested with respect to contact with methylene chloride, ethyl acetate, PLGA polymers, and several agents used as emulsifiers such as polyvinyl alcohol (PVA), polyvinyl pyrolidone (PVP), carboxymethyl cellulose (CMC) and sodium oleate (NaOl). Enzyme stability to temperature and mechanical stirring was also evaluated. Prolidase-loaded PLGA microspheres were prepared and evaluated in terms of protein activity. The results obtained showed that the prolidase-loaded PLGA microspheres can be prepared only upon enzyme stabilization by addition of both BSA and MnCl(2) into its TRIS solution. Methylene chloride was the suitable organic solvent to be used in the double emulsion process, together with PVA as dispersing agent in the outer aqueous phase. Low temperatures during the emulsification step and very short process times are recommended, in order to maintain enzyme activity at its maximum. In these conditions spherical microspheres were obtained, releasing active prolidase for up to 15 days.

Animals↗
PubMed1981

The principles of enzyme stabilization. VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents.

1. The possibility of stabilizing water-soluble enzymes against the inactivation action of organic solvents by means of surfactants has been studied. Several enzymes (alpha-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity. 2. A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of charges in the substrate molecule and on interphase (++, +-, --). 3. The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.

Benzene↗
PubMedDate not supplied

Ca2+-surfactant interactions affect enzyme stability in detergent solutions.

Detergent proteases and amylases generally bind Ca(2+) ions. These bound ions enhance enzyme stability, reducing the rates of degradative reactions such as unfolding and proteolysis. Thus, surfactant aggregates, such as micelles, affect protease and amylase stability indirectly, by competing with the enzymes for Ca(2+) ions. Dissociation constants for Ca(2+) interactions with anionic surfactant micelles are in the 10(-3) to 10(-2) M range. These interactions are weak relative to enzyme-Ca(2+) interactions (K(d) of order 10(-6) M). However, surfactant is typically present at much higher concentration than enzyme, and it is the Ca(2+)-micelle equilibrium that largely determines the amount of free Ca(2+) available for binding to enzymes. The problem of surfactant-mediated Ca(2+) removal from enzymes can be avoided by adding calcium to a detergent formulation in an amount such that the concentration of free Ca(2+) is around 10(-5)M.

Bacillus↗
PubMed1999

Mechanisms of acquired resistance to thymidylate synthase inhibitors: the role of enzyme stability.

Inhibitors of the enzyme thymidylate synthase (TS), such as the fluoropyrimidines 5-fluorouracil and 5'-fluoro-2'-deoxyuridine (FdUrd) or the antifolates AG337, ZD1694, and BW1843U89, are widely used in the chemotherapy of cancer, particularly cancer of the colon and rectum. Numerous studies have shown that TS gene amplification, leading to mRNA and enzyme overproduction, is a major mechanism of resistance to these inhibitors. In the present work, we have isolated and characterized FdUrd-resistant derivatives of several human colon tumor cell lines. Although gene amplification was commonly observed, the increases in mRNA and enzyme were strikingly discordant. In one drug-resistant line, a deficiency of enzyme relative to mRNA was shown to be caused by expression of a metabolically unstable TS molecule. The reduced half-life of TS in this line was caused by a Pro-to-Leu substitution at residue 303 of the TS polypeptide. The mutant enzyme conferred resistance to FdUrd as well as antifolates in transfected cells. In another FdUrd-resistant line, which had an excess of enzyme relative to mRNA, the TS molecule was more stable than in the parent line. However, no amino acid substitutions were detected in the TS polypeptide from this line, which suggests that the stabilization must be caused by changes in one or more cellular factors that regulate TS degradation. The results indicate that changes in the stability of the TS polypeptide accompany, and even contribute to, acquired resistance to TS inhibitors in colon tumor cells.

Amino Acid Substitution↗
PubMed1972

Glycerol as an enzyme-stabilizing agent: effects on aldehyde dehydrogenase.

The potassium-dependent aldehyde dehydrogenase (EC 1.2.1.3), from yeast is markedly altered by the addition of high concentrations of glycerol or other polyhydric alcohols to aqueous buffers. Several lines of evidence suggest that the three-dimensional structure near the active site is involved: (i) The stability of the enzyme when stored at 2 degrees , or when subjected to repeated freezing and thawing, depends upon the presence of at least 30% (v/v) glycerol. (ii) In the same solvent, the K(m) value for DPN and the binding constant for benzaldehyde decrease by 3- and 10-fold, respectively, compared with the values obtained for these substrates in fully aqueous media. (iii) Competitive inhibition by trivalent arsenicals with respect to DPN is no longer observed in glycerol; the inhibition becomes mixed and the K(i) values increase by 5- and 50-fold, respectively, with arsenite and Mapharsen. (iv) Essential sulfhydryl groups, which are easily carboxymethylated in aqueous buffers, are not readily available in either glycerol or mannitol. The data are consistent with a change in topography induced by polyhydric alcohols in which sulfhydryl groups near the DPN-binding site are displaced to a more protected environment, where their reactivity is reduced. Since the stabilizing effects of such alcohols are frequently encountered, these results may have application to other enzymes.

Aldehydes↗
PubMed1983

Choline acetyltransferase. Purification procedure and factors affecting chromatographic properties and enzyme stability.

Purified choline acetyltransferase had a specific activity of 142 mumol of acetylcholine produced min-1 mg-1 and consisted of two proteic forms with Mr = 72,000 and 76,000 on sodium dodecyl sulfate gel electrophoresis. The separation of more than one peak of enzyme activity on CM-cellulose chromatography was shown to reflect the interaction of choline acetyltransferase with other proteins rather than the resolution of different isoenzymes. Purified choline acetyltransferase exhibited a high degree of stability. Enzyme stability was greatly dependent upon the procedures used to reach a given degree of purity, rather than the degree of purity per se, suggesting that specific proteins may be involved in the mechanism of enzyme inactivation. Use of affinity chromatography over Sepharose-blue dextran early in the preparation produced enzyme which was unstable both to storage and to concentration. Although the addition of other proteins such as bovine serum albumin had no significant stabilizing effect, the presence of acetyl coenzyme A during concentration prevented inhibition. Finally, it was shown that the purified enzyme is representative of the total enzyme present in brain in terms of both specific activity and immunochemical properties.

Animals↗
PubMed1994

Effect of polyols on alpha-chymotrypsin thermostability: a mechanistic analysis of the enzyme stabilization.

The influence of the synthetic substrate (N-acetyl-L-tyrosine ethyl ester) and the different polyols (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of alpha-chymotrypsin at 60 degrees C have been studied. The results obtained showed an important stabilizing effect in the presence of both additives. In order to describe the kinetics of enzyme stabilization, the experimental results were analyzed by a four-parameters deactivation model with excellent agreement. In all cases, alpha-chymotrypsin exhibited non-first-order deactivation kinetics, corresponding to a two-step unimolecular mechanism, where the main protective effect of polyols was observed in the first-step of the deactivation profile. Thus, the presence of polyols increased the level of activity stabilization (alpha 1), and decreased the first-order deactivation rate constant (k1). Additionally, the experimental results were analyzed as a function of both, the change in the standard free energy of denaturation (delta(delta Gzero)), and a protective effect, defined as the ratio of alpha-chymotrypsin half-lives (with and without polyols), showing in both cases a clear stabilizing effect of these polyhydroxylic cosolvents for the enzyme. The overall protective effect of polyols was also simultaneously related to their concentration and their water-activity depressing power.

Chymotrypsin↗
PubMed2002

Pressure and heat inactivation of recombinant human acetylcholinesterase. Importance of residue E202 for enzyme stability.

The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild-type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild-type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE.

Acetylcholinesterase↗
PubMed2004

Polymorphisms in human soluble epoxide hydrolase: effects on enzyme activity, enzyme stability, and quaternary structure.

Human soluble epoxide hydrolase (hsEH) has been shown to play a role in regulating blood pressure and inflammation. HsEH consists of an N-terminal phosphatase and a C-terminal epoxide hydrolase domain. In the present study, we examined the effects of polymorphisms in the hsEH gene on phosphatase activity, enzyme stability, and protein quaternary structure. The results showed that mutants Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the Arg103Cys/Arg287Gln (double mutant) have significantly lower phosphatase activity compared to the most frequent allele (MFA) of hsEH. In addition, the Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the double mutant have significantly lower kcat/Km values. The stabilities at 37 degrees C of purified Arg287Gln and Arg103Cys/Arg287Gln mutants were also significantly reduced compared to the MFA. HPLC size-exclusion studies showed that the MFA exists predominantly as a dimer. However, the Arg287Gln and Arg103Cys/Arg287Gln mutants show increased concentration of the monomer. We conclude that the Arg287Gln polymorphism disrupts putative intra- and inter-monomeric salt-bridges responsible for dimerization.

Amino Acid Substitution↗
PubMed2005

Effect of kosmotropicity of ionic liquids on the enzyme stability in aqueous solutions.

This paper examined the effect of several pyridinium and imidazolium-based ionic liquids (ILs) on the protease stability in aqueous solutions. In general, the enzyme was found quite active at low concentrations of hydrophilic ILs. In aqueous environment, the enzyme was stabilized by the kosmotropic anions (such as CF3COO- and CH3COO-) and chaotropic cations (such as [BuPy]+ and [EMIM]+), but was destabilized by chaotropic anions (such as tosylate and BF4-) and kosmotropic cations (such as [BMIM]+).

Acetic Acid↗