[Thermostable enzymes of thermophiles and thermal stability of enzyme protein (author's transl)].
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Bacillus↗
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Yeast alpha-isopropylmalate isomerase. Factors affecting stability and enzyme activity.
Yeast alpha-isopropylmalate isomerase was found to be markedly stabilized by high concentrations of glycerol and (NH4)2SO4. Such conditions of high ionic strength inhibited the enzyme, stabilized the enzyme to heat, and affected kinetic parameters. The isomerase was found to exhibit ionic strength-dependent hysteresis when enzyme, totally but reversibly inhibited by storage under conditions of high ionic strength of (NH4)2SO4, was transferred to a lower concentration of (NH4)2SO4. Alpha-Isopropylmalate isomerase was found to be sensitive to KCN and certain other chelators. The inactivation by KCN was prevented by high concentrations of (NH4)2SO4. These observations implicated a metal involvement but the nature of the metal was not revealed. The metal involvement and some of the other properties of alpha-isopropylmalate isomerase reveal a similarity to aconitase. The similarities in properties between the isomerase and aconitase are summarized. Studies of yeast alpha-isopropylmalate isomerase indicated that it is a single polypeptide of about Mr = 90,000.
Ammonium Sulfate↗
Determination of substrates using poly(ethylene glycol)-stabilized dehydrogenase enzymes by microlitre per minute flow injection.
Flow injection (FI), at a flow rate of microliter min-1, is an effective method for enzymic substrate determination using low concentrations of poly(ethylene glycol) (PEG)-stabilized soluble enzymes. PEG stabilizes dehydrogenase enzymes for at least several days by promoting sub-unit association. Band broadening of knitted open tubular reactors is reduced as flow rate decreases below 300 microliter min-1 and a small tubing diameter is important for a faster rate of absorbance signal increase with residence time. Small (0.5 microliter) sample injections also ensure narrow FI peaks. The determination of several substrates such as pyruvate, lactate, and cortisone using appropriate PEG-stabilized enzymes is demonstrated with this FI instrument at 25 or 50 microliters min-1 with sample throughputs of the order of 2-3 min per sample. The determination of lactate in serum samples is also possible. The advantage of this method, sample throughput, is not sacrificed but enzyme consumption is considerably less, compared to standard ml min-1 FI.
Animals↗
[Stability of enzymes inactivating aminoglycoside antibiotics].
Stability of aminoglycoside phosphotransferases, adenylyltransferases and acetyltransferases isolated from various sources was studied. The enzymes were characterized by different substrate profiles. They were stored at a temperature of -10 degrees C in the form of frozen solutions or at a temperature of 4 degrees C in the lyophilized form. It was shown that lyophilization markedly increased the stability of the enzymes inactivating aminoglycoside antibiotics. Aminoglycoside phosphotransferases and adenylyltransferases with streptomycin as substrate were less stable than aminoglycoside phosphotransferases with neomycin as substrate. Aminoglycoside acetyltransferases from Streptomyces fradiae 918 producing neomycin were least stable among the enzymes studied. Lyophilized enzymes as a possible stabilizer of ATP added to the preparations had no significant effect on their stability during storage.
Acetyltransferases↗
[Long-term stability of enzymes in solution].
Two proteases (thermitase, a thermostable serine protease from Thermoactinomyces vulgaris and subtilisin Carlsberg) and one non-proteolytic enzyme (urate oxidase from Penicillium spp.) were used for revealing the main influences leading to the inactivation of enzyme preparations during their long-time storage at low temperatures. The temperature dependences (0 degree C-60 degrees C) of inactivation resulted in a linear Arrhenius-plot for each of the three native enzymes as well as in the presence of all stabilizing substances tested. Therefore, a method is available which shortens the time considerably needed for the experiments aimed at the discovery of substances stabilizing enzymes under storage conditions (i.e. long time at low temperatures). Because of the linear Arrhenius-plots potential stabilizers can be tested experimentally at suitable higher temperatures and one can extrapolate on their influence on the enzyme at storage temperatures (0 degree C-10 degrees C). By using this method effective combinations of stabilizers for urate oxidase were found, and possible reasons of their stabilizing influence on the enzyme are discussed.
Cold Temperature↗
Subcellular co-localization and potential interaction of glucuronosyltransferases with nascent proteochondroitin sulphate at Golgi sites of chondroitin synthesis.
Microsomal membranes from chick embryo epiphyseal cartilage were fractionated by equilibrium sucrose-density-gradient centrifugation and assayed for GlcA (glucuronic acid) transferase I (the enzyme that transfers GlcA from UDP-GlcA to Gal-Gal-Xyl of proteochondroitin linkage region), for comparison with GlcA transferase II (the GlcA transferase of chondroitin polymerization). Gal(beta1-3)Galbeta1-methyl (disaccharide) and GalNAc(beta1-4)GlcA(beta1-3)GalNAc(beta1-4) GlcA(beta1-3)GalNAc(pentasaccharide) were used respectively as acceptors of [14C]GlcA from UDP-[14C]GlcA. Distributions of the two GlcA transferase activities in the sucrose-density-gradient fractions were compared with each other and with the previously reported distribution of the activities of Gal transferases (UDP-Gal to ovalbumin, and to xylose of the proteochondroitin linkage region) and GalNAc (N-acetylgalactosamine) transferase II of chondroitin polymerization. The linkage-region GlcA transferase I had a dual Golgi distribution similar to that of chondroitin-polymerizing GlcA transferase II and distinctly different from the distribution of linkage-region Gal transferases I and II, which were found exclusively in the heavier fractions. Solubilized GlcA transferase I was partly purified by sequential use of Q-Sepharose, heparin-Sepharose and wheatgerm agglutinin-agarose and was accompanied at each step by some of the GlcA transferase II activity. Both GlcA transferase I and II bound to the Q-Sepharose as though they were highly anionic. However, treatment with chondroitin ABC lyase eliminated the binding while markedly decreasing enzyme stability. The enzyme activities could not be reconstituted by adding chondroitin or chondroitin pentasaccharide to the chondroitin ABC lyase-treated enzymes. Incubation of the partly purified enzymes with both UDP-GlcA and UDP-GalNAc resulted in a 40-fold greater incorporation than with just one sugar nucleotide, indicating the presence of bound, nascent proteochondroitin serving as the acceptor for chondroitin polymerization. These results, together with the membrane co-localization, indicate that GlcA transferase I and GlcA transferase II occur closely together with nascent proteochondroitin at the site of synthesis and that this complex with the nascent proteochondroitin stabilizes both enzymes during purification.
Animals↗
Analysis of enzyme deactivations by a series-type mechanism: influence of modification on the activity and stability of enzymes.
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Asparaginase↗
Effect of mannitol crystallinity on the stabilization of enzymes during freeze-drying.
The stabilizing effect of mannitol during the freeze-drying of proteins was studied using L-lactate dehydrogenase (LDH, rabbit muscle), beta-galactosidase (Escherichia coli) and L-asparaginase (Erwinia chrysanthemi) as model proteins. Crystallization of mannitol was studied by powder X-ray diffraction and differential scanning calorimetry (DSC), in relation to the stabilizing effect. All the enzymes were protected concentration-dependently by amorphous mannitol, but the stabilizing effect was decreased with an increase in mannitol crystallinity. The heat-treatment of frozen solutions above crystallization temperature prior to drying enhanced mannitol crystallization and LDH inactivation. The importance of maintaining excipients in an amorphous state during freeze-drying, previously reported for Aspergillus oryzae beta-galactosidase (K. Izutsu et al., Pharm. Res., 10, 1233 (1993)), was confirmed using three different enzymes.
Animals↗
Deletion of various carboxy-terminal domains of Lactococcus lactis SK11 proteinase: effects on activity, specificity, and stability of the truncated enzyme.
The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.
Caseins↗
Enzyme behavior at surfaces. Site-specific variants of subtilisin BPN' with enhanced surface stability.
Enzyme adsorption and inactivation at the solid/liquid interface for subtilisin BPN' show a strong dependence on the nature of the solid surface. Adsorption of BPN' at the solid/liquid interface is considerably greater for a hydrophobic surface than for a hydrophilic one. Likewise, the rate of inactivation of the wild-type BPN' is over five times greater when equilibrated with a hydrophobic surface than with a hydrophilic surface. The rate data from these enzyme inactivation experiments performed at 50 degrees C are best fit by a second-order kinetic equation, suggesting a bimolecular pathway to inactivation. The role of increased surface adsorption on this bimolecular inactivation is discussed in terms of two different mechanisms. Several site-specific variants of subtilisin BPN' have been made in an attempt to alter the surface-inactivation of the wild-type enzyme. The extent of adsorption on the model surfaces is significantly lowered by certain lysine to phenylalanine changes in BPN'. Consequently, the surface autolytic stability shows a 4-fold improvement. The change in surface autolytic stability is achieved even though the basic kinetic parameters (kcat and KM) of the variant enzymes are not significantly different on a soluble substrate. The results provide insights into the use of mutagenesis to probe the mechanism of protein interactions with surfaces.
Adsorption↗
Stabilization of enzymes by their specific antibodies.
In nature, increased stability of enzymes has often been found to be associated with noncovalent protein-protein interactions. Specific antibodies should be suitable for this purpose. To test this hypothesis, we used a number of model enzymes, complexed them with their specific antibodies, and exposed them and the free enzymes to low and high temperature, lyophilization, oxidation, and alcohol. The retained activity of the antibody-complexed enzymes was substantially, and in some cases dramatically, higher. In general mechanistic terms, stabilization may have been accomplished either by noncovalent antibody crosslinking of discontinuous oligopeptide chains on the surface of the enzyme, thereby increasing resistance to unfolding of the enzyme, or by physical shielding by the antibodies of vulnerable sites on the surface of the enzyme.
Animals↗
Salt-dependent studies of NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii.
The salt-dependent stability of recombinant dimeric isocitrate dehydrogenase [ICDH; isocitrate: NADP oxidoreductase (decarboxylating), EC 1.1.1.42] from the halophilic archaeon Haloferax volcanii (Hv) was investigated in various conditions. Hv ICDH dissociation/deactivation was measured to probe the respective effect of anions and cations on stability. Surprisingly, enzyme stability was found to be mainly sensitive to cations and very little (or not) sensitive to anions. Divalent cations induced a strong shift of the active/inactive transition towards low salt concentration. A high resistance of Hv ICDH to chemical denaturation was also found. The data were analysed and are discussed in the framework of the solvation stability model for halophilic proteins.
Anions↗
Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 2. Specific activity and thermal stability of enzymes bound weakly and strongly to Sepharose CL 4B.
Ribonuclease A (EC 3.1.4.22) and alpha-chymotrypsin (EC 3.4.21.1) have been covalently coupled, by a varying number of bonds, to Sepharose CL 4B which was activated with different amounts of CNBr. Upon increasing the number (1-8) of points of attachment between the enzyme and the matrix, the specific activities of immobilized ribonuclease A relative to its soluble counterpart decreased from 60 to 15% while the amount of protein coupled increased from 5 to 37 mg per g of sucked gel. Differential scanning calorimetry was used to determine whether the immobilization caused any changes in the physicochemical properties of the enzyme. Ribonuclease A, weakly bound to the matrix, showed almost the same behavior as the soluble enzyme. By contrast strongly immobilized enzyme exhibited a higher transition temperature (by about 5 degrees C) and a broader endotherm. Similar results were found for alpha-chymotrypsin.
Animals↗
Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment*
Epoxy supports (Eupergit C) may be very suitable to achieve the multipoint covalent attachment of proteins and enzymes, therefore, to stabilize their three-dimensional structure. To achieve a significant multipoint covalent attachment, the control of the experimental conditions was found to be critical. A three-step immobilization/stabilization procedure is here proposed: 1) the enzyme is firstly covalently immobilized under very mild experimental conditions (e.g. pH 7.0 and 20 degrees C); 2) the already immobilized enzyme is further incubated under more drastic conditions (higher pH values, longer incubation periods, etc.) to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; 3) the remaining groups of the support are blocked to stop any additional interaction between the enzyme and the support. Progressive establishment of new enzyme-support attachments was showed by the progressive irreversible covalent immobilization of several subunits of multi-subunits proteins (all non-covalent structures contained in crude extracts of different microorganism, penicillin G acylase and chymotrypsin). This multipoint covalent attachment enabled the significant thermostabilization of two relevant enzymes, (compared with the just immobilized derivatives): chymotrypsin (5-fold factor) and penicillin G acylase (18-fold factor). Bearing in mind that this stabilization was additive to that achieved by conventional immobilization, the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative. These stabilizations were observed also when the inactivations were promoted by the enzyme exposure to drastic pH values or the presence of cosolvents.
Journal Article↗
Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. I. Enzyme activity and calorimetric studies.
Stabilization of labile proteins during lyophilization requires protection of the protein against both freezing and dehydration stresses. Solutions of 1-10% (wt/vol) polyethylene glycol (PEG) fully protected both lactate dehydrogenase and phosphofructokinase during freezing and thawing, but did not stabilize the proteins during freeze-drying. Thus, with this lyophilization system a second compound could be tested for its capacity to stabilize dried proteins, independent of its ability to provide cryopreservation. In the presence of low concentrations of glucose or trehalose (which alone provided minimal protection) and 1% PEG (wt/vol), almost full enzyme activity was recovered after freeze-drying and rehydration. Differential scanning calorimetry indicated that the PEG was crystalline and the sugars were amorphous in the dried samples. Experiments with lactose and mannitol demonstrated that if these compounds also crystallized during freeze-drying, protein stabilization was reduced or abolished. PEG stabilizes the proteins during freezing, due to preferential exclusion of PEG from the protein's surface. The sugars protect the proteins during dehydration by hydrogen bonding to the dried protein, thus serving as water substitutes. This report provides the first example of stabilization of proteins during lyophilization through separate, specific treatments of the fundamentally different stresses of freezing and dehydration.
Animals↗
Heparan N-sulfatase: in vitro mutagenesis of potential N-glycosylation sites.
Heparan N-sulfatase cDNA contains five potential N-glycosylation sites at Asn positions 41, 142, 151, 264, and 413. We used site-directed mutagenesis, substituting the codon of asparagine for glutamine, to eliminate selected glycosylation sites and then performed expression studies in COS-7 cells to determine the influence on the catalytic activity, lysosomal targeting, and glycosylation-phosphorylation of the enzyme. Elimination of site 5 did not affect significantly enzyme activity; elimination of sites 2 and 4 gave a partial reduction, while elimination of sites 1 and 3 resulted in drastic reduction of catalytic activity (25 and 14%, respectively, of normal values), indicating that glycosylation of asparagine 41 and asparagine 151 is essential for catalysis and/or enzyme stability. Wild type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for acquisition of enzyme activity and/or for enzyme stability. Metabolic labeling of each mutant cDNA, transiently transfected into COS cells, showed that enzyme from mutants N142Q, N264Q, and N413Q appeared to be properly folded, as judged by its ability to be proteolytically processed to a lower molecular weight form, while enzyme from mutants N41Q and N151Q did not reach lysosomes. These studies confirm that the five glycosylation sites of heparan N-sulfatase are all functional and show that Asn 41 and Asn 151 have a role in protein folding and/or stability.
Amino Acid Substitution↗
Crosslinking of enzymes for improved stability and performance.
Stabilization of enzymes without diminishing and diluting their activity is crucial for their use as industrial catalysts. One practically proven approach involves crystallization of the enzyme (size 1-200 microm) followed by subsequent crosslinking. In many cases, the resulting crosslinked enzyme crystals exhibit increased storage stability as well as stability in applications. The technology is complementary to protein engineering methods that aim at boosting the inherent stability of the enzyme.
Biotechnology↗
Development of small-size tubular-flow continuous reactors for the analysis of operational stability of enzymes in low-water systems.
A very small-scale continuous flow reactor has been designed for use with enzymes in organic media, particularly for operational stability studies. It is constructed from fairly inexpensive components, and typically uses 5 mg of catalyst and flow rates of 1 to 5 mL/h, so only small quantities of feedstock need to be handled. The design allows control of the thermodynamic water activity of the feed, and works with temperatures up to at least 80 degrees C. The reactor has been operated with both nonpolar (octane) and polar (4-methyl-pentan-2-one) solvents, and with the more viscous solvent-free reactant mixture. It has been applied to studies of the operational stability of lipases from Chromobacterium viscosum (lyophilized powder or polypropylene-adsorbed) and Rhizomucor miehei (Lipozyme) in different experimental conditions. Transesterification of geraniol and ethylcaproate has been adopted as a model transformation.
Acyclic Monoterpenes↗