Inclusion bodies of the thermophilic endoglucanase D from Clostridium thermocellum are made of native enzyme that resists 8 M urea.

Endoglucanase D from Clostridium thermocellum was purified from inclusion bodies formed upon its overproduction in Escherichia coli, using 5 M urea as a solubilizing solution. We examined the effects of denaturing agents upon the stability of the pure soluble enzyme as a function of the temperature. At room temperature, guanidinium chloride induces an irreversible denaturation. By comparison, we observed no structural or functional effects at room temperature using high concentrations of urea as denaturing agent. The irreversible denaturation process observed with guanidinium chloride also occurs with urea but only at elevated temperature (greater than or equal to 60 degrees C); in 6 M urea, the activation energy of the denaturation reaction is decreased by a factor of only 1.8. We interpret the high resistance of this protein to urea as reflecting a reduced flexibility of its structure at normal temperatures which should be correlated to the thermophilic origin of this protein.     

Proteolytic activity from a thermophilic Streptomyces megasporus strain SDP4

Multiple proteases secreted by a thermophilic actinomycete Streptomyces megasporus SDP4 after 18 h of growth at 55 °C are reported. The enzyme preparation exhibited activity over a broad pH and temperature range of pH 6–12 and 25–85 °C, respectively. Optimum activity was observed at pH 8·0, pH 10·0 and 55 °C and was calcium independent. Thermostability was enhanced in the presence of 0·01 mol l⁻¹ calcium ions and half-life was 30 min at 85 °C. The enzyme was active in the presence of SDS. Both, EDTA and PMSF were partially inhibitory, indicating the presence of serine and metal requiring proteases. Three active zones in the range of 90–30 kDa were detected post-electrophoretically.     

Temperature- and denaturant-induced unfolding of two thermophilic esterases.

We studied the temperature- and denaturant-induced denaturation of two thermophilic esterases, AFEST from Archeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, by means of circular dichroism measurements. Both enzymes showed a very high denaturation temperature: 99 degrees C for AFEST and 91 degrees C for EST2. They also showed a remarkable resistance against urea; at half-completion of the transition the urea concentration was 7.1 M for AFEST and 5.9 M for EST2. On the contrary, both enzymes showed a weak resistance against GuHCl; at half-completion of the transition the GuHCl concentration was 2.0 M for AFEST and 1.9 M for EST2. The thermodynamic parameters characterizing urea- and GuHCl-induced denaturation of the studied enzymes have been obtained by both the linear extrapolation model and the denaturant binding model. The dependence of the thermal stability on NaCl concentration for both esterases has also been determined. A careful analysis of the data, coupled with available structural information, has allowed the proposal of a reliable interpretation.     

Comparison of the wild-type alpha-amylase and its variant enzymes in Bacillus amyloliquefaciens in activity and thermal stability, and insights into engineering the thermal stability of bacillus alpha-amylase

The starch hydrolysis activity and thermal stability of Bacillus amyloliquefaciens alpha-amylase (wild-type enzyme or WT) and its variant enzymes, designated as M77, M111, and 21B, were compared. All have an optimal pH at around 6, as well as almost the same reaction rates and Km and kcat values. The optimal temperature in the absence of Ca2+ ions is 60 degrees C for WT and M77 and 40 degrees C for M111 and 21B. Those of M111 and 21B rose to 50-60 degrees C upon the addition of 5 mM CaCl2, while those of WT and M77 did not change. The dissociation constants Kd for Ca2+ to WT and M77 are much lower than those of M111 and 21B. Asp233 in WT is replaced by Asn in M111 and 21B, while it is retained in M77, suggesting that Asp233 is involved in the thermal stability of the enzyme through Ca2+ ion binding. These findings provide insight into engineering the thermal stability of B. amyloliquefaciens alpha-amylase, which would be useful for its applications in the baking industry and in glucose manufacturing.     

The role of bound calcium ions in thermostable, proteolytic enzymes. I. Studies on thermomycolase, the thermostable protease fron the fungus Malbranchea pulchella.

Thermomycolase, the thermostable, extracellular, serine protease of the fungus Malbranchea pulchella (G. Voordouw, G. M. Gaucher, and R. S. Roche (1974), Can J. Biochem. 52, 981-990), binds one calcium ion with an apparent binding constant of 5 X 10(5) M-1 at 25degreesC, pH 7.50, and ionic strength 0.1. There is very little change in the overall conformation of thermomycolase upon binding of this calcium ion: no change can be detected, beyond experimental error, in the sedimentation coefficient or the aromatic and peptide circular dichroism of the enzyme. However, binding of calcium changes the absorption spectrum, the ultraviolet difference spectrum being characterized by a strong band at 237 nm and smaller bands at 280 and 295 nm. The difference molar extinction coefficient at 237 nm parallels the calcium-binding isotherm. The small changes in equilibrium properties are constrasted by large calcium-dependent changes in the rates of autolytic degradation and thermal and urea denaturation. The dependence of the second-order rate constant for autolytic degradation on the free calcium ion concentration can be quantitatively accounted for by a model in which only conformers with an unoccupied calcium binding site serve as substrates in the reaction. The calcium dependence of the first-order rate constant for thermal denaturation at 70degreesC and pH 7.0 can also be accounted for quantitatively by a model in which the critically activated intermediate has a smaller calcium-binding constant than the native form of the enzyme under these conditions. The same model also accounts for the denaturation in 8 M urea at 50degreesC, pH 7.0. Rates of hydrogen-tritium exchange are shown to decrease when the calcium ion is bound. Irrespective of the occupancy of the calcium-binding site 33% of the backbone peptide hydrogens of thermomycolase do not exchange within 24 hr at 25degreesC, pH 8.0, and ionic strength 0.1.     

Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation

The conformational stability of RNase Rs was determined with chemical and thermal denaturants over the pH range of 1-10. Equilibrium unfolding with urea showed that values of D(1/2) (5.7 M) and DeltaG(H(2)O) (12.8 kcal/mol) were highest at pH 5.0, its pI and the maximum conformational stability of RNase Rs was observed near pH 5.0. Denaturation with guanidine hydrochloride (GdnHCl), at pH 5.0, gave similar values of DeltaG(H(2)O) although GdnHCl was 2-fold more potent denaturant with D(1/2) value of 3.1 M. The curves of fraction unfolded (f(U)) obtained with fluorescence and CD measurements overlapped at pH 5.0. Denaturation of RNase Rs with urea in the pH range studied was reversible but the enzyme denatured irreversibly >pH 11.0. Thermal denaturation of RNase Rs was reversible in the pH range of 2.0-3.0 and 6.0-9.0. Thermal denaturation in the pH range 4.0-5.5 resulted in aggregation and precipitation of the protein above 55 degrees C. The aggregate was amorphous or disordered precipitate as observed in TE micrographs. Blue shift in emission lambda(max) and enhancement of fluorescence intensity of ANS at 70 degrees C indicated the presence of solvent exposed hydrophobic surfaces as a result of heat treatment. Aggregation could be prevented partially with alpha-cyclodextrin (0.15 M) and completely with urea at concentrations >3 M. Aggregation was probably due to intermolecular hydrophobic interaction favored by minimum charge-charge repulsion at the pI of the enzyme. Both urea and temperature-induced denaturation studies showed that RNase Rs unfolds through a two-state F right arrow over left arrow U mechanism. The pH dependence of stability described by DeltaG(H(2)O) (urea) and DeltaG (25 degrees C) suggested that electrostatic interactions among the charged groups make a significant contribution to the conformational stability of RNase Rs. Since RNase Rs is a disulfide-containing protein, the major element for structural stability are the covalent disulfide bonds.     

The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes.     

Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.     

Structural basis of the thermostability of monomeric malate synthase from a thermophilic Bacillus.

Malate synthases from a thermophilic Bacillus and Escherichia coli have been isolated in a high state of purity. Molecular weights of these two proteins determined in the native state and after denaturation in sodium dodecyl sulfate-mercaptoethanol show that the enzymes are monomeric. This conclusion is supported, for the thermophile enzyme, by the result of an electrophoretic analysis of that protein after treatment with dimethylsuberimidate and denaturation. The thermophilic Bacillus malate synthase is considerably more thermostable than its mesophilic counterparts from E. coli, Bacillus licheniformis, and Pseudomonas indigofera. It is, however, markedly labilized by an increase in the ionic strength of the medium brought about by the addition of 0.2 M potassium chloride or in pH above 9. Increased ionic strength has little effect on the thermostability of the mesophilic bacterial malate synthases. These observations provide strong support for the idea that monomeric proteins in thermophiles owe their unusual heat stability to the presence of salt bridges in their tertiary structure.     

Influence of synthetic peptide inhibitors on the thermal stability of thermitase, a serine proteinase from Thermoactinomyces vulgaris

The influence of chloromethyl ketones and methyl ketones of N-acylated peptides on the thermal denaturation of thermitase was investigated in the presence and the absence of calcium ions. The chloromethyl ketone derivatives are known to react irreversibly with the enzyme, whereas the corresponding methyl ketones are reversible inhibitors. Both groups of inhibitors offer a broad variety of affinity constants. The irreversible inhibition of thermitase causes a marked stabilization against thermal denaturation. On the other hand, the enzyme stability is not influenced by the binding of reversible inhibitors. The stabilizing effect of calcium ions is not dependent on the inhibitor binding. The importance of bivalent interaction (bridge formation) in the active site region of the enzyme for its thermal stability is discussed.     

Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101

Summary An alkalophilic Bacillus sp. no. AH-101, which produced extremely thermostable alkaline protease, was isolated among 200 soil samples. The enzyme production reached its maximum level of 1500 units/ml after about 24 h in alkaline medium (pH 9.5). The enzyme was most active toward casein at pH 12–13 and stable to 10 min incubation at 60° C from pH 5–13. Calcium ions were effective in stabilizing the enzyme especially at higher temperatures. The optimum and stable temperatures were about 80° C and below about 70° C respectively in the presence of 5 mM calcium ions. The enzyme was completely inactivated by phenylmethane sulphonyl fluoride, but little affected by ethylenediaminetetraacetic acid, urea, sodium dodecylbenzenesulphonate and sodium dodecyl sulphate. The molecular weight and sedimentation constant were approximately 30 000 and 3.0S respectively, and the isoelectric point was at pH 9.2. These results indicte that no. AH-101 alkaline protease is more stable against both temperature and highly alkaline conditions than any other protease so far reported.     

Unfolding-refolding behaviour of chicken egg white ovomucoid and its correlation with the three domain structure of the protein

The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and its four fragments representing domains I, II + III, I + II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min-1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.     

The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets

The basal apparatus, consisting of an array of interconnected basal bodies bearing bifurcating striated rootlets encompassing a nucleus, has been isolated from hypertonically deciliated columnar gill epithelial cells of the bay scallop Aequipecten irradians through gentle lysis with Triton X-100. The rootlets, 8-10 mum in length, were not easily preserved with conventional electron microscope fixatives, suggesting that the extent of their contribution to cellular architecture has been somewhat underestimated, even though Englemann described many of the structural details of the basal apparatus in 1880. The striated rootlets were soluble at high but not at low pH, in 2 M solutions of sodium azide and potassium thiocyanate but not sodium or potassium chloride, in 1% deoxycholate but not digitonin, and in the denaturing solvents 6 M guanidine-HC1, 8 M urea, and 1% sodium dodecylsulfate at 100 degrees C. The protein found consistently when rootlets were solubilized migrated on SDS-polyacrylamide gels as a closely spaced doublet with apparent molecular weights of 230,000 and 250,000 daltons. This unique protein, distinct from tropocollagen or various muscle components, has been named ankyrin because of the rootlet's anchor-like function in the cell.     

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.     

Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation

Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation.     

Thermal stability of lactate dehydrogenase in the complex with the anion polyelectrolyte poly(styrene sulfonate)

The temperature stability of the cytoplasmic enzyme of the glycolysis of lactate dehydrogenase from a pig muscle (isoenzyme M4) in a complex with the anion polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, the own protein fluorescence, and circular dichroism. Calorimetric investigations of complex of lactate dehydrogenase with poly(styrenesulphonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulphonate)/lactate dehydrogenase, though at 20 degrees C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulphonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

Temperature stability of lactate dehydrogenase in complex with anionic polyelectrolyte poly(styrenesulfonate)

The temperature stability of the cytoplasmic enzyme of glycolysis, lactate dehydrogenase from pig muscle (isoenzyme M4) in complex with anionic polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, own protein fluorescence, and circular dichroism. Calorimetric investigations of the complex of lactate dehydrogenase with poly(styrenesulfonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulfonate)/lactate dehydrogenase, though at 20°C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulfonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

Complete conformational stability of kinetically stable dimeric serine protease milin against pH,temperature, urea,and proteolysis

Spectroscopic, calorimetric, and proteolytic methods were utilized to evaluate the stability of the kinetically stable, differentially glycosylated, dimeric serine protease milin as a function of pH (1.0–11.0), temperature, urea, and GuHCl denaturation in presence of 8 M urea at pH 2.0. The stability of milin remains equivalent to that of native at pH 1.0–11.0. However, negligible and reversible alteration in structure upon temperature transition has been observed at pH 2.0 and with 1.6 M GuHCl. Irreversible and incomplete calorimetric transition with apparent T _m > 100°C was observed at basic pH (9.0 and 10.0). Urea-induced unfolding at pH 4.0, and at pH 2.0 with GuHCl, in presence of 8 M urea also reveals incomplete unfolding. Milin has been found to exhibit proteolytic resistant in either native or denatured state against various commercial proteases. These results imply that the high conformational stability of milin against various denaturating conditions enable its potential use in protease-based industries.     

States of tryptophyl residues and stability of recombinant human matrix metalloproteinase 7 (matrilysin) as examined by fluorescence

States of tryptophyl residues and stability of human matrilysin were studied. The activation energy for the thermal inactivation of matrilysin was determined to be 237 kJ/mol, and 50% of the activity was lost upon incubation at 69 degrees C for 10 min. The activity was increased by adding NaCl, and was doubled with 3 M NaCl. Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues. Half of the change was observed at 2.2-2.7 M GdnHCl, whereas no change was observed even with 8 M urea. Half of the inactivation was induced at 0.8 M GndHCl and at 2 M urea. The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denaturation. Matrilysin contains four tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide. No tryptophyls in the native enzyme were accessible to I(-) and Cs(+), and 2.4 residues were accessible to acrylamide. Based on the crystallographic study, Trp154 is water-accessible, but it should be in a crevice not to contact with I(-) and Cs(+). All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one.     

Denaturation ofBacillus stearothermophilus α-amylase by urea and detergents

Denaturation ofBacillus stearothermophilus -amylase by urea and detergents was investigated for the purpose of understanding the mechanism of denaturation of this enzyme. The enzyme was extremely resistant to denaturation by detergents at 60° C, either in the presence or absence of added calcium. Addition of EDTA was necessary to obtain denaturation by detergents. The rate of denaturation of the -amylase by urea was strongly dependent on the incubation pH and presence or absence of calcium ions. Calcium-binding groups were shown to have pKa values of 5.5 for exogenous calcium and 4.7 to 4.8 for endogenous calcium. A mechanism is proposed for the denaturation ofBacillus stearothermophilus -amylase.