Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes

Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines. It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures. Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation. This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization. The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed. Inactivation of the protein by autolysis did not occur. This paper reports the use of a series of polyol osmolytes to stabilize PA. Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent. Addition of NaCl to glycerol solution further enhanced the thermal stability of PA. An increase in the T(1/2) value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20 degrees C was observed. The half-life (t(1/2)) of PA thermal inactivation at 40 degrees C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control. This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax.     

Gln277 and Phe554 residues are involved in thermal inactivation of protective antigen of Bacillus anthracis

Protective antigen (PA) is the main component of all the vaccines against anthrax. The currently available vaccines have traces of other proteins that contribute to its reactogenicity. Thus, purified PA is recommended for human vaccination. PA loses its biological activity within 48h at 37 degrees C and its thermolability has been a cause of concern as accidental exposure to higher temperatures during transportation or storage could decrease its efficacy. In the present study, we have used protein engineering approach to increase the thermostability of PA by mutating amino acid residues on the surface as well as the interior of the protein. After screening several mutants, the mutants Gln277Ala and Phe554Ala have been found to be more thermostable than the wild-type PA. Gln277Ala retains approximately 45% and Phe554Ala retains approximately 90% activity, even after incubation at 37 degrees C for 48h while in the same period wild-type PA loses its biological activity completely. It is the first report of increasing thermostability of PA using site-directed mutagenesis. Generation of such mutants could pave the way for better anthrax vaccines with longer shelf life.     

Thermostabilization of protective antigen—the binding component of anthrax lethal toxin

Protective antigen (PA) is the binding component of anthrax lethal toxin produced by Bacillus anthracis, and constitutes a major ingredient of the vaccine against anthrax. PA and lethal factor when added together are cytolytic to mouse macrophages and J774G8 macrophage cell line. This in vitro lethal toxicity assay is very useful in understanding the molecular mechanism of action of lethal toxin. Effective utilization of PA is, however, hampered due to its thermolability. On prolonged storage at 37 ° C, PA was found to lose its activity almost completely. The effect of solvent additives like trehalose, sorbitol, xylitol, sodium citrate and magnesium sulphate on the thermal stabilization of PA was examined. The results indicated an increase in the stability of PA when the incubation at 37 ° C was carried out in the presence of solvent additives used in the 1–3 M range. Magnesium sulphate helped retain the activity up to 82.7% against the control in which no additive was used, as judged by cytolytic assay using J774G8 macrophage cell line. Trehalose or sodium citrate also showed an appreciable protection of PA activity, while sorbitol or xylitol were not very effective. Competitive binding assay using radiolabeled PA showed that PA had lost capacity of binding to macrophage cells on prolonged incubation at 37 ° C. Circular dichroism results at 4, 18 and 37 ° C indicated an increase in secondary structure at 37 ° C relative to that at 4 or 18 ° C, supporting the activity data.     

A study of the physiology of Bacillus anthracis Sterne during manufacture of the UK acellular anthrax vaccine

AIM: To analyse the growth of Bacillus anthracis during simulations of the UK anthrax vaccine manufacturing process. METHODS AND RESULTS: Simulated vaccine production runs were performed using the toxigenic, acapsulate Sterne 34F(2) strain of B. anthracis in semi-defined medium. After rising during the logarithmic growth phase, the pH of the culture starts to fall at about 18 h from pH 8.7 to reach <7.6 at 26 h, coincident with consumption of glucose and optimal production of protective antigen (PA; 7.89 g ml(-1), SD 1.0) and lethal factor (LF; 1.85 g ml(-1), SD 0.29). No increased breakdown of toxin antigens was seen over the 26-32 h period. When glucose was exhausted, amino acids (principally serine) were utilized as an alternative carbon source. Sporulation was not observed during the 32 h. CONCLUSIONS: PA and LF, the principal constituents in the UK anthrax vaccine, undergo little degradation during vaccine fermentation. The vaccine manufacturing process is robust and reproducible. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed analysis of the manufacturing process used for the UK acellular anthrax vaccine; insight gained into the process will support continued and safe vaccine manufacture.     

Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.     

Comparison of starch hydrolysis activity and thermal stability of two Bacillus licheniformis alpha-amylases and insights into engineering alpha-amylase variants active under acidic conditions

Bacillus licheniformis alpha-amylase (BLA) is widely used in various procedures of starch degradation in the food industry, and a BLA species with improved activity at higher temperature and under acidic conditions is desirable. Two BLA species, designated as PA and MA, have been isolated from the wild-type B. licheniformis strain and a mutant strain, respectively. In this study, their starch-hydrolysis activity and thermal stability were examined. MA showed higher activity than PA, especially at acidic pH (pH 5.0-5.5), and even after 1 h of treatment at 90 degrees C. MA was active in the range of pH 4.0-8.0, which is much wider than that (pH 4.5-7.5) of PA. It was shown that the proton dissociation constants on the acidic and alkaline sides (pKa1 and pKa2) were shifted to more acidic and basic values, respectively, by the mutation of PA to MA. The activation energy and thermodynamic parameters for their thermal inactivation indicate that MA is more thermally stable and catalytically active than PA, suggesting that MA could be useful for glucose-production process coupled with reactions catalyzed by beta-amylase.     

Thermobarostability of alpha-chymotrypsin in reversed micelles of aerosol OT in octane solvated by water-glycerol mixtures

Thermostability of alpha-chymotrypsin at normal pressure in reversed micelles depends on both an effective surfactant solvation degree and glycerol content in the system. The difference in alpha-chymotrypsin stability in reversed micelles at various glycerol concentrations [up to 60% (v/v)] was more pronounced at high surfactant degrees of solvation, R >/= 16. After a 1-h incubation at 40 degrees C in "aqueous" reversed micelles (in the absence of glycerol), alpha-chymotrypsin retained only 1% of initial catalytic activity and 10, 22, 59, and 48% residual activity in glycerol-solvated micelles with 20, 30, 50, and 60% (v/v) glycerol, respectively. The explanation of the observed effects is given in the frames of micellar matrix structural order increasing in the presence of glycerol as a water-miscible cosolvent that leads to the decreasing mobility of the alpha-chymotrypsin molecule and, thus the increase of its stability. It was found that glycerol or hydrostatic pressure could be used to stabilize alpha-chymotrypsin in reversed micelles; a lower pressure is necessary to reach a given level of enzyme stability in the presence of glycerol.     

Stability of domain 4 of the anthrax toxin protective antigen and the effect of the VWA domain of CMG2 on stability

The major immunogenic component of the current anthrax vaccine, anthrax vaccine adsorbed (AVA) is protective antigen (PA). We have shown recently that the thermodynamic stability of PA can be significantly improved by binding to the Von‐Willebrand factor A (VWA) domain of capillary morphogenesis protein 2 (CMG2), and improvements in thermodynamic stability may improve storage and long‐term stability of PA for use as a vaccine. In order to understand the origin of this increase in stability, we have isolated the receptor binding domain of PA, domain 4 (D4), and have studied the effect of the addition of CMG2 on thermodynamic stability. We are able to determine a binding affinity between D4 and CMG2 (～300 nM), which is significantly weaker than that between full‐length PA and CMG2 (170–300 pM). Unlike full‐length PA, we observe very little change in stability of D4 on binding to CMG2, using either fluorescence or ¹⁹F‐NMR experiments. Because in previous experiments we could observe a stabilization of both domain 4 and domain 2, the mechanism of stabilization of PA by CMG2 is likely to involve a mutual stabilization of these two domains.     

Development of acetylcholinesterase silica sol-gel immobilized biosensor--an application towards oxydemeton methyl detection

An amperometric silica sol-gel film immobilized biosensor doped with acetylcholinesterase was fabricated in the laboratory finding application in organophosphate detection based on enzyme inhibition. The substrate used was acetylthiocholine chloride and thiocholine released from the enzymatic hydrolysis was electrochemically oxidized giving larger anodic current at 0.5-0.6 V (versus Ag/AgCl reference). The dependencies of the current response on pH, enzyme loading and thermal stability of the developed biosensor were evaluated. The analytical performance of enzyme electrode towards substrate and inhibitor was investigated. Oxydemeton methyl was taken as a model compound for the inhibition studies. Linear calibration for oxydemeton methyl was obtained in the range of 2-200 ppb under the optimized conditions following an incubation time of 20 min. Treatment of the inhibited enzyme with pyridine-2-aldehyde restored 92% of its original activity. The sensor stored at -20 degrees C had a good storage and operational stability retaining 85% of its original activity for 60 successive measurements.     

The dissociation of glucose oxidase by sodium n-dodecyl sulphate.

1. The enzymic activity of glucose oxidase was determined as a function of pH and sodium n-dodecyl sulphate (SDS) concentration. 2. Glucose oxidase is not deactivated by SDS at pH 6 even after prolonged incubation, but is deactivated at pH 4.3 and 3.65. 3. Sedimentation-rate analysis showed that glucose oxidase dissociates into its two subunits at pH 5 and below, and sedimentation-equilibrium experiments in the presence of SDS gave a subunit molecular weight of 73,500. 4. SDS binds to glucose oxidase in acid solutions; specific binding occurs ap pH 3.65, but at pH 6 only co-operative binding was observed. 5. Glucose oxidases in which some of the carboxy groups were blocked with glycine methyl ester were deactivated by SDS at pH 6.0; the rate of deactivation increased with the extent of esterification. 6. Deactivation of esterified glucose oxidases correlated with thermal analysis of the initial SDS interaction, the exothermicity of the interaction increasing with the extent of esterification. 7. The results show that carboxy groups confer resistance to deactivation by SDS on glucose oxidase by screening cationic residues and inhibiting specific interactions that facilitate dissociation into subunits.     

Anthrax toxin receptor 2 determinants that dictate the pH threshold of toxin pore formation

The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH approximately 6.0 or below when it is bound to ANTXR1, but only at pH approximately 5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation.     

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.     

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.     

Surface Plasmon Resonance Biosensor for Detection of Bacillus anthracis, the Causative Agent of Anthrax from Soil Samples Targeting Protective Antigen

Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, K_D (equilibrium constant) and B_max (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb’s free energy (∆G = −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation.     

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.     

Stabilization of immobilized glucose oxidase against thermal inactivation by silanization for biosensor applications

An important requirement of immobilized enzyme based biosensors is the thermal stability of the enzyme. Studies were carried out to increase thermal stability of glucose oxidase (GOD) for biosensor applications. Immobilization of the enzyme was carried out using glass beads as support and the effect of silane concentration (in the range 1-10%) during the silanization step on the thermal stability of GOD has been investigated. Upon incubation at 70 degrees C for 3h, the activity retention with 1% silane was only 23%, which increased with silane concentration to reach a maximum up to 250% of the initial activity with 4% silane. Above this concentration the activity decreased. The increased stability of the enzyme in the presence of high silane concentrations may be attributed to the increase in the surface hydrophobicity of the support. The decrease in the enzyme stability for silane concentrations above 4% was apparently due to the uneven deposition of the silane layer on the glass bead support. Further work on thermal stability above 70 degrees C was carried out by using 4% silane and it was found that the enzyme was stable up to 75 degrees C with an increased activity of 180% after 3-h incubation. Although silanization has been used for the modification of the supports for immobilization of enzymes, the use of higher concentrations to stabilize immobilized enzymes is being reported for the first time.     

Studies on yellow fever vaccine. II--Stability of the reconstituted product

This work evaluated the stability of diluted yellow fever vaccine in order to determine conditions that maintain the minimum of 3 log10 of 17D virus per human dose as required by WHO. The vaccines were held at 0 degrees C or at 37 degrees C and were diluted either with distilled water, with 0.15 M saline or with 0.15 M PBS at pH 5.5, 7.2 and 8.0. In a next step, stabilizer substances such as gelatin and peptone were added to the vaccines. Dilution of the vaccines in distilled water maintained the virus titre for up to three hours at 37 degrees C and this diluent has been adopted for routine use in Brazil.     

Unfolding transitions of Bacillus anthracis protective antigen

Protective antigen (PA) is an 83kDa protein which, although essential for toxicity of Bacillus anthracis, is harmless and an effective vaccine component. In vivo it undergoes receptor binding, proteolysis, heptamerisation and membrane insertion. Here we probe the response of PA to denaturants, temperature and pH. We present analyses (including barycentric mean) of the unfolding and refolding behavior of PA and reveal the origin of two critical steps in the denaturant unfolding pathway in which the first step is a calcium and pH dependent rearrangement of domain 1. Thermal unfolding fits a single transition near 50 degrees C. We show for the first time circular dichroism (CD) spectra of the heptameric, furin-cleaved PA63 and the low-pH forms of both PA83 and PA63. Although only PA63 should reach the acidic endosome, both PA83 and PA63 undergo similar acidic transitions and an unusual change from a beta II to a beta I CD spectrum.     

重组PA4复制子病毒颗粒疫苗免疫原性的分析

目的:构建携带炭疽毒素保护性抗原第四结构域(PA4)基因的重组Semliki森林病毒(SFV)复制子病毒颗粒,并对其免疫原性进行研究。方法:将编码炭疽PA4的SFV复制子DNA载体pSCAR-SPA4,与辅助SFV DNA载体pSHCAR共转染BHK21细胞,制备表达PA4的重组复制子病毒颗粒;用重组复制子病毒颗粒疫苗免疫小鼠,并采用ELISA法检测其血清抗体水平和细胞因子IFN-γ和IL-4。结果:免疫小鼠血清中检测到较高的抗体水平,免疫小鼠的脾淋巴细胞经特异性抗原刺激后产生了明显的T细胞增殖反应并分泌产生了IFN-γ和IL-4。结论:重组PA4复制子病毒颗粒疫苗免疫小鼠后能够产生特异性的抗体反应和细胞免疫反应。制备的重组PA4复制子病毒颗粒极有潜力作为人用炭疽候选疫苗,为进一步研究新型炭疽疫苗奠定了基础。     

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.