The Effect of pH and NaCl Levels on the Physicochemical and Emulsifying Properties of a Cruciferin Protein Isolate

The influence of pH (3.0, 5.0, and 7.0) and ionic strength (0, 50, 100 mM NaCl) on the physicochemical and emulsifying properties of a cruciferin-rich protein isolate (CPI) was investigated. Surface charge on the CPI was found to substantially reduced in the presence of NaCl. Surface hydrophobicity was found to be the lowest for CPI at pH 7.0 with 100 mM NaCl, and highest at pH 3.0 without NaCl. Solubility was found to be lowest at pH 5.0 and 7.0 without NaCl (<20 %), however greatly improved for all other pH and NaCl conditions (>80 %). Interfacial tension was found to be lowest at 10–11 mN/m for pH 5.0–0 mM NaCl and pH 7.0–50/100 mM NaCl, whereas under all other conditions interfacial tension was higher (15+ mN/m). Overall, NaCl has no effect on EAI at pH 3.0 where it ranged between 18.8 and 19.4 m²/g. At pH 5.0, EAI decreased from 21.1 to 12.8 m²/g as NaCl levels increased from 0 to 100 mM. At pH 7.0, EAI values were found to decrease from 14.9 to 5.2 m²/g as NaCl levels were raised from 0 to 100 mM. Overall, ESI was reduced with the addition of NaCl from ~15.7 min at 0 mM NaCl to ~11.6 min and ~12.0 min for the 50 and 100 mM NaCl levels, respectively.     

Thermal stability of fatty acid-serum albumin complexes studied by differential scanning calorimetry.

Differential scanning calorimetry has been used to study the thermal stability of bovine serum albumin as affected by binding of fatty acids (lauric acid and stearic acid) and sodium dodecyl sulfate (SDS). All the ligands stabilized the protein molecules in a similar manner, but to different levels. A maximum increase in denaturation temperature of 30 degrees C was obtained with lauric acid. The thermograms indicate the presence of several ligand-albumin complexes having different heat stabilities. Variations in pH in 0.9% NaCl affected the heat stability of both ligand-poor and ligand-rich albumin, the former being more sensitive to variations in pH within the physiological range. Variations in NaCl concentration affected the thermal stabilities at neutral pH, expecially at low salt concentrations. While ligand-rich albumin was somewhat destabilized by increasing NaCl concentrations, ligand-poor albumin was strongly stabilized. The potential use of differential scanning calorimetry in ligand-albumin research is discussed.     

The influence of alkali and heat treatment on yeast protein

The soluble components in disintegrated cells of Saccharomyces cereivisiae have been characterized by means of extraction, centrifugation, dialysis, and gel filtration. The influence of alkali and heat treatment on the protein and RNA in the soluble fraction from disintegrated yeast cells and on functional properties of protein concentrates have been studied. After water extraction and centrifugation at 100000 g 42% of the nitrogen containing components of the disintegrated cells were recovered in the supernatant. By extraction at pH 11.5 an additional 31% of the nitrogen was solubilized. Half of the water-soluble nitrogen-containing components has a molecular weight lower than 5000. In the water- and alkali-soluble fractions about 80% of each amino acid was recovered The water-soluble protein was separated into 3 fractions by gel filtration on Sephadex G 200. The major portion of the protein had a molecular weight about 100,000. The amount of protein in this fraction was decreased after treatment at increasing pH and temperature. No degradation of protein to low molecular peptides occurred. The amount of RNA in the soluble fraction was only slightly influenced by alkali treatment and by heat treatment at pH 7.5 in the presence of 5% NaCl. RNA was not degraded to low molecular components of the treatments. The solubility of protein concentrates decreased after treatment at alkaline pH and after heat precipitation.     

Differential Thermal Analysis of Milk Proteins

Differential thermal analysis (DTA) was used for study of milk protein denaturation. Protein solutions produced an endothermic peak of characteristic shape and temperature of peak minimum. The peak minimum is considered the coagulation temperature of the protein.

The influence of pH and additives such as sugars and NaCl was clearly observed on the thermograms of β-lactoglobulin solution. Addition of κ-casein to β-lactoglobulin solution showed an inhibitory effect on the heat coagulation.

Solid proteins produced two-stage exothermic peaks between 200°C and 400°C.

DTA was a useful method in the study of heat denaturation and degradation of protein.     

Heat-Induced Whey Protein Gels: Effects of pH and the Addition of Sodium Caseinate

The effects of pH (6.7 or 5.8), protein concentration and the heat treatment conditions (70 or 90 °C) on the physical properties of heat-induced milk protein gels were studied using uniaxial compression, scanning electron microscopy, differential scanning calorimetry, and water-holding capacity measurements. The systems were formed from whey protein isolate (10–15% w/v) with (5% w/v) or without the addition of caseinate. The reduction in pH from 6.7 to 5.8 increased the denaturation temperature of the whey proteins, which directly affected the gel structure and mechanical properties. Due to this increase in the denaturation temperature of the β-lactoglobulin and α-lactalbumin, a heat treatment of 70 °C/30 min did not provide sufficient protein unfolding to form self-supporting gels. However, the presence of 5% (w/v) sodium caseinate decreased the whey protein thermo stability and was essential for the formation of self-supporting gels at pH 6.7 with heat treatment at 70 °C/30 min. The gels formed at pH 6.7 showed a fine-stranded structure, with great rigidity and deformability as compared to those formed at pH 5.8. The latter had a particulate structure and exuded water, which did not occur with the gels formed at pH 6.7. The addition of sodium caseinate led to less porous networks with increased gel deformability and strength but decreased water exudation. The same tendencies were observed with increasing whey protein concentration.     

Emulsifying behaviour and rheological properties of the extracellular polysaccharide produced by Pseudomonas oleovorans grown on glycerol byproduct

The functional properties of a novel extracellular polysaccharide (EPS) produced by Pseudomonas oleovorans grown on glycerol byproduct, generated by the biodiesel industry, were investigated. The EPS is a high molecular weight (4.6 × 10⁶) heteropolysaccharide, composed by neutral sugars (galactose, 68%; mannose, 17%; glucose, 13%; rhamnose, 2%; fucose, 4%) and acyl groups (3.04%). This biopolymer has pseudoplastic fluid behaviour in aqueous media. The apparent viscosity was stable for the pH range 2.9–7.1 and NaCl concentrations up to 1.0 M. Though the apparent viscosity decreased at high temperatures, at alkaline conditions and at NaCl concentrations of 2.0 M, pseudoplastic fluid behaviour was retained. The EPS was capable of stabilizing water emulsions with several hydrophobic compounds, including hydrocarbons, vegetable and mineral oils. It retained its emulsifying activity during exposure to wide temperature (30–50 °C) and pH (2–12) variations, as well as to the presence of NaCl at concentrations as high as 2.0 M.     

Compatible solutes contribute to heat resistance and ribosome stability in Escherichia coli AW1.7

This study investigated the mechanisms of heat resistance in Escherichia coli AW1.7 by quantification of cytoplasmic solutes, determination of ribosome denaturation, and by determination of protein denaturation. To assess the contribution of heat shock proteins and compatible solutes, experiments were conducted after exposure to sublethal heat shock, and with cultures grown at NaCl concentrations ranging from 0 to 6%. Heat resistance of E. coli AW1.7 was compared to the heat sensitive E. coli GGG10 and a plasmid-cured, heat sensitive derivative of E. coli AW1.7 named E. coli AW1.7ΔpHR1. Sublethal heat shock improved survival at 60°C of E. coli GGG10 and AW1.7ΔpHR1 but not of E. coli AW1.7. Addition of NaCl increased the heat resistance of all three strains, but only E. coli AW1.7 exhibited high heat resistance when grown in NaCl concentrations ranging from 2 to 6%. E. coli AW1.7 and GGG10 accumulated 16.1±0.8 and 8.8±0.8mmolL(-1) amino acids when grown at 0% NaCl, and 1.47±0.07 and 0.78±0.06mmolL(-1) carbohydrates when grown at 6% NaCl, respectively. Ribosome denaturation was determined by differential scanning calorimetry. After growth in the presence of 0% NaCl, the 30S subunit denatured at 63.7±0.8°C and 60.7±0.3°C in E. coli AW1.7 and GGG10, respectively. Fourier-transformed-infrared-spectroscopy did not indicate differences in protein denaturation between the strains during heating. In conclusion, heat resistance in E. coli AW1.7 correlates to ribosome stability at 60°C and is dependent on accumulation of cytoplasmic solutes.     

A new approach for determining the stability of recombinant human epidermal growth factor by thermal Fourier transform infrared (FTIR) microspectroscopy

Based on Fourier transform infrared (FTIR) microspectroscopy, the conformation of rhEGF under the influence of pH, heat treatment, chaotropic salts, concentration of salt and protein structure perturbants was studied. The FTIR spectrum of rhEGF showed that major secondary structures from amide I bands composed of 40.6% beta-sheets, 25.0% reverse turns, 16.5% random coils, 13.0% loops and 4.9% side-chain structures. At extreme pH conditions (pH < 4 and pH > 8), there were changes in intensity of the bands attributed to loop (1658 cm(-1)) and random coil structures, and these bands shifted to lower wavenumbers, indicating changes in protein conformation. Thermal denaturation of rhEGF occurred at 40-76 degrees C and the formation of intermolecular beta-aggregates was revealed by the FTIR spectra. Thermal-irreversible property of rhEGF after second-heating treatment suggested that rhEGF has a poor thermal stability. While investigating the stability of rhEGF in the presence of chaotropic salts, anions induced protein unfolding of rhEGF more significantly than cations. The optimal stabilizing effect was found at the 2 M NaCl added to rhEGF, and expressed the structure of rhEGF more stable on the many components. The bands of loop structure (1654 cm(-1)), beta-sheet (1638 cm(-1)) and intermolecular antiparallel beta-aggregation formation (1694, 1619 and 1612 cm(-1)) seem to be "marked" to be more sensitive in determining environmental changes of rhEGF for FTIR microspectroscopy.     

Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.     

pH-dependent aggregation of oligomeric Artocarpus hirsuta lectin on thermal denaturation

The pH dependence of the activity, aggregation, and secondary structure of Artocarpus hirsuta lectin was studied using intrinsic and extrinsic fluorescence, light scattering, and circular dichroism. The lectin is more stable in the neutral and acidic than in the alkaline pH range, which is also reflected in the binding constants of the lectin to methyl alpha-galactopyranoside (me alpha-gal). The aggregation of the protein due to heat denaturation is prevented at both extremes of pH. The binding of hydrophobic dye to the lectin takes place at pH 1-2, which increases with increasing temperature. The exposure of hydrophobic patches at pH 1 is reversible. The secondary structure of the lectin is intact in the pH range of 1-8 and is distorted above pH 9. Aggregation of the protein due to heat denaturation is also prevented in the presence of guanidine hydrochloride (GdnHCl).     

Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10 mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.     

Influence of Physico-Chemical Factors on the Oligomerization and Biological Activity of Bacteriocin AS-48

Bacteriocin AS-48 forms a mixture of monomers and oligomers in aqueous solutions. Such oligomers can be clearly differentiated by SDS-PAGE after formaldehyde crosslinking, and we have verified that these associates are stable to acid treatment after fixation. In addition, they show antimicrobial activity and are recognized by anti-AS-48 antibodies. AS-48 oligomers can be dissociated by the detergents SDS and Triton X-100. The degree of oligomerization of AS-48 depends on the pH of the solution and the protein concentration. At pH below 5, AS-48 is in the monomeric state at protein concentrations below 0.55.mg ml⁻¹, but it also forms dimers above this protein concentration. This bacteriocin forms oligomers at pH values above 5, in agreement with the observation that it is also more hydrophobic at neutral pH. AS-48 is stable to mild heat treatments irrespectively of pH. At 120°C it is more heat resistant under acidic conditions, but it inactivates at neutral pH. Activity of AS-48 against E. faecalis is highest at neutral pH, but it is highest at pH 4 for E. coli. The influence of pH on bacteriocin activity could be owing to changes in the conformation/oligomerization of the bacteriocin peptide as well as to changes in the surface charge of the target bacteria. Received: 3 July 2000\t/\tAccepted: 11 August 2000     

Thermal denaturation and regeneration of japanese-radish peroxidase.

Thermal denaturation of Japanese-radish peroxidase [EC 1.11.1.7] was investigated with respect to its spectrophotometric properties and effect on the enzymatic activity. Inactivation of the peroxidase occurred at temperatures higher than 60degrees and involved three processes, i.e., dissociation of protohemin from the holoperoxidase, a conformation change in the apperoxidase, and the modification or degradation of protohemin. The splitting process of protohemin from holoperoxidase as followed by the change in the absorption spectrum at high temperatures coincided with the degrease in the activity, and it was found to be at least biphasic. The regeneration of peroxidase on cooling to room temperature was essentially reversible at neutral pH, while at pH 5 and pH 9 these processes were irreversible. The irreversibility at acidic pH was mainly due to an irreversible change in the conformation of the apoenzyme. The difference spectrum of heat-treated apoperoxidase exhibited a denaturation blueshift with negative maxima at 287 and 294 nm, and the total protein fluorescence quantum yield. qprotein, increased by 20% compared to that of the untreated apoenzyme. On the other hand, the irreversibility at alkaline pH was largely attributable to the modification of protohemin. Apoperoxidase was more resistnat to heat denaturation but the modification or degradation of protohemin in heated enzyme was greater at alkaline pH than at acidic pH. The pyridine-ferrohemochrome spectrum of peroxidase exhibited slight shifts of the maxima of the alpha-band to shorter wavelength on heat treatment, and the paper chromatogram showed the presence of a new derivative other than protohemin. The modified product is probably (2(4)-vinyl-4(2)-hydroxyethyldeuterohemin.     

pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).     

Growth of Halotolerant Food Spoiling Yeast Debaryomyces nepalensis NCYC 3413 Under the Influence of pH and Salt

Debaryomyces nepalensis, a halotolerant food-spoiling yeast could grow in complex (YEPD) medium at different pHs ranging between 3.0 and 11.0 in the absence of salt and at pH 3.0–9.0 in the presence of different concentrations of NaCl and KCl. The specific growth rate of D. nepalensis was not affected by the initial pH of the medium in the absence of salts, whereas it was affected in the presence of salts. At 2 M NaCl and KCl, the organism exhibited a synergistic effect on pH and salt stress, which was unique in the Debaryomyces species. Irrespective of the initial pH and salt, the intracellular pH of D. nepalensis was ~7.0. Significant organic acid was produced at neutral and alkaline pH and organic acid production increased with the increase in pH and salt. Very specific organic acids are produced in the presence of NaCl and KCl. Our observation would contribute to a better understanding of the physiological phenomenon of halotolerance in D. nepalensis.     

Biosurfactant production by free and alginate entrapped cells of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Production of biosurfactant by free and alginate-entrapped cells of Pseudomonas fluorescens Migula 1895-DSMZ was investigated using olive oil as the sole carbon and energy source. Biosurfactant synthesis was followed by measuring surface tension and emulsifying index E24 over 5 days at ambient temperature and at neutral pH. Diffusional limitations in alginate beads affected the kinetics of biosurfactant production when compared to that obtained with free cells culture. Nevertheless, the emulsion stability was improved and fewer by-products interfered with the biosurfactant activity. A decrease in pH down to 5 in the case of immobilized cells was observed during the first 3 days, after which it returned to its initial value. The minimum values of surface tension were 30 and 35 dynes cm⁻¹ achieved after 40 and 72 h with free and immobilized cells, respectively, while the corresponding maximum E24 values were 67 and 62%, respectively. After separation by acetone precipitation, the biosurfactant showed a rhamnolipid-type in nature, and had a good foaming and emulsifying activities. The critical micellar concentration was found to be 290 mg l⁻¹. The biosurfactant also showed good stability during exposure to high temperatures (up to 120 °C for 15 min), to high salinity (10% NaCl) and to a wide range of pH (4–9).     

Effects of ribonucleic acid removal methods on proteolytic activity and protein solubility in Candida utilis

Candida utilis was grown under controlled conditions and nucleic acids were removed from whole cells and homogenates by alkaline hydrolysis techniques, en-zymatic methods, and washing with buffer. Homogenization released hydrolytic enzymes and proteolytic activity increased with incubation at elevated temperatures under acidic conditions. Slight proteolysis occurred in all incubated samples and this may contribute to protein insolubilization. Very little protein was lost during incu-bation when compared to similar processes using bakers' yeast. This can be due to lower levels of protease activities in C. utilis. Alkaline hydrolysis methods resulted in hydrolysis of some proteins and irreversible insolubilization of the protein. These methods also destroyed any residual enzymatic activities. Heat denaturation studies suggest that protein insolubilization occurs at neutral pH when heat treatments equivalent to or greater than 85° C for 15 min are used. SDS-PAGE methods were used to characterize and monitor changes in protein. Eighteen proteins and/or sub-units were present at levels of 1% or greater. Results may help to explain changesin functional properties of sample preparations which accompany RNA removal.     

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

Nucleic acid can catalyze the conversion of α‐helical cellular prion protein to β‐sheet rich Proteinase K resistant prion protein oligomers and amyloid polymers in vitro and in solution. Because unfolding of a protein molecule from its ordered α‐helical structure is considered to be a necessary step for the structural conversion to its β‐sheet rich isoform, we have studied the unfolding of the α‐helical globular 121–231 fragment of mouse recombinant prion protein in the presence of different nucleic acids at neutral and acid pH. Nucleic acids, either single or double stranded, do not have any significant effect on the secondary structure of the protein fragment at neutral pH; however the protein secondary structure is modified by the nucleic acids at pH 5. Nucleic acids do not show any significant effect on the temperature induced unfolding of the globular prion protein domain at neutral pH which, however, undergoes a gross conformational change at pH 5 as evidenced from the lowering of the midpoint of thermal denaturation temperatures, Tm, of the protein. The extent of Tm decrease shows a dependence on the nature of nucleic acid. The interaction of nucleic acid with the nonpolar groups exposed from the protein interior at pH 5 probably contributes substantially to the unfolding process of the protein.     

The Effect of pH and Heat Pre-Treatments on the Physicochemical and Emulsifying Properties of β-lactoglobulin

The overall goal of this research was to investigate structure-function mechanisms associated the emulsifying properties β-lactoglobulin (β-LG). Specifically the physicochemical (i.e., surface charge and hydrophobicity, size and interfacial tension) and emulsifying (i.e., emulsification activity (EAI) and stability indices (ESI)) properties of β-LG were investigated in response to changes in pH (3.0, 5.0 and 7.0) and heat pre-treatment conditions (25, 55 and 85 °C). Hydrophobicity was found to be greatest at pH 5.0/85 °C, whereas at all conditions it was significantly lower. Surface charge on β-LG was found to be neutral at?~?pH 3.9, regardless of conditions. Aggregate size was also found to be highest at pH 5.0/85 °C (avg. hydrodynamic radii of ~714 nm), corresponding to a reduced net surface charge and high hydrophobicity. Little size dependence of aggregates was observed at pH 3.0 regardless to the temperature pre-treatments (radii ~120 nm). In contrast, at pH 7.0 slight temperature dependence was apparent, where treatments at 25, 55 and 85 °C led to radii of 412.8, 307.2 and 232.3 nm, respectively. Overall, the addition of β-LG to a canola oil–water system resulted in a decline in interfacial tension from ~28 mN/m to ~18 mN/m, however the effect of pH/temperature conditions was minimal. EAI was found to be highest when β-LG solutions displayed high surface charge combined with moderate hydrophobicity. In contrast, ESI was higher under conditions where β-LG solutions remained in a native (25 °C) or fully denatured state (85 °C) versus one in where partially unravelling may be occurring (55 °C).     

Combined effect of growth medium,age of cells and phase of sporulation on heat resistance and recovery of Hansenula anomala

Effects of two growth media, age of cells and phase of sporulation on heat resistance of Hansenula anomala were determined. Cells were grown on two solid media, McClary's acetate and V8 juice agars, at 21 ° C for 16 days. Heat resistance of cells was determined in 0.06 M potassium phosphate buffer at 48 ° C. Heat-stressed cells were plated on four recovery media: yeast extract-malt extract-peptone-glucose (YMPG), pH 7.0; YMPG, pH 3.5; YMPG containing 6% NaCl, pH 7.0; and YMPG containing 20% sucrose, pH 7.0. The composition of sporulation medium influenced the extent of sporulation and the relative heat resistance of sporulating cells. One-day-old cells were the most sensitive to heat. The heat resistance of cells was generally increased as the incubation time was extended to 16 days. Heat treatment caused a greater increase in sensitivity to NaCl than to sucrose or acid pH in recovery media. Young cells were more sensitive to NaCl than were older cells.