Effect of heat‐assisted pulsed electric fields and bacteriophage on enterohemorrhagic Escherichia coli O157:H7

Pulsed electric fields (PEF), heat‐assisted PEF (H‐PEF), and virulent bacteriophage (VP) are non‐thermal techniques for pathogen inactivation in liquids that were investigated individually, and in combination (PEF/VP, H‐PEF/VP) to control enterohemorrhagic Escherichia coli (EHEC) O157:H7 in Luria‐Bertani broth (LBB) and Ringer's solution (RS). Treated cells were subsequently incubated at refrigeration (4°C) and temperature‐abuse conditions (12°C) for 5 days. When EHEC cells grown in LBB were subjected to non‐thermal processing and subsequently stored at 12°C for 5 days, reductions in count of between 0.1 and 0.6 log cycles were observed and following storage at 4°C the decrease in counts varied between 0.2 and 1.1 log₁₀. For bacteria cells suspended in RS values ranged from 0.1 to ≥3.9 log cycles at both storage temperatures. The most effective treatments were H‐PEF and H‐PEF/VP, both producing a >3.4 log cycle reduction of cells suspended in non‐nutrient RS. Analysis of EHEC recovery on selective and non‐selective media indicated no occurrence of sub‐lethal damage for VP, PEF/VP, and H‐PEF/VP‐treated cells. The findings indicate that combining PEF and lytic phage may represent a suitable alternative to conventional fluid decontamination following further process optimization. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:110–118, 2015     

Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis: effect of calcium, pH and temperature

The first step of starch hydrolysis, i.e. liquefaction has been studied in this work. Two commercial α-amylases from Bacilllus licheniformis, known as Termamyl and Liquozyme have been used for this purpose. Using starch as the substrate, kinetics of both enzymes has been determined at optimal pH and temperature (pH 7, T = 80 °C) and at 65 °C and pH 5.5. Michaelis–Menten model with uncompetitive product inhibition was used to describe enzyme kinetics. Mathematical models were developed and validated in the repetitive batch and fed-batch reactor. Enzyme inactivation was described by the two-step inactivation model. All experiments were performed with and without calcium ions. The activities of both tested amylases are approximately one hundred times higher at 80 °C than at 65 °C. Lower inactivation rates of enzymes were noticed in the experiments performed at 65 °C without the addition of calcium than in the experiments at 80 °C. Calcium ions in the reaction medium significantly enhance amylase stability at 80 °C and pH 7. At other process conditions (65 °C and pH 5.5) a weaker calcium stabilizing effect was detected.     

Utilization of cross‐linked laccase aggregates in a perfusion basket reactor for the continuous elimination of endocrine‐disrupting chemicals

A perfusion basket reactor (BR) was developed for the continuous utilization of insolubilized laccase as cross‐linked enzyme aggregates (CLEAs). The BR consisted of an unbaffled basket made of a metallic filtration module filled with CLEAs and continuously agitated by a 3‐blade marine propeller. The agitation conditions influenced both the apparent laccase activity in the reactor and the stability of the biocatalyst. Optimal laccase activity was obtained at a rotational speed of 12.5 rps and the highest stability was reached at speeds of 1.7 rps or lower. The activity and stability of the biocatalyst were affected drastically upon the appearance of vortices in the reaction medium. This reactor was used for the continuous elimination of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA), and triclosan (TCS). Optimization of EDC elimination by laccase CLEAs as a function of temperature and pH was achieved by response surface methodology using a central composite factorial design. The optimal conditions of pH and temperature were, respectively, 4.8 and 40.3°C for the elimination of p353NP (a branched isomer of NP), 4.7 and 48.0°C for BPA, and 4.9 and 41.2°C for TCS. Finally, the BR was used for the continuous elimination of these EDCs from a 5 mg L^?1 aqueous solution using 1 mg of CLEAs at pH 5 and room temperature. Our results showed that at least 85% of these EDCs could be eliminated with a hydraulic retention time of 325 min. The performances of the BR were quite stable over a 7‐day period of continuous treatment. Furthermore, this system could eliminate the same EDCs from a 100 mg L^?1 solution. Finally, a mathematical model combining the Michaelis–Menten kinetics of the laccase CLEAs and the continuous stirred tank reactor behavior of the BR was developed to predict the elimination of these xenobiotics. Biotechnol. Bioeng. 2009;102: 1582–1592. © 2008 Wiley Periodicals, Inc.     

Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules

Turbidity measurements have been used to study the in vitro assembly and disassembly of porcine neurotubules. All measurements were carried out with tubulin with a purity higher than 80%. Tubules formed by in vitro assembly of this protein are so long that the turbidity is insensitive to length and is a function only of the total mass of high molecular weight material. Porcine tubulin shows a critical concentration for assembly of about 0.2 mg/ml under optimal conditions, pH 6.6, 0.1m-2-(N-morpholino)ethane sulfonic acid, 26 to 37 °C. Under these conditions assembly and disassembly are essentially fully reversible in the presence of excess GTP. The kinetics of assembly show an initial lag and initial rates which are strongly temperature dependent. Our samples show a concentration dependence of no more than second order. The apparent activation enthalpy of assembly is 25 kcal/mol; the apparent reaction enthalpy of assembly for the chain propagation step is 21 kcal/mol. Disassembly kinetics show an apparent negative activation enthalpy of ?28 kcal/mol. They are independent of tubule length implying a slow activation step followed by rapid depolymerization. At 20 °C, cycles of polymerization and depolymerization show hysteresis effects in the assembly kinetics though not in disassembly rates or final states. This is most easily explained by postulating a slow reversible inactivation at 4 °C of the initiation complex for tubule assembly. Conditions are reported for producing tubulin in a state which cannot assemble in aqueous buffer unless nucleotides are added. GTP, ATP and ADP, but not GDP, are effective in promoting tubule assembly. An adenylate kinase impurity in our preparation may be the cause of this unusual effect. Whether or not it is actually associated with tubulin or tubules is unknown.     

The carp M1 muscle‐specific creatine kinase subisoform is adaptive to the synchronized changes in body temperature and intracellular pH that occur in the common carp Cyprinus carpio

The three previously cloned Cyprinus carpio muscle‐specific subisoforms of creatine kinase (CK, EC 2.7.3.2) designated M1‐, M2‐ and M3‐CK were examined. At temperatures <15° C and at pH >7·7, specific activities of M1‐CK were three to eight‐fold higher than specific activities of M3‐ and rabbit (R) M‐CK. At pH 8·0, M1‐CK exhibited its highest specific activity at 15° C. Michaelis constants of PCr () and ADP () of M1‐CK were relatively stable at pH between 7·1–8·0 and 25–5° C. Its calculated activation energy of catalysis (E_a) at pH 8·0 was lower than at pH 7·1. Circular dichroism spectroscopy results showed that changes in secondary structures in M1‐CK at the pH and temperatures studied were much less than in the cases of RM‐ and M3‐CK. The M1‐CK enzyme seemed to have evolved to adapt to the synchronized changes in body temperature and intracellular pH of C. carpio.     

“Setting paint” analogy for the hydrophobic self‐association of tropoelastin into elastin‐like hydrogel

Alkaline tropoelastin solutions (pH 11) were optically clear at low temperatures, but a firm gel formed when the temperature was raised to 37°C. Reversion to a clear solution took place if the temperature was lowered to below 20°C within less than 2 h, but not if 37°C was maintained for several hours. The precipitated elastin‐like hydrogel thus formed did not visually redissolve at low temperatures. Tropoelastin hydrogel was stable to subsequent washings with alkaline solution at 37°C, but at 4°C some hydrogel redissolved showing that association is at least partly reversible. Washing the hydrogel with neutral 8M urea solution at 4°C dissolved less than 10% of tropoelastin in 24 h. We characterized this phenomenon by combining temperature‐controlled light microscopy analysis, ¹H NMR spectroscopy (temperature, diffusion, and relaxation time studies), and UV‐absorption‐based concentration measurements. The self‐association of tropoelastin at pH 11 is due to hydrophobic interactions in an emulsion‐like system in which the spherules coalesce in a manner like a water‐based latex paint that forms a durable hydrophobic sheet as water and the organic solvent evaporate. In the present case, the sedimentation and entanglement of the tropoelastin porous sheets means that reverse dissolution is a kinetically slow process. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 321–330, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

High stability of self‐assembled peptide nanowires against thermal,chemical, and proteolytic attacks

Understanding the self‐assembly of peptides into ordered nanostructures is recently getting much attention since it can provide an alternative route for fabricating novel bio‐inspired materials. In order to realize the potential of the peptide‐based nanofabrication technology, however, more information is needed regarding the integrity or stability of peptide nanostructures under the process conditions encountered in their applications. In this study, we investigated the stability of self‐assembled peptide nanowires (PNWs) and nanotubes (PNTs) against thermal, chemical, proteolytic attacks, and their conformational changes upon heat treatment. PNWs and PNTs were grown by the self‐assembly of diphenylalanine (Phe–Phe), a peptide building block, on solid substrates at different chemical atmospheres and temperatures. The incubation of diphenylalanine under aniline vapor at 150°C led to the formation of PNWs, while its incubation with water vapor at 25°C produced PNTs. We analyzed the stability of peptide nanostructures using multiple tools, such as electron microscopy, thermal analysis tools, circular dichroism, and Fourier‐transform infrared spectroscopy. Our results show that PNWs are highly stable up to 200°C and remain unchanged when incubated in aqueous solutions (from pH 1 to 14) or in various chemical solvents (from polar to non‐polar). In contrast, PNTs started to disintegrate even at 100°C and underwent a conformational change at an elevated temperature. When we further studied their resistance to a proteolytic environment, we discovered that PNWs kept their initial structure while PNTs fully disintegrated. We found that the high stability of PNWs originates from their predominant β‐sheet conformation and the conformational change of diphenylalanine nanostructures. Our study suggests that self‐assembled PNWs are suitable for future nano‐scale applications requiring harsh processing conditions. Biotechnol. Bioeng. 2010; 105: 221–230. © 2009 Wiley Periodicals, Inc.     

Optimization of pH,Temperature and Inoculum Ratio for the Production of β‐D‐Galactosidase by Kluyveromyces marxianus Using a Lactose‐free Medium

The pH, temperature and inoculum ratio for the production of β‐galactosidase by Kluyveromyces marxianus CDB 002 were optimized using sugar‐cane molasses (100 g/l) in a lactose‐free medium. The temperature optimum was evaluated in the range from 28–37 °C. Lactase production was initiated after substrate consumption indicating a reversible enzyme inhibition or catabolic repression. The specific enzyme activity after 45 h was between 456.3 U/g cell mass (37 °C) and 733.3 U/g (34 °C), whereas the highest volumetric activity was obtained at 30 °C: 21.8 U/ml. This is generally consistent with results from other authors that used whey as a carbon source. Ethanol as a by‐product reached its maximum concentration after 10–14 h (31.1–40.5 g/l), but was completely consumed afterwards. A pH of 5.5 without further control gave the best production rate for lactase (484.4 U/l × h). In this process, the pH was stable during cell growth at 5.5 and then went up to pH 7.2 after 45 h. At a fixed pH of 5.5 or 6.5, the production rates achieved 313.3 U/l × h and 233.3 U/ l × h, respectively. These results differed from those of other authors, who suggested a fixed pH at 7.0 using whey as a carbon source. There were no significant differences between inoculum ratios of 1% [v/v] and 10% [v/v] so that 1% is the preferable ratio as it is cheaper. Yeast extract (10 g/l) and peptone (20 g/l) were used as the vitamin and nitrogen source, respectively, for the studies of temperature and pH. These were substituted by corn steep liquor (100 g/l) for inoculum ratio experiments. Production of lactase using sugar cane molasses in a lactose‐free medium gave better enzyme productivity rates than obtained by other authors using whey. The optimum conditions for β‐galactosidase synthesis were a temperature of 30–34 °C and an inoculum ratio of 1% [v/v], an initial pH of 5.5 without any further control or a control of 5.5 during cell growth. Then the pH was raised up to 7.     

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl₂. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl₂ producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)₂‐Man₁ was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)₂‐Man₁ can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP‐mannose regenerating cascade and can further be used to study coupling of the GDP‐mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell‐free production of LLOs as precursors for in vitro glycoengineering of proteins.     

C. botulinum inactivation kinetics implemented in a computational model of a high‐pressure sterilization process

High‐pressure, high‐temperature (HPHT) processing is effective for microbial spore inactivation using mild preheating, followed by rapid volumetric compression heating and cooling on pressure release, enabling much shorter processing times than conventional thermal processing for many food products. A computational thermal fluid dynamic (CTFD) model has been developed to model all processing steps, including the vertical pressure vessel, an internal polymeric carrier, and food packages in an axis‐symmetric geometry. Heat transfer and fluid dynamic equations were coupled to four selected kinetic models for the inactivation of C. botulinum; the traditional first‐order kinetic model, the Weibull model, an nth‐order model, and a combined discrete log‐linear nth‐order model. The models were solved to compare the resulting microbial inactivation distributions. The initial temperature of the system was set to 90°C and pressure was selected at 600 MPa, holding for 220 s, with a target temperature of 121°C. A representation of the extent of microbial inactivation throughout all processing steps was obtained for each microbial model. Comparison of the models showed that the conventional thermal processing kinetics (not accounting for pressure) required shorter holding times to achieve a 12D reduction of C. botulinum spores than the other models. The temperature distribution inside the vessel resulted in a more uniform inactivation distribution when using a Weibull or an nth‐order kinetics model than when using log‐linear kinetics. The CTFD platform could illustrate the inactivation extent and uniformity provided by the microbial models. The platform is expected to be useful to evaluate models fitted into new C. botulinum inactivation data at varying conditions of pressure and temperature, as an aid for regulatory filing of the technology as well as in process and equipment design. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Influence of pH and sodium chloride on kinetics of thermal inactivation of the bacteriocin-like substance P34

Aims: To investigate the kinetics of thermal inactivation of the bacteriocin‐like substance P34 at different pH and sodium chloride concentration. Methods and Results: Samples of bacteriocin were treated at different time–temperature combinations in the range of 0–300 min and 90–120°C and the kinetic parameters for bacteriocin inactivation were calculated. For all treatments, the thermal inactivation reaction fitted adequately to first‐order model. D‐ and k‐values were smaller and higher, respectively, for pH 4·5 than for 6·0 or 7·0, indicating that bacteriocin P34 was less thermostable at lower pH. At 120, 115 and 100°C, the addition of sodium chloride decreased thermal stability. For other temperatures, addition of NaCl increased stability of the peptide. The presence of greater amount of the salt (50 g l^?1) resulted in a higher thermal stability of bacteriocin P34, suggesting that the reduction in water activity of the solution interfered on the stability of the peptide. Conclusions: Based on an isothermal experiment in the temperature range of 90–120°C, and by thermal death time models, bacteriocin P34 is less heat stable at low pH and has increased thermal stability in the presence of NaCl. Addition of NaCl improved the stability of the peptide P34 at high temperatures. Significance and Impact of the Study: Studies on kinetics of thermal inactivation of bacteriocins are essential to allow their proper utilization in the food industry.     

Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

Improved assembly of bispecific antibodies from knob and hole half‐antibodies

A process was developed for large‐scale assembly of IgG₁ and IgG₄ bispecific antibodies from knob and hole half‐antibodies. We optimized assembly conditions such as pH, temperature, stabilizers, and reducing agent. We also identified and exploited structural changes unique to knob and hole half‐antibodies with the result of improving assembly outcome, specifically storing half‐antibodies at higher pH will condition them to assemble more rapidly and produce fewer high molecular‐weight species (HMWS). Application of heat to the assemblies resulted in an acceleration of assembly rate, with optimal formation of bispecific achieved at 37°C. IgG₄ half‐antibodies were unusually sensitive to temperature‐dependent formation of HMWS in pre‐assembly conditioning as well as during assembly. We selected l ‐histidine and Polyvinylpyrrolidone (PVP) as stabilizers to prevent HMWS formation in IgG₄, and achieved rapid and high‐efficiency assemblies. Using optimized assembly conditions, we developed and scaled up a method for assembling bispecific antibody with 90% assembly efficiency over 6 h with minimal impact to product quality, generating a pool with bispecific antibody for downstream processing. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1315–1322, 2015     

Investigating the potential of Bacillus subtilis α‐amylase as a pressure‐temperature‐time indicator for high hydrostatic pressure pasteurization processes

The potential of Bacillus subtilis α‐amylase (BSA) as a pressure‐temperature‐time indicator (pTTI) for high pressure pasteurization processing (400–600 MPa; T_i 10–40°C; 1–15 min) was investigated. A stepwise approach was followed for the development of an enzyme‐based, extrinsic, isolated pTTI. First, based on literature data on the pressure stability, BSA was selected as a candidate indicator. Next to the accuracy and ease of the measurement of the indicator's response (residual activity) to the pressure treatment, the storage and handling stability of BSA at atmospheric pressure was verified. Second, the stability of BSA at a constant temperature (T) and time in function of pressure (p) was investigated. Solvent engineering was used to shift the inactivation window of BSA in the processing range of interest. Third, the enzyme (1 g/L BSA—MES 0.05 M pH 5.0) was kinetically calibrated under isobaric‐isothermal conditions. Time dependent changes in activity could be modeled best by a first‐order model. Except for low pressures and high temperatures, a synergistic effect between pressure and temperature could be observed. Based on the model selected to describe the combined p,T‐dependency of the inactivation rate constant, an elliptically shaped isorate contour plot could be constructed, illustrating the processing range where BSA can be used to demonstrate temperature gradients. Fourth, the validity of the kinetic model was tested successfully under dynamic conditions similar to those used in food industry. Finally, the indicator was found suitable to demonstrate nonuniformity in two‐sectional planes of a vertical, single vessel system. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Cationic oligopeptides with the repeating sequence L‐lysyl‐L‐alanyl‐L‐alanine: conformational and thermal stability study using optical spectroscopic methods

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L ‐Lys‐L ‐Ala‐L ‐Ala)_n (n = 1, 2, and 3) were measured over a range of temperatures (20–90 °C) in aqueous solution at near‐neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I′ region indicate the presence of extended helical polyproline II (PPII)‐like conformation at room temperature. UV‐ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII‐like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles ? and ψ are within the range ?82 to ?107° and 143–154°, respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII‐like conformation of the Lys‐Ala‐Ala sequence is still preferred, being in equilibrium with an unordered conformer at near‐neutral pH values within the range of temperatures from 20 to 90 °C. This finding was obtained from analysis of the temperature‐dependent spectra using the singular value decomposition method. The study presents KAA‐containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Purification and characterization of intracellular and extracellular α‐glucosidases from Geobacillus toebii strain E134

Two different α‐glucosidase‐producing thermophilic E134 strains were isolated from a hot spring in Kozakli, Turkey. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, the strain was proposed to be a species of G. toebii. Its thermostable exo‐α‐1,4‐glucosidases also were characterized and compared, which were purified from the intracellular and extracellular fractions with estimated molecular weights of 65 and 45 kDa. The intracellular and extracellular α‐glucosidases showed optimal activity at 65 °C, pH 7·0, and at 70 °C, pH 6·8, with 3·65 and 0·83 K_m values for the pNPG substrate, respectively. Both enzymes remained active over temperature and pH ranges of 35–70 °C and 4·5–11·0. They retained 82 and 84% of their activities when incubated at 60 °C for 5 h. Their relative activities were 45–75% and 45–60% at pH 4·5 and 11·0 values for 15 h at 35 °C. They could hydrolyse the α‐1,3 and α‐1,4 bonds on substrates in addition to a high transglycosylation activity, although the intracellular enzyme had more affinity to the substrates both in hydrolysis and transglycosylation reactions. Furthermore, although sodium dodecyl sulfate behaved as an activator for both of them at 60 °C, urea and ethanol only increased the activity of the extracellular α‐glucosidase. By this study, G. toebii E134 strain was introduced, which might have a potential in biotechnological processes when the conformational stability of its enzymes to heat, pH and denaturants were considered. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis and conformational analysis of salivary proline‐rich peptide P‐B

The 57‐amino acid human salivary polypeptide P‐B has been synthesized by the solid‐phase method using 9‐fluorenylmethoxycarbonyl (Fmoc) strategy. The circular dichroism (CD) spectroscopy, Fourier‐transform infrared spectroscopy (FTIR) and molecular modeling methods have been used for conformational studies of P‐B. Examination of the CD spectra of P‐B showed the content of the secondary structure to be independent of temperature over the range 0–60 °C at pH = 7 as well as over the pH range of 2–12 at 37 °C. P‐B adopts predominantly unordered structure with locally appearing β‐turns. The cumulative results obtained using the CD and FTIR spectroscopic techniques indicate the percentage of the polyproline type‐II (PPII) helix being as low as about 10%. Similarly, the molecular dynamics (MD) simulations reveal only a short PPII helix in the C‐terminal fragment of the peptide (Pro⁵¹–Pro⁵⁴), which constitutes 7%. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Nitric oxide‐mediated inhibition of NFκB regulates hyperthermia‐induced apoptosis

Ascertaining the upstream regulatory mechanisms of hyperthermia‐induced apoptosis is important to understand the role of hyperthermia in combined modality cancer therapy. Accordingly, we investigated whether (i) hyperthermia‐induced apoptosis is mediated through the nitric oxide (NO) signaling pathway and (ii) inhibition of post‐translational modification of IκBα and down regulation of NFκB‐DNA binding activity is an intermediate step in NO‐dependent apoptosis in MCF‐7 breast cancer cells. For hyperthermia treatment, the cells were exposed to 43°C. Intracellular NO levels measured by the fluorescent intensity of DAF‐2A and iNOS expression by immunobloting revealed an increased level of iNOS dependent NO production after 43°C. Apoptosis measured by Annexin V expression and cell survival by clonogenic assay showed a 20% increase in apoptosis after 43°C treatments. EMSA analysis showed a dose‐dependent inhibition of NFκB‐DNA binding activity. The hyperthermia‐mediated inhibition of NFκB was persistent even after 48 h. Inhibition of NO by L ‐NAME rescued the NFκB‐DNA binding activity and inhibits heat‐induced apoptosis. Similarly, over‐expression of NFκB by transient transfection inhibits heat‐induced apoptosis. These results demonstrate that apoptosis upon hyperthermia exposure of MCF‐7 cells is regulated by NO‐mediated suppression of NFκB. J. Cell. Biochem. 106: 999–1009, 2009. © 2009 Wiley‐Liss, Inc.     

Hydride‐Enhanced CO2 Methanation: Water‐Stable BaTiO2.4H0.6 as a New Support

Catalytic CO₂ hydrogenation to CH₄ provides a promising approach to producing natural gas, and reducing the emissions of global CO₂. However, the efficiency of catalytic CO₂ methanation is limited by slow kinetics at low temperatures. This study first demonstrates that an air‐ and water‐stable perovskite oxyhydride BaTiO_2.4H_0.6 could function as an active support material for Ni‐, Ru‐based catalysts for CO₂ methanation at 300–350 °C, a relatively lower temperature. With the oxyhydride support, the activity for Ni and Ru increases by a factor of 2–7 when compared to the BaTiO₃ oxide support. Kinetic analysis shows reduced H₂ poisoning probably due to spillover, implying that the activity change is due to the kinetics being influenced by hydride. Furthermore, the oxyhydride‐supported Ni catalyst is also durable with its catalytic performance preserved for at least 10 h under a humid environment at elevated temperatures. It is anticipated that these perovskite oxyhydrides will shed new light on the design of high‐efficiency metal‐based catalysts for water‐involved catalytic reactions.     

In vitro folding of methionine‐arginine human lyspro‐proinsulin S‐sulfonate—Disulfide formation pathways and factors controlling yield

We investigated the in vitro folding of an oxidized proinsulin (methionine‐arginine human lyspro‐proinsulin S‐sulfonate), using cysteine as a reducing agent at 5°C and high pH (10.5–11). Folding intermediates were detected and characterized by means of matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS), reversed‐phase chromatography (RPC), size‐exclusion chromatography, and gel electrophoresis. The folding kinetics and yield depended on the protein and cysteine concentrations. RPC coupled with MALDI‐MS analyses indicated a sequential formation of intermediates with one, two, and three disulfide bonds. The MALDI‐MS analysis of Glu‐C digested, purified intermediates indicated that an intra‐A‐chain disulfide bond formed first among A6, A7, and A11. Various non‐native intra‐A (A20 with A6, A7, or A11), intra‐B (between B7 and B19), and inter‐A‐B disulfide bonds were observed in the intermediates with two disulfide bonds. The intermediates with three disulfide bonds had mainly the non‐native intra‐A and intra‐B bonds. At a cysteine‐to‐proinsulin‐SH ratio of 3.5, all intermediates with the non‐native disulfide bonds were converted to properly folded proinsulin via disulfide bond reshuffling, which was the slowest step. Aggregation via the formation of intermolecular disulfide bonds of early intermediates was the major cause of yield loss. At a higher cysteine‐to‐proinsulin‐SH ratio, some intermediates and folded MR‐KPB‐hPI were reduced to proteins with thiolate anions, which caused unfolding and even more yield loss than what resulted from aggregation of the early intermediates. Reducing protein concentration, while keeping an optimal cysteine‐to‐protein ratio, can improve folding yield significantly. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010