Thermostable Structure Research Articles

DUTP pyrophosphatases from hyperthermophilic eubacterium and archaeon: Structural and functional examinations on the suitability for PCR application.

Deoxyuridine triphosphate pyrophosphatase (DUT) suppresses incorporation of uracil into genomic DNA during replication. Thermostable DUTs from hyperthermophilic archaea such as Thermococcus pacificus enhance PCR amplification by preventing misincorporation of dUTP generated by spontaneous deamination of dCTP. However, it is necessary to elucidate whether DUTs do not cause dNTP imbalances during PCR by unwanted side activity. Moreover, it has been unknown what structural features define the thermostability of those DUTs. Here, DUT from a hyperthermophilic eubacterium, Aquifex aeolicus (Aa-DUT), was characterized together with those from T. pacificus (Tp-DUT). Aa-DUT was as thermostable as Tp-DUT up to at least 95°C. The crystal structures of the two thermostable enzymes were determined, which revealed that the structures of Aa-DUT and Tp-DUT resembled those of monofunctional and bifunctional DUTs, respectively. Generally, bifunctional DUTs harbor the dCTP deaminase activity in addition to the DUT activity. However, not only Aa-DUT but also Tp-DUT showed poor activity towards dCTP, indicating both enzymes are monofunctional. We further examined eight types of parameters related to thermostability of protein structure and found that the thermostability of Aa-DUT and Tp-DUT might be accomplished by increased numbers of ion pairs on the protein surface. Finally, we verified that Aa-DUT promoted PCR amplification with Pfu DNA polymerase to the same extent as Tp-DUT. Collectively, we conclude that both DUTs from hyperthermophiles maintain the enzymatic activity at high temperatures without consuming dCTP due to the lack of the deaminate activity.

An in-depth investigation of molecular interaction in zeaxanthin/corn silk glycan complexes and its positive role in hypoglycemic activity

Glycans in corn silk could interact with co-existing small molecules during its absorption, digestion, and biological process. In order to understand the exact mechanism of action of zeaxanthin, it is critical to investigate the biomolecular interactions, which were necessary to form a glycan-small molecule complex and yet produce the bioactive effect. So far, the in-depth study of these natural interactions has not been fully elucidated. Here, we probed that the molecular interaction between zeaxanthin (ZEA) and glycans from corn silk (CSGs) was driven by enthalpy. More importantly, it was the first time found that CSGs can bind to lipid-soluble ZEA could be binded with CSGs. It was the first report on the thermostability of insulin structure and natural glycans. This study should facilitate our understanding of the interaction between lipid soluble molecules and glycans, and provide a more comprehensive understanding of the nutrient base in food.

TEMPERATURE DEPENDENCE OF HEAT CAPACITY AND CHANGES IN THERMODYNAMIC FUNCTIONS OF ALLOYS OF THE Pb – Te SYSTEM

Lead, in comparison with other metals, has low chemical activity and high corrosion resistance. Complex alloying of lead with antimony, tellurium and copper in optimal concentrations made it possible to obtain highly effective alloys for protective cable sheaths. Pb – Sb – Cu – Te lead alloy provides the cable sheath with high resistance to fatigue, creep and active deformation in a wide temperature range, as well as good manufacturability. The basis for such a complex of positive characteristics is a specific fine-grained thermostable structure, which determines the stability of properties in operation. Alloys of this composition are at the level of world standards: they have the best complex of operational and technological characteristics compared to the most promising domestic and foreign analogues. In the work, the heat capacity of the alloys of the Rb – Te system was determined in the "cooling" mode by the known heat capacity of a reference sample made of copper grade M00. Polynomials describing the temperature dependence of the heat capacity and changes in the thermodynamic functions of alloys are obtained. It isfound that with increasing temperature and tellurium content, the heat capacity, enthalpy and entropy of lead increase slightly, and the Gibbs energy values decrease.

Kinetic study on the molecular mechanism of light-driven inward proton transport by schizorhodopsins

Schizorhodopsins (SzRs) are light-driven inward proton pumping membrane proteins. A H+ is released to the cytoplasmic solvent from the chromophore, retinal Schiff base (RSB), after light absorption, and then another H+ is bound to the RSB at the end of photocyclic reaction. However, the mechanistic detail of H+ transfers in SzR is almost unknown. Here we studied the deuterium isotope effect and the temperature dependence of the reaction rate constants of elementary steps in the photocycles of SzRs. The former indicated that deprotonation and reprotonation of RSB is mainly accomplished by H+ hopping between heavy atoms with similar H+ affinity. Furthermore, the temperature dependence of the rate constants revealed that most of H+ transfer events have a high entropy barrier. In contrast, the activation enthalpy and entropy of extremely thermostable SzR (MsSzR) are significantly higher than other types of SzRs (SzR1 and MtSzR) suggesting that its highly thermostable structure is optimized with at the cost of slower reaction rates at ambient temperatures.

The Influence of Transglutaminase on Minced Muscular Fish Tissue Structure Formation After the Application of Various Protein Substrates

This research aimed to examine the effect of a microbial transglutaminase preparation (Activa® GS, Ajinomoto Co., Inc, Japan) on the structure formation of the myofibrillar protein system of a deep-sea fish species – the giant grenadier – after the addition of various protein substrates. The low content of proteins and their low water-holding capacity in this fish, subjected to various processing methods, leads to significant losses in the initial mass and decreased gelation ability in the muscle tissue system. Various concentrations of transglutaminase were used but these did not ensure the restructuring of the initial muscle tissue of the grenadier. Additional protein substrates with different molecular weights and amino acid composition were added, including gelatin, milk casein, hydrolysates of the skin and milt of the fish, and whole bivalves, which were used to create a firm structure. It was shown that the introduction of gelatin and casein at a concentration of 5% led to the formation of a firm, thermostable structure under the action of the enzyme, while hydrolyzed proteins with low molecular weight at their various concentrations enhanced the expression of water and formation of the fluid consistency. The ability of gastrointestinal tract proteases (pepsin and trypsin) to digest did not depend on the formation of protein-to-protein cross-linking in these combined products. The influence on the growth of the Tetrahymena pyriformis ciliates test culture also showed the high degree of product availability. The technology of molded products based on fermented minced muscle tissue of grenadier with added casein, both in the form of semi-finished products and in the form of ready-to-eat products, was developed.
 Keywords: transglutaminase, muscle tissue, structure formation, deep-sea fish

Optimizing Human Epidermal Growth Factor for its Endurance and Specificity Via Directed Evolution: Functional Importance of Leucine at Position 8

Epidermal growth factor regulates cell proliferation and migration by binding and activating the extracellular region of its receptor. Many engineered EGFs with enhanced agonist activity are produced, yet the short half-life and poor environmental tolerance of EGF limits its physiological application. By using a more stable porcine EGF as the template in the staggered extension process, two mutants of human and porcine EGF, hL8P and pP8L, were obtained with improved targeting towards normal cells. The comparative studies of hEGF and mutants were conducted to evaluate their activities as agonists. The mutants showed specificity for the proliferation and migration of normal and cancer cells, i.e. both pP8L and hL8P promoted the proliferation and migration of normal cells while delayed that of cancer cells. Both mutants preserved the structure thermostability of hEGF and pP8L showed improved acidic tolerance than hEGF. For the metabolism analysis in HEK293T cells, hL8P and pP8L demonstrated the lower rate of both mutants-EGFR internalization and biodegradation, but higher recycling to the cell surface. This study not only enhanced EGF agonist activity without loss of endurance but also provided a new application foreground for trauma therapy and cancer targeting therapy.

Mitogenome analysis of dwarf pufferfish (Carinotetraodon travancoricus) endemic to southwest India and its implications in the phylogeny of Tetraodontidae

The Tetraodontidae (pufferfishes), is primarily a family of marine and estuarine fishes with a limited number of freshwater species. Freshwater invasions can be observed in South America, Southeast Asia and central Africa. In the present study, we have analysed the complete mitogenome of freshwater pufferfish, Carinotetraodon travancoricus (dwarf pufferfish or Malabar pufferfish) endemic to southwest India. The genome is 16487 bp in length and consist of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region like all the other vertebrate mitogenomes. The protein-coding genes ranged from 165 bp (ATP synthase subunit 8) to 1812 bp (NADH dehydrogenase subunit 5) and comprised of 11310 bp in total, constituting 68.5% of the complete mitogenome. Some overlaps have been observed in protein-coding genes by a total of 7 bp. The AT skew (0.032166) and GC skew (-0.29746) of the mitogenome indicated that heavy strand consists equal amount of A and T, but the overall base composition was mainly C skewed. The noncoding D-loop region comprised 869 bp. The conserved motifs ATGTA and its complement TACAT associated with thermostable hairpin structure formation were identified in the control region. The phylogenetic analysis depicted a sister group relationship of C. travancoricus with euryhaline species Dichotomyctere nigroviridis and D. ocellatus with 100% bootstrap value rather than with the other freshwater members of Carinotetraodon species from Southeast Asia. The data from this study will be useful for proper identification, genetic differentiation, management and conservation of the dwarf Indian pufferfish.

Physicochemical and Structural Properties of Recovered Elastin from Jumbo Squid (Dosidicus gigas) By-Products

ABSTRACTPhysicochemical and structural properties of soluble jumbo squid (Dosidicus gigas) elastin recovered from skin by-products were evaluated. The molecular weight of isolated elastin was ~40 kDa with an isoelectric point (pI) between 9 and 10. Aspartic, glutamic, arginine, proline, glycine, and lysine amino acids were the most abundant in squid elastin, whereas the hydroxyproline absence, ~0.7% cysteine content, and the calculated 0.35 isoleucine/leucine ratio were used as purity index. Total and reactive sulfhydryl contents were similar (247.0 ± 5.1 vs 242.0 ± 7.5 μmol mg−1 of protein, P ≥ 0.05) in purified squid elastin but surprisingly higher than previously reported in other elastins. On the other hand, the secondary structures of squid elastin analyzed by Fourier transform infrared spectroscopy (FTIR) were ~45% β-sheets, ~15% α-helices, ~10% β-turns, and ~30% undefined structures. In addition, squid elastin experienced glass transition at 82.01 ± 0.01ºC, denaturation temperature at 110.45 ± 0.64ºC, and aggregation at 197.5 ± 0.23ºC. In conclusion, the prevalence of charged amino acids and pI of squid elastin can facilitate its solubilization in hydrophilic systems, whereas the secondary structure profile and thermostability are desirable features in proteins used for biopolymer designs such as food biofilms or barrier systems.

Influence of Calcium Ions on the Thermal Characteristics of α-amylase from Thermophilic Anoxybacillus sp. GXS-BL.

Background: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotech-nology.Objective:In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL.Methods: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (half-life, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template.Results:With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly high-er than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α-helical contents increasing, and the transition from α-helices to β-sheet structures, which was obviously dif-ferent in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure.Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

Emulsifying properties of glycation or glycation-heat modified egg white protein

To investigate the potential of glycation and/or heat modified egg white protein (EWP) as stabilizer for oil-in-water emulsions, glycated EWP (GEWP) was prepared by a dry-heated Maillard reaction using EWP and isomalto-oligosaccharide. Heat-induced GEWP nanoparticles (HGN) were then fabricated by heating GEWP at 90 °C for 30 min at pH 6.0–9.0. The physicochemical characteristics (particle diameter, ζ-potential and surface hydrophobicity) and emulsifying capacity of GEWP and HGN were investigated. In addition, the storage, oxidation, thermal treatment and salt stabilities of emulsions prepared with GEWP and HGN were evaluated. The results indicated that the emulsions prepared with modified EWPs exhibited higher protein adsorption on the oil-water interface and smaller particle size. The GEWP- and HGN-stabilized emulsions were stable against droplet aggregation during storage and exhibited better oxidation stability, which may be related to the enhanced antioxidant activity and absorption ability of the modified EWPs. Additionally, the emulsions prepared with GEWP and HGN exhibited higher heat and salt stabilities due to the increase in surface hydrophobicity and net charge. The thermostable structure of the proteins induced by glycation and heat treatment also contributed to the improvement in heat stability. Overall, the HGN-stabilized emulsions exhibited the best physicochemical stability among the modified EWPs.

Substitution of iron with cobalt in the prosthetic group of bacterial cytochrome P450: Effects on the stability and structure of the protein

The origin of thermostability of the heme monooxygenase from Thermus thermophilus, CYP175A1 has been investigated using multi-wavelength circular dichroism (CD) spectroscopy at different temperatures. The far-UV and visible CD studies were carried out on WTCYP175A1, reduced WTCYP175A1 (i.e. Fe(II)-WTCYP175A1), apoCYP175A1, and the cobalt protoporphyrin (CoP) bound apoprotein (CoPCYP175A1) to determine the thermostability of the secondary and tertiary structure of the variants of the enzyme. Non-linear least squares analyses of the multi-wavelength CD data were used to determine the midpoint unfolding temperature (Tm), and the associated thermodynamic parameters for unfolding of the enzyme. The midpoint temperature (Tm) for thermal unfolding of WTCYP175A1 was much higher than that of the reduced enzyme (Fe(II)-WTCYP175A1), which was higher than that of the CoP bound analogue, while the Tm for the apoprotein was even lower than the CoPCYP175A1. The temperature for maximum stability (TS) also followed the order WTCYP175A1 > Fe(II)-WTCYP175A1 ≈ CoPCYP175A1 > apoCYP175A1 supporting that the binding of the metal prosthetic group indeed have important role on the overall stability of the protein. However, the maximum free energy of unfolding (ΔGS) of the secondary structure of the protein remained almost the same for all variants of CYP175A1. These studies thus show that weak non-bonding interactions such as hydrogen bonding and metal coordination play critical role in imparting high thermostability of the overall structure of the thermostable enzyme, CYP175A1. The thermostability of tertiary structure of the active site of the enzyme was also shown to depend on the nature of binding of the metal cofactor to the protein matrix.

Gene cloning, expression, molecular modeling and docking study of the protease SAPRH from Bacillus safensis strain RH12

The sapRH gene, which encodes the serine alkaline protease SAPRH, from Bacillus safensis RH12, was isolated and its DNA sequence was determined. The deduced amino-acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with SAPB from B. pumilus CBS, with only 9 amino-acids of difference. The region, encoding SAPRH was heterologously expressed in E. coli BL21-AI™ cells using GATEWAY™ pDEST™17 expression-vector. The recombinant (His)6-tag enzyme (His6-rSAPRH) was purified in a single affinity chromatography step and its biochemical properties were determined and compared to those of SAPRH and rSAPB. Interestingly, His6-rSAPRH showed improved thermostability compared to SAPRH and rSAPB. The molecular dynamics of SAPRH compared to SAPB revealed a more thermostable structure, thus confirming the in vitro results showing that His6-rSAPRH has a t1/2 of 120 min against 90 and 30 min for SAPRH and rSAPB, respectively, at 70 °C and different kinetic parameters to synthetic peptides. The docking simulations data allow in getting an insight into the involvement of some key amino-acids in substrate binding and account for the selectivity. Overall, this is the first report of a sapRH gene cloned from B. safensis which can be a promising potential candidate for future applications in detergent formulations.

Phase Transition of As-Milled and Annealed CrCuFeMnNi High-Entropy Alloy Powder

The CrCuFeMnNi high entropy alloy (HEA) powder was synthesized by mechanical alloying. The effects of milling time and subsequent annealing on the structure evolution, thermostability and magnetic property were investigated. After 50[Formula: see text]h of milling, the CrCuFeMnNi HEA powder consisted of a major FCC phase and a small amount of BCC phase. The crystallite size and strain lattice of 50[Formula: see text]h-ball-milled CrCuFeMnNi HEA powder were 12[Formula: see text]nm and 1.02%, respectively. The powder exhibited refined morphology and excellent chemical homogeneity. The supersaturated solid solution structure of the as-milled HEA powder transformed into FCC1, FCC2, a small amount of BCC and [Formula: see text] phase in annealed state. Most of the BCC phase decomposed into FCC (mainly FCC2 phase) and [Formula: see text] phases, and the dynamic phase transition was almost in equilibrium at 900[Formula: see text]C. The saturated magnetization and coercivity force of the 50[Formula: see text]h-ball-milled CrCuFeMnNi HEA powder were respectively 16.1[Formula: see text]emu/g and 56.2[Formula: see text]Oe.

Bioinformatics Analysis of Upstream Region and Protein Structure of Fungal Phytase Gene.

Phytase increases the bioavailability of phytate phosphorus in seed-based animal feeds and reduces the phosphorus pollution of animal waste. Since most animal feeds for pellets are heated up to 65-80 °C, the production of a thermostable structure for phytase can be useful. In this study, we sought to perform bioinformatics analysis of the upstream region and protein structure of fungal phytase to improve its expression and thermostability properties. We used bioinformatics methods such as similarity search, multiple alignment, statistical analysis of physicochemical properties of amino acids, pattern recognition, and protein modeling to find out the effective factors in heat resistance of phytase. Change in Gibbs free energy (ΔG) of the best pattern promoter resulting from the interaction between RNA polymerase and the promoter sequences of modified genes of phytase was equal to -9 kcalmol-1, which is lower compared to other interactions. The evaluation of the three-dimensional structure of new phytases showed that amino acid substitutions aimed at improving thermostability did not change the form and structure of the protein. The results of Prochek, Whatcheck, and ERRAT for structural analysis and verification were 84, 72, and 70, respectively, that were satisfactory.

Lanthanide(III) complex metal-organic frameworks with a phenanthroline-carboxylate derivate and 2,5-thiophenedicarboxylate coligand: hydrothermal synthesis, crystal structure, and high thermostability

Abstract Based on 2-(4-carboxyphenyl)imidazo[4,5-f]-1,10-phenanthroline (HNCP) and 2,5-thiophenedicarboxylate (TDC2−) ligands, three new lanthanide-containing (Sm, Nd, and Pr) compounds, [Sm(NCP)(TDC)] n (1), [Nd(NCP)(TDC)] n ·2n(H2O)0.5 (2), and [Pr(NCP)(TDC)] n ·n(H2O)0.5 (3), have been synthesized using the hydrothermal method and structurally characterized using single-crystal X-ray diffraction. Structural analyses have revealed that compounds 1–3 are 3D isostructural metal-organic frameworks in which the [Ln2(COO)4] dimers can be regarded as 6-connecting nodes, and the TDC2− and NCP− ligands are simplified as connectors to achieve the double interspersed 3D networks with the point symbol {412·63}. Thermogravimetric analysis has illustrated that the rigid architecture contributes to superior thermal stability with a thermal decomposition temperature of more than 400°C for the resulting metal-organic frameworks.

Transformation of a Thermostable G-Quadruplex Structure into DNA Duplex Driven by Reverse Gyrase.

Reverse gyrase is a topoisomerase that can introduce positive supercoils to its substrate DNA. It is demonstrated in our studies that a highly thermal stable G-quadruplex structure in a mini-plasmid DNA was transformed into its duplex conformation after a treatment with reverse gyrase. The structural difference of the topoisomers were verified and analyzed by gel electrophoresis, atomic force microscopy examination, and endonuclease digestion assays. All evidence suggested that the overwinding structure of positive supercoil could provide a driven force to disintegrate G-quadruplex and reform duplex. The results of our studies could suggest that hyperthermophiles might use reverse gyrase to manipulate the disintegration of non-B DNA structures and safekeep their genomic information.

Quantitative Structure-Thermostability Relationship of Late Transition Metal Catalysts in Ethylene Oligo/Polymerization

Quantitative structure–thermostability relationship was carried out for four series of bis(imino)pyridine iron (cobalt) complexes and α-diimine nickel complexes systems in ethylene oligo/polymerization. Three structural parameters were correlated with thermal stability, including bond order of metal-nitrogen (B), minimum distance (D) between central metal and ortho-carbon atoms on the aryl moiety and dihedral angle (α) of a central five-membered ring. The variation degree of catalytic activities between optimum and room temperatures (AT) was calculated to describe the thermal stability of the complex. By multiple linear regression analysis (MLRA), the thermal stability presents good correlation with three structural parameters with the correlation coefficients (R2) over 0.95. Furthermore, the contributions of each parameter were evaluated. Through this work, it is expected to help the design of a late transition metal complex with thermal stability at the molecular level.

Oxidation of coal in industrial conditions. 3. Mechanical strength of coke

The change in mechanical strength of coke is investigated as a function of the storage time of the coal from which it is produced. Considerable variability is noted in the limiting coal-storage time due to deterioration in strength of the coke produced. Accordingly, care must be taken in specifying different storage times for different coal stocks. When oxidized coal is employed, wear of the coke is aggravated, because its strength is reduced. Analysis of graphical and mathematical relationships indicates that, in both summer and winter, coal of moderate metamorphic development may be stored for the longest times in terms of loss of coke strength. The change in the coking properties of coal is assumed to depend on the ease of penetration of the plastic mass through the surface layer of rigid thermostable structure formed on oxidation. Improvement in the coking properties of bituminous coal on natural oxidation is attributed to the increase in expansion pressure.

Effect of temperature on lignin-derived inhibition studied with three structurally different cellobiohydrolases

Non-productive enzyme adsorption onto lignin inhibits enzymatic hydrolysis of lignocellulosic biomass. Three cellobiohydrolases, Trichoderma reesei Cel7A (TrCel7A) and two engineered fusion enzymes, with distinctive modular structures and temperature stabilities were employed to study the effect of temperature on inhibition arising from non-productive cellulase adsorption. The fusion enzymes, TeCel7A-CBM1 and TeCel7A-CBM3, were composed of a thermostable Talaromyces emersonii Cel7A (TeCel7A) catalytic domain fused to a carbohydrate-binding module (CBM) either from family 1 or from family 3. With all studied enzymes, increase in temperature was found to increase the inhibitory effect of supplemented lignin in the enzymatic hydrolysis of microcrystalline cellulose. However, for the different enzymes, lignin-derived inhibition emerged at different temperatures. Low binding onto lignin and thermostable structure were characteristic for the most lignin-tolerant enzyme, TeCel7A-CBM1, whereas TrCel7A was most susceptible to lignin especially at elevated temperature (55 °C).

High-temperature dilatometry of Ti-46Al-8Nb refractory alloy

The temperature dependence of the linear thermal expansion coefficient K L of the intermetallic Ti- 46Al-8Nb (at %) alloy is experimentally determined for the first time within the temperature range from 373 to 1773 K (solidus point). The determined boundaries of phase fields are compared with the results of differential thermal analysis and the calculated phase diagram of the alloy. The hightemperature limit (1384 K) of the alloy structure thermostability is detected from signs of the α2 + γ α + γ phase transition in dilatometric curves. The restructuring mechanism in the α + γ field is studied by scanning electron microscopy. It is shown that the α2 + γ α + γ phase transition is accompanied by selective structural degradation of singlecrystalline α2 lamellae and the related destruction of a fine lamellar α2�Ti 3Al(Nb) + γ�TiAl(Nb) texture. The average values of KL of the alloy are calculated within 100�K ranges in the lowtemperature α2 + γ phase field, which is of inter� est from a practical viewpoint, according to the State Standard GOST 8.018-2007.