INFLUENCE OF G187W/K188F/D189Y MUTATION IN THE SUBSTRATE BINDING POCKET OF TRYPSIN ON β-CASEIN PROCESSING

Undertaking to modulate the catalytic properties of trypsin, highly conserved G187, K188 and D189 were replaced with aromatic amino acid residues in order to perturb the electrostatics and to amplify hydrophobic interactions of the substrate binding site. The kinetic parameters of the wild-type trypsin and G187W/K188F/D189Y mutant were determined with synthetic tetrapeptide substrates and β-casein at different pHs. Compared with trypsin, the mutant G187W/K188F/D189Y exhibits 1.3-fold increase in K_m values for the substrates containing arginine and lysine. This mutant shows a 30- to 40-fold decrease of its kcat and its second-order rate constant k_cm/k_m decreases = 40- and 55-fold for substrates containing arginine and lysine, respectively. The kinetic analysis reveals that the mutant conserves the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. Surprisingly, as demonstrated only by proteolysis of a natural substrate (β-casein), the mutant cleaves also peptide bonds involving asparagine and glutamine.     

Efficient adsorption of super greenhouse gas (tetrafluoromethane) in carbon nanotubes

Light membranes composed of single-walled carbon nanotubes (SWNTs) can serve as efficient nanoscale vessels for encapsulation of tetrafluoromethane at 300 K and operating external pressure of 1 bar. We use grand canonical Monte Carlo simulation for modeling of CF4 encapsulation at 300 K and pressures up to 2 bar. We find that the amount of adsorbed CF4 strongly depends on the pore size in nanotubes; at 1 bar the most efficient nanotubes for volumetric storage have size R = 0.68 nm. This size corresponds to the (10,10) armchair nanotubes produced nowadays in large quantities. For mass storage (i.e., weight %) the most efficient nanotubes have size R = 1.02 nm corresponding to (15,15) armchair nanotubes. They are better adsorbents than currently used activated carbons and zeolites, reaching approximately equal to 2.4 mol kg(-1) of CF4, whereas, the best activated carbon Carbosieve G molecular sieve can adsorb 1.7 mol kg(-1) of CF4 at 300 K and 1 bar. We demonstrate that the high enthalpy of adsorption cannot be used as an only measure of storage efficiency. The optimal balance between the binding energy (i.e., enthalpy of adsorption) and space available for the accommodation of molecules (i.e., presence of inaccessible pore volume) is a key for encapsulation of van der Walls molecules. Our systematic computational study gives the clear direction in the timely problem of control emission of CF4 and other perfluorocarbons into atmosphere.     

Effects of pH and temperature on metmyoglobin solubility in a model system

From a series of experiments heating metmyoglobin solutions at pH 5.0 through 7.0, the effects of temperature and pH on the thermal stability of metmyoglobin were investigated. The percent metmyoglobin denatured at temperatures from 25 to 80 °C was determined. pHs lower than 6.5 caused metmyoglobin denaturation at various temperatures from 25 to 80 °C, but it was particularly apparent when pH was < 5.6. Thermal stability of metmyoglobin increased as pH increased. Metmyoglobin denaturation occurred at 55 °C at pH 5, however, denaturation did not occur until 60 °C at pHs from 5.3 to 7.0. A slower heating rate (0.9 °C/min) resulted in more metmyoglobin thermal denaturation than a faster heating rate (1.3 °C/min) when the temperature was above 55 to 60 °C. The denaturation caused by low pH alone was reversible, while that caused by high temperature was not. Techniques which increase muscle pH, such as the injection of sodium bicarbonate, could effectively improve the color condition of PSE meat.     

Improvement of stability of oil-in-water emulsions containing caseinate-coated droplets by addition of sodium alginate

ABSTRACT:  The potential of sodium alginate for improving the stability of emulsions containing caseinate-coated droplets was investigated. One wt% corn oil-in-water emulsions containing anionic caseinate-coated droplets (0.15 wt% sodium caseinate) and anionic sodium alginate (0 to 1 wt%) were prepared at pH 7. The pH of these emulsions was then adjusted to 3.5, so that the anionic alginate molecules adsorbed to the cationic caseinate-coated droplets. Extensive droplet aggregation occurred when there was insufficient alginate to completely saturate the droplet surfaces due to bridging flocculation, and when the nonadsorbed alginate concentration was high enough to induce depletion flocculation. Emulsions with relatively small particle sizes could be formed over a range of alginate concentrations (0.1 to 0.4 wt%). The influence of pHs (3 to 7) and sodium chloride (0 to 500 mM) on the properties of primary (0 wt% alginate) and secondary (0.15 wt% alginate) emulsions was studied. Alginate adsorbed to the droplet surfaces at pHs 3, 4, and 5, but not at pHs 6 and 7, due to electrostatic attraction between anionic groups on the alginate and cationic groups on the adsorbed caseinate. Secondary emulsions had better stability than primary emulsions at pH values near caseinate's isoelectric point (pHs 4 and 5). In addition, secondary emulsions were stable up to higher ionic strengths (< 300 mM) than primary emulsions (<50 mM). The controlled electrostatic deposition method utilized in this study could be used to extend the range of application of dairy protein emulsifiers in the food industry.     

Adsorption of transgenic insecticidal Cry1Ab protein to SiO2. 2. Patch-controlled electrostatic attraction

Adsorption governs the fate of Cry proteins from genetically modified Bt crops in soils. The effect of ionic strength (I) on the adsorption of Cry1Ab (isoelectric point IEP(Cry1Ab) ≈ 6) to negatively charged quartz (SiO(2)) and positively charged poly-L-lysine (PLL) was investigated at pH 5 to 8, using quartz crystal microbalance with dissipation monitoring and optical waveguide lightmode spectroscopy. Cry1Ab adsorbed via positively and negatively charged surface patches to SiO(2) and PLL, respectively. This patch controlled electrostatic attraction (PCEA) explains the observed increase in Cry1Ab adsorption to sorbents that carried the same net charge as the protein (SiO(2) at pH > IEP(Cry1Ab) and PLL at pH < IEP(Cry1Ab)) with decreasing I. In contrast, the adsorption of two reference proteins, BSA and HEWL, with different adsorption mechanism, were little affected by similar changes of I. Consistent with PCEA, Cry1Ab desorption from SiO(2) at pH > IEP(Cry1Ab) increased with increasing I and pH. Weak Cry1Ab-SiO(2) PCEA above pH 7 resulted in reversible, concentration dependent adsorption. Solution depletion experiments showed that PCEA also governed Cry1Ab adsorption to SiO(2) particles at environmentally relevant concentrations (a few ng mL(-1)). These results imply that models describing Cry1Ab adsorption to charged surfaces in soils need to account for the nonuniform surface charge distribution of the protein.     

Examination of hydrophobic contaminant adsorption in mineral micropores with grand canonical Monte Carlo simulations

A molecular level understanding of the interactions between hydrophobic organic contaminants (HOCs) and sediments is needed in order to assess contaminant fate in the environment. Grand canonical Monte Carlo simulations were performed to investigate water and trichloroethylene (TCE) adsorption in slit micropores confined by charged and uncharged silica surfaces. Gas-phase single-sorbate simulations with water or TCE were performed as well as mixture simulations of bulk water containing TCE at 1% of its saturation concentration. Gas-phase isosteric heats for water adsorption in the uncharged pores ranged from -40 to -52 kJ/mol, and the densities of the adsorbed water phases were always less than that for bulk water. Gas-phase isosteric heats for water adsorption in the charged pores ranged from -79 to -170 kJ/mol, and the densities of the adsorbed water phases were close to that for bulk water. The isosteric heats and water densities indicated that the uncharged pores were mildly hydrophobic, and the charged pores were very hydrophilic. In mixture simulations of adsorption from solution, the presence of water promoted TCE adsorption in uncharged pores with widths between 14 and 20 A. The isosteric heats for TCE adsorption from solution ranged from -14 to -27 kJ/mol in the uncharged pores and from -9.3 to -50 kJ/mol in the charged pores. Strong attractions to the pore surfaces were significantly diminished after adsorption of the first two monolayers of either adsorbate. Aqueous-phase TCE at a concentration equal to 1% of its saturation concentration was able to completely displace adsorbed water in uncharged pores. Even in highly hydrophilic pores, TCE at this concentration was able to displace up to 50% of the adsorbed water. Apparent differential enthalpies of adsorption determined from the temperature dependence of TCE adsorption isotherms underestimated the magnitude of the true isosteric heats of adsorption by up to 30 kJ/mol. This shows that HOC adsorption enthalpies determined from the temperature dependence of their adsorption isotherms underestimate the true strength of HOC-adsorbent interactions.     

Amino Acid Additives Effects on Sweet Potato Starch Thermal Properties

This study assessed the effects of amino acid additives, aspartic acid, leucine, lysine, and methionine, on the thermal characteristics of white‐fleshed and orange‐fleshed Beauregard sweet potato starches. The orange‐fleshed sweet potato (OFSPS) starch began to gelatinize at a lower temperature (56.8°C) than the white‐fleshed sweet potato starch (WFSPS) (70.1°C), but the two starches needed the same amount of energy to gelatinize. Lysine increased the gelatinization temperature of the OFSPS. The addition of leucine, aspartic acid, and methionine had no apparent impact on the OFSPS. The addition of lysine or aspartic acid increased the gelatinization temperatures of the WFSPS. Overall, the two starches used were more affected by charged amino acids than by the neutral ones.     

柑橘柠檬苦素大孔树脂分离纯化方法

以柑橘柠檬苦素为考察指标,研究大孔树脂分离纯化柠檬苦素的工艺条件。结果表明,D101大孔树脂对柑橘柠檬苦素有较好的吸附分离性能,是分离纯化柑橘柠檬苦素的适宜大孔树脂;D101大孔树脂分离纯化柑橘柠檬苦素的最佳工艺条件为：上样流速1 mL/min、上样质量浓度0.5 mg/mL、用 pH 6、80%的乙醇溶液作洗脱剂、洗脱流速2 mL/min。通过树脂回收重复使用,发现D101树脂通过再生处理后,其吸附性能未有明显降低,可以重复使用。采用上述方法得到D101大孔树脂对柠檬苦素的吸附率为88.53%,解吸率为93.47%,得率为82.75%。高效液相色谱检测可知,柠檬苦素的含量达到了92.79%。     

Adsorption characteristics of various proteins to a titanium surface

Adsorption characteristics of 18 proteins, with different sizes and isoelectric points, to a titanium oxide surface were studied. The adsorption isotherms were categorized based on protein type and pH: type 1, irreversible adsorption; type 2, Langmuir-type reversible adsorption; and type 3, reversible and irreversible adsorption. Most of the proteins tested were irreversibly adsorbed in the pH range of 3-8, whereas most adsorbed reversibly at pH 8.5-9.4. Protamine, with a pI value of 12, adsorbed reversibly in the pH range of 3-9. pH values that gave maximal sums of irreversibly and reversibly adsorbed proteins were in the pH range of 3-8 and tended to increase slightly with the pI value of the corresponding protein. pH values that gave maximal quantities of irreversibly adsorbed protein ranged between 4-6 and were nearly independent of pI.     

Sodium caseinate/carboxymethylcellulose interactions at oil–water interface: Relationship to emulsion stability

The effect of carboxymethylcellulose (CMC) on the properties of oil-in-water emulsions prepared with sodium caseinate (CN) was studied at different pHs (4–7). At pH 7, the surface protein coverage increased gradually with increasing CMC concentration, followed by a preferential adsorption of β-casein. While at pH 4, a sharp decrease in surface protein coverage was noted between 0 and 0.3 wt.% CMC, and no obvious difference in protein composition was observed. ζ-Potential measurements indicated that CMC adsorbed onto the CN-coated droplets at pH 4–5, but not at pH 6–7. As a result, the excess of non-adsorbed CMC induced depletion flocculation in the neutral emulsions. However, the acidic emulsions containing high levels of CMC (>0.3 wt.%) remained stable after 60 days of storage due to the formation of multilayer structures. At pH 4, CMC desorbed from the droplet surfaces at high NaCl concentrations, leading to lower emulsion stability.     

EFFECTS OF ACETYLATION ON PHYSICOCHEMICAL PROPERTIES OF J1S SOY PROTEIN

Acetylation of ε-amino groups of lysine residues changed conformation of glycinin isolated from soybean, the extent of which depended upon the degree of modification. Soy glycinin with lysine residue modifications of 0%, 28%, 65%, 85%, and 95% were used for the experiment. Accessibility of tyrosine and tryptophan residues, which were shown to increase as modification percent increased, were detected using uv absorption spectra and fluorescence spectra, respectively. Surface hydrophobicity was found to increase more than two times over native glycinin, when lysine residues were excessively modified to above 95%. Masking of charged lysine residues, exposure of hydrophobic interior, and subunit dissociation should have contributed to the increase. Enthalpy, as obtained from differential scanning calorimetry, dropped from 3.6 callg native protein to 0 callg protein, when lysine residues were acetylated above 65%. implicating complete denaturation. The hydrolytic rates, using α-chymotrypsin, increased initially, then decreased at more than 65% lysine modification.     

Altering pasting characteristics of sweet potato starches through amino acid additives

ABSTRACT:  This study assessed the effects of amino acid additives, aspartic acid, leucine, lysine, and methionine, on the pasting and thermal characteristics of white- and orange-fleshed Beauregard sweet potato starches. A rapid visco analyzer 3D was used to determine pasting properties. In comparing pasting characteristics, starch from orange-fleshed sweet potato was found to be easier to cook, had a lower potential for retrogradation, and was less stable during heating than the white-fleshed sweet potato starch. The RVA analysis showed that the charged amino acids, aspartic acid and lysine, altered pasting characteristics of the 2 starches more than the neutral amino acids, leucine and methionine. Aspartic acid had similar effects on both starches, making them less stable during cooking and lowering the potential for retrogradation. Lysine, when added to the orange-fleshed sweet potato starch, decreased the breakdown, allowing for more stability during cooking. This study showed that pasting properties of sweet potato starches can be altered by the addition of amino acids.     

陈窖豆豉粑类黑精提取及骨架肽段氨基酸组成分析

发酵期18个月的陈窖豆豉粑,其类黑精含量按Hashiba方法以干基计为4.76%。采用优化的常温分离提取工艺获得的类黑精组分经氨基酸分析表明,陈窖豆豉粑类黑精骨架由肽段构成,并且肽段中最具类黑精形成活性的氨基酸残基主要是天冬氨酸(Asp)、谷氨酸(Glu)、精氨酸(Arg)、赖氨酸(Lys)和脯氨酸(Pro)。     

Replacement of His12 or His119 of bovine pancreatic ribonuclease A with acidic amino acid residues for the modification of activity and stability

In an attempt to produce a bovine pancreatic ribonuclease A (RNase A) with increased activity and stability, the catalytic pair of His12 and His119 was substituted with aspartic acid or glutamic acid, and aspartic acid, respectively, to evaluate the role of the two histidine residues in the activity and stability. Kinetic analysis revealed that k(cat)/K(m) values were significantly reduced for all mutant enzymes due to a decreased k(cat) rather than an increased K(m): the k(cat) values for both CpA and C>p of H12D and H12E decreased to about 1/1000; the k(cat) values of H119D decreased by 1/3300 for CpA and 1/80 for C>p. Thus, neither Asp nor Glu is able to act solely as an efficient catalytic residue of RNase A. Alkylation with iodoacetic acid (IAA) revealed that mutant enzymes had reduced reaction rates and that no modification was evident at Glu12 and Asp12 of H12E and H12D, respectively. This indicates that the low catalytic activity of mutant enzymes could be due to low basicity of Asp12 and Glu12. While the T(m) of H119D was almost the same as that of the wild-type enzyme, the T(m) of both H12D and H12E markedly decreased. It became apparent that His12 located at the bottom of the active site cleft contributes significantly to the structural stability of RNase A.     

糖碳源和氨基酸氮源对苍白杆菌属发酵产生物表面活性剂的影响

该实验为优化苍白杆菌（Ochrobactrum sp.）产生物表面活性剂的发酵条件,比较了不同糖碳源（蔗糖、乳糖、甘油、木糖、果糖）、氨基酸氮源（谷氨酸、天冬氨酸、赖氨酸、精氨酸和缬氨酸）、碳氮比（C/N）（1、10、20、30、40）对生物表面活性剂发酵的影响。结果表明,最优碳源为蔗糖,发酵5 d达最大乳化活性56.30%;最优氮源为天冬氨酸,发酵4 d达最大乳化活性54.00%;最优C/N为10,发酵4 d达最大乳化活性55.75%。     

The effect of vitamins A and E on the amino acid composition of proteins in different segments of the brain in aged animals]

The amino acids protein composition was studied in different segments of the brain in young (1.5 month old) and old (23-months) rats. The research covered also a short-lived action of vitamins A and E on the amino acids protein composition in the brain of 23-months old rats. The levels of the glutamic, aspartic acids, lysine, serine, of the sum of valine+methionine was found to be down, whereas the content of other amino acids proved invariable. After vitamins A and E loads the glutamic and aspartic acids in the brain segments under investigation was found to be up. In individual segments the content of lysine, thyrosine, valine+methionine experienced a certain normalization.     

Interfacial properties of deamidated wheat protein in relation to its ability to stabilise oil-in-water emulsions

Isolated wheat protein (IWP) is an acidic deamidated wheat protein. The deamidation process enhances the protein solubility at pHs greater than 6, and therefore its potential ability to act as a food emulsifier. The interfacial properties and the mechanism by which this protein stabilises oil-in-water emulsions were investigated by measuring the protein's absorbed layer thickness on latex particles, its interfacial rheology, and the colloidal and thermal stability of IWP stabilised emulsions. IWP forms a relatively thick interfacial layer of 18 nm upon adsorption onto latex beads, suggesting that the protein adsorbed with the long axis perpendicular to the surface, i.e. end-on, at a full protein coverage. The interfacial rheology measurement showed that IWP formed a relatively weak fluid-like interface. Similar to other protein emulsifiers, the colloidal stability of IWP emulsions is provided largely through electrostatic repulsion. Although IWP emulsions were sensitive to salt induced flocculation, the presence of excess protein in the aqueous phase (e.g. 4 wt%) was able to reduce the effect of salt screening (50 mM CaCl₂) on a 25 wt% oil-in-water emulsion completely. The emulsions underwent minimal coalescence when droplets were in close contact, e.g. flocculated, because the interfacial layer of IWP provides a barrier to droplet coalescence, even in high salt environments. IWP emulsions were resistant to thermal treatment with no changes in particle size observed when the emulsions were heated (up to 90 °C for 20 min) in the absence or the presence of 150 mM NaCl. The heat stability of IWP emulsions is thought to arise from the structure of IWP at the interface. A lack of free cysteines combined with few hydrophobic regions meant that there were minimal interactions between protein molecules adsorbed onto the same droplet or on neighbouring droplets. The unique interfacial properties of IWP, e.g. its physical layer thickness and the structure provide enhanced stability for emulsions against coalescence and heating.     

磁性金属有机框架材料对水中林可霉素的去除研究

目的 基于磁性金属有机框架材料(metal-organic framework, MOF)的磁固相萃取(magnetic solid phase extraction, MSPE)技术吸附去除环境水样中残留的林可霉素, 有效预防抗生素污染。方法 采用溶剂热法预制Fe3O4纳米颗粒, 然后与铜基金属离子和均苯三甲酸有机配体结合制备Fe3O4-MOF199复合材料。通过扫描电子显微镜(scanning electron microscopy, SEM)、X射线衍射(X-ray diffraction, XRD)、傅里叶红外光谱(Fourier transform infrared spectroscopy, FTIR)、差热热重分析曲线(thermogravimetric differential thermal analysis, TG-DTA)和N2吸附-解吸实验等表征手段对材料的形貌、晶体结构及理化性质进行分析。通过等温吸附平衡和吸附动力学实验评估该材料对林可霉素吸附性能, 并对吸附后洗脱剂进行优化。结果 所制备的Fe3O4-MOF199呈规则的正八面体形态, 并具有较高的比表面积(749.19 m2/g)和优越的热稳定性。吸附实验结果表明该吸附过程符合Freundlich吸附动力学方程和伪二阶动力学模型。Fe3O4-MOF199材料用于海河水和津河水中低浓度的林可霉素吸附, 去除率可达80%以上。结论 Fe3O4-MOF199材料具有良好的吸附性能和吸附效果, 结合MSPE工艺可以有效应用于抗生素残留的吸附, 所建立的方法具有简单、有效、准确的林可霉素吸附去除性能。     

Speciation of the ionizable antibiotic sulfamethazine on black carbon (biochar)

Adsorption of ionizable compounds by black carbon is poorly characterized. Adsorption of the veterinary antibiotic sulfamethazine (SMT; a.k.a., sulfadimidine; pK(a1) = 2.28, pK(a2) = 7.42) on a charcoal was determined as a function of concentration, pH, inorganic ions, and organic ions and molecules. SMT displayed unconventional adsorption behavior. Despite its hydrophilic nature (log K(ow) = 0.27), the distribution ratio K(d) at pH 5, where SMT(0) prevails, was as high as 10(6) L kg(-1), up to 10(4) times greater than literature reported K(oc). The K(d) decreases at high and low pH but not commensurate with the decline in K(ow) of the ionized forms. At pH 1, where SMT(+) is predominant and the surface is positive, a major driving force is π-π electron donor-acceptor interaction of the protonated aniline ring with the π-electron rich graphene surface, referred to as π(+)-π EDA, rather than ordinary electrostatic cation exchange. In the alkaline region, where SMT(-) prevails and the surface is negative, adsorption is accompanied by near-stoichiometric proton exchange with water, leading to the release of OH(-) and formation of an exceptionally strong H-bond between SMT(0) and a surface carboxylate or phenolate, classified as a negative charge-assisted H-bond, (-)CAHB. At pH 5, SMT(0) adsorption is accompanied by partial proton release and is competitive with trimethylphenylammonium ion, signifying contributions from SMT(+) and/or the zwitterion, SMT(±), which take advantage of π(+)-π EDA interaction and Coulombic attraction to deprotonated surface groups. In essence, both pK(a1) and pK(a2) increase, and SMT(±) is stabilized, in the adsorbed relative to the dissolved state.     

Effects of monovalent cations on the competitive adsorption of perfluoroalkyl acids by kaolinite: experimental studies and modeling

Our hypothesis that longer-chained perfluoroalkyl acids (PFAAs) outcompete shorter-chained PFAAs during adsorption was tested in this study, wherein the adsorption interactions of six frequently detected PFAAs with kaolinite clay were modeled and examined experimentally using various suspension compositions. Competitive adsorption of PFAAs on the kaolinite surface was observed for the first time, and longer-chained PFAAs outcompeted those with a shorter chain. The electrostatic repulsion between adsorbed PFAA molecules is a primary inhibitory factor in PFAA adsorption. An increase in aqueous sodium or hydrogen ion concentration weakened electrostatic repulsions and changed the adsorption free energy. Therefore, the adsorption of a shorter-chained PFAA with weaker hydrophobicity could occur at high sodium or hydrogen ion concentrations. The experimental and modeling data suggest that the adsorption of shorter-chained PFAAs (≤4 perfluorinated carbons) in freshwater with a typical ionic strength of 10(-2.5) is not thermodynamically favorable. Furthermore, by measuring the electrokinetic potential of kaolinite suspension in the presence of PFAAs, we found that the kaolinite surface became more negatively charged because of the adsorption of PFAAs. This observation indicates that the adsorbed PFAA molecules were within the electrical double layer of the kaolinite surface and that they contributed to the potential at the slipping plane. The possible alignments of adsorbed PFAA molecules on the kaolinite surface were then proposed.