反胶团系统及其在蛋白质分离复性中的应用研究进展

Reverse micelles bring mild and effective microenvironments in organic solvent that contain bitmolecules, which have attracted immense attention for application in the isolation of proteins, protein refolding, and enzymatic reaction. In this review, the application of reverse micelles for protein separation and refolding has been briefly summarized and various reverse micellar systems composed of different surfactants, including ionic, non- ionic, mixed, and affinity-based reverse micelles, have been highlighted. It illustrates especially the potential application of the novel affinity-based reverse micelles consisting of biocompatible surfactant coupled with affinity ligands. Moreover, the importance to develop universal affinity-based reverse micelles for protein separation and refolding in the downstream processing of biotechnology has been pointed out.     

XPS and SEM study of UV laser surface modification of polymers

Several commercial polymers—poly(ethylene) (PE), poly(propylene) (PP), poly(vinylidene fluoride) (PVF₂), poly(vinyl chloride) (PVC) and polystyrene (PS)—were treated in air with an argon-fluorine UV excimer laser (λ = 193 nm). The polymer etch rate was investigated by two methods: quartz crystal microbalance (QCM) and piercing of films. X-ray photoelectron spectroscopy (XPS) analysis was performed on the modified surfaces after laser exposure at various fluences. Samples were subsequently analysed by scanning electron microscopy (SEM). From our results, polymers may be classified into two categories concerning their reactivity towards UV laser light. — the weakly absorbing polymers (e.g. PE, PP, PVF₂) where a photothermal process (thermal degradation) dominates the interaction. — the strongly absorbing polymers (e.g. PVC, PS) where a photochemical process (photoablation) dominates the interaction.     

Extraction of IgG4 Fab Fragments Using HDEHP-Isooctane and -Corn Oil Reverse Micelles

The forward extraction (FE) and backward extraction (BE) of humanized IgG4 Fab fragments using HDEHP-isooctane and -corn oil reverse micelles (RMs) with/without a non-ionic (Brij 30) and counterionic (TOMAC) surfactant, respectively, was evaluated, and both solvents were equally effective. Interphase precipitate formation during FE caused Fab fragment loss; however, acetone precipitation with an ethanol wash step enabled true percentage extractions to be determined. Brij 30 and TOMAC influenced extraction; TOMAC mainly increased BE at pH 8 with Brij 30 in FE. The highest extraction yields were without Brij 30 and TOMAC for isooctane and with TOMAC for corn oil. Water content analysis showed RMs formed in both solvents with/without Brij 30 and TOMAC, confirming extraction feasibility.     

Effect of a Mutant Danbaekkong Allele on Soybean Seed Yield,Protein, and Oil Concentration

There is a known negative correlation between soybean [Glycine max [L.] Merr.] seed protein and oil and between protein and yield. This challenges breeders to increase protein concentration while maintaining oil concentration and yield. The objective of this study was to determine if marker-assisted selection for the Danbaekkong (Dan) protein allele on chromosome 20 influences seed yield and quality traits in near isogenic genetic backgrounds. A population of 24 F₇-derived near isogenic lines (NIL) of soybean was created by crossing G03-3101 × LD00-2817P. The 24 NIL consisted of 12 wild type (WT) and 12 mutant Dan type lines. These NIL were grown in 2016 and 2017 field seasons in replicated field trials in nine environments, with six in Tennessee and one each in Arkansas, Missouri, and North Carolina. There were significant (P < 0.05) differences in yield, protein, and oil concentrations between the two experimental groups. The Dan group had significantly (P < 0.05) more protein (421 g kg⁻¹), less oil (192 g kg⁻¹), and lower yield (3143 kg ha⁻¹) than the WT group (390 g kg⁻¹ protein, 210 g kg⁻¹ oil, and 3281 kg ha⁻¹ yield). These results support previous research and corroborate the overall negative genetic correlations. Nevertheless, seed yield of several higher-protein Dan lines MC-13, MC-16, MC-19, and MC-24 exceeded seed yield of lower protein WT lines MC-2, MC-3, MC-6, and MC-10. The higher-protein lines represent genetic resources for reducing the negative correlation between protein and yield.     

Extraction of Human IgG4 Monoclonal Antibodies Using AOT- and HDEHP-Isooctane Reverse Micelles

The extraction of 0.05 and 1 mg mL^?1 human IgG4 using reverse micelles (RMs) formed with anionic surfactants AOT or HDEHP in isooctane was evaluated. For both surfactants the use of 1 mg mL^?1 IgG4 resulted in higher forward extraction (FE), and generally better backward extraction (BE) yields than 0.05 mg mL^?1 IgG4, achieving optimum FE and BE yields at FE pHs of 5 at 3.13 mM AOT and 6 at 1.56 mM HDEHP. IgG4 precipitation at the interface was observed at the lower pHs during FE which appeared to cause low overall extraction yields. Water content analysis revealed AOT-RMs were much bigger than HDEHP-RMs.     

Chemical interaction of D9 alloy clad with B4C in liquid sodium: Studies employing XPS,XRD, and SEM

A cell representing typical control rod subassembly of the prototype fast breeder reactor is designed to probe the liquid sodium-mediated chemical interaction of boron carbide (B₄C) control rod with the D9 alloy clad. The cell was equilibrated at 973 K for 5000 hours with liquid sodium in the annular gap. XRD shows the formation of Cr₃C₂ and Fe₂B along with oxides of Ni, Cr, and Fe. XPS studies reveal the diffusion of boron and carbon up to a depth of about 160 and 120 μm, respectively. A boron-rich region is observed up to a depth of about 40 μm which consists of B³⁺ arising from oxides and a nearly constant elemental boron region, extending up to a depth of 160 μm. The highly reacted zone extends up to a depth of about 40 μm consisting of oxides of most of the elements of D9 clad exhibiting a higher valence states. As the cell is helium leak tested, oxygen out gassed from B₄C at 973 K played a major role in chemical oxidation of the constituents of the D9 alloy which can be minimized by using high-density B₄C.     

Reverse Micellar Extraction of Bromelain from Pineapple (Ananas comosus L. Merryl) Waste: Scale-up,Reverse Micelles Characterization and Mass Transfer Studies

Scale-up studies for phase transfer mode of reverse micellar extraction are attempted for the separation and primary purification of bromelain (EC 3.4.22.33) from pineapple (Ananas comosus L. Merryl) waste. Characterization of reverse micelles and mass transfer studies for the real system has been attempted for the first time. Scale-up of the extraction process employing commercial grade surfactant cetyltrimethylammonium bromide (CTAB) and solvent isooctane resulted in purification of 2.43 fold with an activity recovery 81.3%. The reverse micellar size estimated using empirical and geometrical models indicated that the reverse micelles are large enough (R_m = 7.2–9.6 nm) to host bromelain molecules that are relatively smaller in size (～1.67 nm). The studies on the kinetics of mass transfer indicated a relatively slower rate (by ～34%) of mass transfer in case of back extraction compared to forward extraction. Process scale-up did not significantly affect the extraction efficiency whereas purity of phase components played a major role. The mass transfer across the phases was high in the initial period of mixing for both forward and back extractions.     

Impact of Free Fatty Acids and Phospholipids on Reverse Micelles Formation and Lipid Oxidation in Bulk Oil

Association colloids such as phospholipid reverse micelles could increase the rate of lipid oxidation in bulk oils. In addition to phospholipids, other surface active minor components in commercial oils such as free fatty acids may impact lipid oxidation rates and the physical properties of reverse micelles. In this study, the effects of free fatty acids on changes in the critical micelle concentration (CMC) of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) in stripped corn oil (SCO) were determined by using the 7,7,8,8-tetracyanoquinodimethane solubilization technique. Different free fatty acids including myristoleic, oleic, elaidic, linoleic and eicosenoic were added at 0.5 % by wt along with the DOPC into the bulk oils. There was no significant effect of free fatty acids with different chain length, configuration and number of double bonds on the CMC value for DOPC in bulk oil. However, increasing concentrations of oleic acid (0.5 to 5 % by wt) caused the CMC value for DOPC in bulk oils to increase from 400 to 1,000 μmol/kg oil. Physical properties of DOPC reverse micelles in the presence of free fatty acids in bulk oils were also investigated by the small angle X-ray scattering technique. Results showed that free fatty acids could impact on the reverse micelle structure of DOPC in bulk oils. Moreover, free fatty acid decreased pH inside reverse micelle as confirmed by the NMR studies. The oxidation studies done by monitoring the lipid hydroperoxide and hexanal formation revealed that free fatty acids exhibited pro-oxidative activity in the presence and absence of DOPC. Different types of free fatty acids had similar pro-oxidative activity in bulk oil.     

Effects of Emulsification of Fat on the Surface Tension of Protein Solutions and Surface Properties of the Resultant Spray-Dried Particles

To examine the effect of protein adsorption on the fat–water interface on the surface composition of spray-dried particles, whey, hydrolyzed whey, and soy protein isolate emulsions were prepared at three different protein to fat ratios of 1:1, 1:5, and 1:10 and spray dried. Non-hydrolyzed whey protein isolate (WPI) and the more hydrolyzed whey protein solutions at 20.2% degree of hydrolysis (DH) had significantly lower surface tension values with fat than without fat. The correlation between the reduction of surface tension value of an emulsion and the increase in protein surface composition of powder particles was observed for WPI and HWP406 but was not observed for the other protein isolate types. It was clear that the spray-dried emulsions had fat as the dominant component on the surface of the powder particles and that the amount of protein on the surface became severely depressed at higher fat addition levels. In terms of its powder morphology, the unique powder structures such as the indentations and folds usually found on the surface of protein containing powders were not evident because they were compromised by the presence of high surface fat. The powder with higher surface fat had more crumpled particle structures and dimpled surfaces.     

Active state of model cobalt foil catalyst studied by SEM, TPR/TPO, XPS and TG

Four states of the cobalt foil catalyst, corresponding to different redox treatment and activity, were defined: oxidised, reduced, active and deactivated. They were investigated by scanning electron microscopy (SEM), temperature-programmed reduction (TPR), temperature-programmed oxidation (TPO), X-ray photoelectron spectroscopy (XPS) and thermogravimetric (TG) methods and in the hydrogenation of ethylene used as a test reaction. Particular emphasis was laid on the study of the active state, achieved after the catalyst reduction at moderate temperatures. It was shown that the catalyst preactivated by a series of redox cycles is built of a cobalt oxide layer of a characteristic size and dispersion, which is stuck to the metallic bulk. Reduction at a moderate temperature, prolonged even to several hours, converts only a small fraction of the oxide layer into metallic Co. XPS, TPR and TPO methods distinguished various states of oxygen and cobalt on the surface of the activated or partially activated samples. The results were interpreted in terms of the mechanism of autocatalytic reduction. The deactivation was associated with the structural reconstruction of the surface, taking place either in the reaction mixture during the hydrogenation of ethylene or in hydrogen atmosphere. Formation of the inactive carbon deposit was experimentally excluded.     

Surface chemistry of SnO2 nanowires on Ag-catalyst-covered Si substrate studied using XPS and TDS methods

In this paper we investigate the surface chemistry, including surface contaminations, of SnO₂ nanowires deposited on Ag-covered Si substrate by vapor phase deposition (VPD), thanks to x-ray photoelectron spectroscopy (XPS) in combination with thermal desorption spectroscopy (TDS). Air-exposed SnO₂ nanowires are slightly non-stoichiometric, and a huge amount of C contaminations is observed at their surface. After the thermal physical desorption (TPD) process, SnO₂ nanowires become almost stoichiometric without any surface C contaminations. This is probably related to the fact that C contaminations, as well as residual gases from air, are weakly bounded to the crystalline SnO₂ nanowires and can be easily removed from their surface. The obtained results gave us insight on the interpretation of the aging effect of SnO₂ nanowires that is of great importance for their potential application in the development of novel chemical nanosensor devices.     

Microstructural characterisation of geopolymers synthesised from kaolinite/stilbite mixtures using XRD, MAS-NMR, SEM/EDX, TEM/EDX, and HREM

When crystalline aluminosilicates partially dissolve in a concentrated alkaline medium, an amorphous geopolymeric gel is formed interspersed with undissolved crystalline particles. Some aluminosilicates dissolve more readily than others to give an equilibrium ratio of aluminium to silicon in the gel. In this case study, kaolinite and stilbite mixtures were used to investigate the relative reactivity of different minerals when present in different ratios. XRD and ²⁹Si and ²⁷Al MAS-NMR were used to determine when a specific mineral was completely transferred into the gel phase. Electron diffraction using transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HREM) were employed to establish the amorphous nature of the gel phase. Scanning electron microscopy (SEM)/energy dispersive X-ray (EDX) and TEM/EDX were then used to determine the composition of the gel. By using simple mass balance assumptions, the quantity of gel and the extent of partial dissolution of an aluminosilicate could then be calculated. It was found that a geopolymer containing a higher weight percentage of CaO in its gel, a lower ratio of (average surface area)/gel, and where the undissolved crystalline particles have a higher hardness had higher mechanical strength. The method developed in this paper is also applicable to other cementitious materials.     

大豆分离蛋白的性质测定与改性研究

采用POCl3对大豆分离蛋白进行改性.通过正交实验,得到影响蛋白质溶解性、乳化性、起泡性等的最佳工艺条件.     

The structural (FTIR,XRD, and XPS) and biological studies of thermosensitive chitosan chloride gels with β‐glycerophosphate disodium

In this article, the properties of thermosensitive chitosan hydrogels (structural and biological) prepared using chitosan chloride and β‐glycerophosphate are presented. The porous structure of the gels was observed with a scanning electron microscopy (SEM). Rheological and other structural and morphological investigations were carried out by infrared spectroscopy and X‐ray photoelectron spectroscopy. Crystallinity of the gel was determined by X‐ray diffraction analysis. Biocompatibility of the chitosan gels was investigated by contact with bone cells (line Saos‐2) for 7 and 21 days. The cells were encapsulated and seeded onto the gel. A live/dead test showed no cytotoxic reaction. The structure of the gels, before and after the cell culture, was tested using SEM (including ESEM), to select the most suitable method of preparation and reliable visualization of the gel with cells. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46459.     

XPS,TDS, and AFM studies of surface chemistry and morphology of Ag-covered L-CVD SnO2 nanolayers

This is well known that the selectivity and sensitivity of tin dioxide (SnO₂) thin film sensors for the detection of low concentration of volatile sulfides such as H₂S in air can be improved by small amount of Ag additives. In this paper we present the results of comparative X-ray photoelectron spectroscopy (XPS), thermal desorption spectroscopy (TDS), and atomic force microscopy (AFM) studies of the surface chemistry and morphology of SnO₂ nanolayers obtained by laser-enhanced chemical vapor deposition (L-CVD) additionally covered with 1 monolayer (ML) of Ag. For as deposited SnO₂ nanolayers, a mixture of tin oxide (SnO) and tin dioxide (SnO₂) with the [C]/[Sn] ratio of approximately 1.3 was observed. After dry air exposure, the [O]/[Sn] ratio slightly increased to approximately 1.55. Moreover, an evident increasing of C contamination was observed with [C]/[Sn] ratio of approximately 3.5. After TDS experiment, the [O]/[Sn] ratio goes back to 1.3, whereas C contamination evidently decreases (by factor of 3). Simultaneously, the Ag concentration after air exposure and TDS experiment subsequently decreased (finally by factor of approximately 2), which was caused by the diffusion of Ag atoms into the subsurface layers related to the grain-type surface morphology of Ag-covered L-CVD SnO₂ nanolayers, as confirmed by XPS ion depth profiling studies. The variation of surface chemistry of the Ag-covered L-CVD SnO₂ after air exposure observed by XPS was in a good correlation with the desorption of residual gases from these nanolayers observed in TDS experiments.     

Structure,Protein Interactions and In Vitro Protease Accessibility of Extruded and Pressurized Full-Fat Soybean Flakes

The objectives of the present study were to determine how extrusion (barrel temperature of 100 °C) and high-pressure processing (HPP, 200 and 500 MPa, 15 min, 25 °C) of full-fat soybean flakes (FFSF) modified the structure of soybean cotyledon cells, the protein interactions and the in vitro protease accessibility. Cellular disruption of the cotyledon cells was only observed for extruded FFSF. Extrusion and HPP at 500 MPa favored formation of insoluble protein aggregates, in which oil was entrapped. High pressure size exclusion chromatography (HPSEC) and extraction methods using buffers containing SDS and 2-mercaptoethanol suggested that noncovalent interactions were the main forces in protein aggregate formation during HPP 500 MPa and extrusion. Intermolecular cross-linking by disulfide bonding was also involved in insoluble aggregates, but at a lesser extent than noncovalent interactions. Extrusion and HPP 500-MPa treatment enhanced the proteolytic attack, while treatment at 200 MPa had no impact. Drastic changes in the peptide profile of the extracted proteins were, however, only observed for the enzyme-treated 500-MPa FFSF. Optimal oil and protein extraction yields required cellular disruption of cotyledon cells and hydrolysis of protein aggregates, which were obtained with enzyme-assisted aqueous extraction of extruded FFSF.     

Investigation of Relationship between Surface Tension of Feed Solution Containing Various Proteins and Surface Composition and Morphology of Powder Particles

The surface tension of freshly created food protein powder isolates was measured in aqueous solutions as a function of concentration, hydrolysis, and temperature. The surface tension of the solutions was measured immediately to best predict their surface-active behavior in a spray-drying scenario, where instantaneous values are more relevant than equilibrium surface tension measurements. Whole whey protein, hydrolyzed whey proteins (degrees of hydrolysis of 4, 9.5, 12, 17, and 20.2%), soy protein, pea protein isolates, and gelatin powders were diluted in a range of concentrations (0.04–2 g/L) and their surface tension values were reported at 23 ± 1°C. It was found that at higher concentrations hydrolyzed whey proteins at degrees of hydrolysis of 9.5 and 12%, and soy protein isolates in particular, showed excellent surface activity (shown through a decrease in surface tension) compared to nonhydrolyzed whey protein and gelatin. When comparing the influence of the degree of hydrolysis of whey proteins, the reverse was observed at lower concentrations (0.04–0.1 g/L), with the nonhydrolyzed whey protein reducing surface tension values more effectively than their hydrolyzed counterparts.

Additionally, the protein solutions (2 g/L) were maintained at higher temperatures of 40, 50, and 60°C and the surface tension values were measured. There was a general improvement of surface activity of proteins indicated by the reduced surface tension of solutions at these temperatures compared with the pure water values. The protein solutions were also spray dried with maltodextrin (MD30) and the powder particle surface composition and structures were analyzed via X-ray photoelectron spectroscopy and scanning electron micrography. There was a trend of correlation between the surface activities of protein in solution with that of the surface composition of protein found on the powder particles. However, there were morphological indicators that corresponded well to the amount of protein present on the surface.     

Effect of Extraction and Precipitation Conditions During Soybean Protein Isolate Production on the Genistein Series Content

The influence of the conditions for isolation of soy protein on the content of genistein and its conjugated forms was studied. The major components of the genistein series isolated from soybean flour were malonyl genistin (54.3%), genistin (36.9%), and equal amounts (4.4%) of genistein and acetyl genistin. A modification in the conjugation profile of genistein between pH 4.5 and 8.0 and above pH 10 was attributed to the action of β-glucosidase and the saponification reaction, respectively. A decrease in the content of total genistein in the insoluble flour residue and in the soy protein isolate (SPI) with increasing extraction pH was detected, while in the whey, the total genistein content was not affected by pH. The effect of pH variation during acid precipitation on the content of genistein and its conjugated forms, at a constant extraction pH (8.0), was also studied. Under these conditions, the highest absolute content of total genistein in the SPI was obtained at pH 3.5 and the lowest was obtained at pH 5.6. The total genistein content in the whey followed an inverse trend compared with that of the protein yield. A temperature increase did not substantially affect the distribution of the different genistein forms or their total contents. The content of total genistein was higher in the glycinin than in the β-conglycinin protein fraction. The effect of the pH during the alcohol/water extraction of the isoflavones was also analyzed. The efficiency of the extraction was lower at pH values between 3.25 and 3.5 than at other pH values.     

中华绒螯蟹组织蛋白中变应原组分的分析与提取

目的对中华绒螯蟹组织蛋白中的变应原组分进行分析鉴定和分离提取,为研制蟹类食物过敏反应的检测试剂和脱敏制剂奠定基础。方法中华绒螯蟹粗提蛋白经SDS-PAGE分离,用蟹过敏症患者血清经Westernblot分析其变应原组分。粗提蛋白经硫酸铵分段盐析和DEAE-52离子交换层析,分离提取中华绒螯蟹的主要变应原成分,并用蟹过敏症患者血清对层析产物进行ELISA分析。结果SDS-PAGE分析显示,中华绒螯蟹粗提蛋白有20多条可辨蛋白带,相对分子质量在15700～118000之间。Westernblot分析显示,7份蟹过敏患者血清与粗提蛋白均呈阳性反应,主要有9条反应带,其中相对分子质量为35000、41000、48000和66000的4条蛋白带与7份患者血清均呈阳性反应。经DEAE-52离子交换层析分离后,变应原主要出现在30%盐析段0.2mol/L洗脱峰、50%盐析段0.2和0.4mol/L洗脱峰、70%盐析段0.2和0.3mol/L洗脱峰、90%盐析段0.2mol/L洗脱峰内。结论中华绒螯蟹组织蛋白中的相对分子质量为35000、41000、48000和66000的组分是其主要变应原。