Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM

In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning turmeling microscope（STM） was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic sodium （AOT）/iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of extraction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol·L^-1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca^2＋, Na^＋, K^＋ which were in the range of 0.2-1.0mol.L^-1, and influence of concentration of Ca^2＋ is greater than that of Na^＋, and K^＋ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were： AOT reversed micelles 0.05mol·L^-1, trypsin concentration in crude pancreatin solution 3mg·ml^-1, pH 5.2-- 5.3, ratio （by volume） of extraction phase to strip-extraction phase 1 ： 1, and time of 5min. The corresponding percentage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginlne ethyl ester （BAEE） U·mg^-1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol·L^-1 ceryl-trimethyl-ammonium bromide （CTAB）, total extraction percentage of trypsin was 74.18% and specific activity was 3148.3 BAEE U·mg^-1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sulfate-polyacryl amide gel electrophoresis （SDS-PAGE） and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.

LIQUID-LIQUID EXTRACTION OF BSA BY USING CTAB-HEXANOL-OCTANE REVERSED MICELLAR SYSTEM

1 INTRODUCTIONIn biotechnology there is a need for new protein recovery process,which combines a highselectivity for the desired product with substantial concentration increased and easy to scale-up.In this context,liquid-liquid extraction with reversed micellar phase might serve this purpose.Reversed micelles are aggregates of surfactant molecules containing an inner core of water mole-cules,dispersed in a continuous organic solvent medium.These systems are opticallytransparent and thermodynamically stable.It has beendemonstrated [1,2]that under certainconditions proteins can be transferred from an aqueous phase towards a reversed micellarphase or vice versa(Fig.1)

Factors Affecting Trypsin Extraction by AOT Reversed Micelles and Observation by STM

In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning tunneling microscope(STM) was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic so- dium (AOT)/ iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of ex- traction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol·L-1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca2+, Na+, K+ which were in the range of 0.2—1.0mol·L-1, and influence of concentration of Ca2+ is greater than that of Na+, and K+ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were: AOT reversed micelles 0.05mol·L-1, trypsin concentration in crude pancreatin solution 3mg·ml-1, pH 5.2— 5.3, ratio (by volume)of extraction phase to strip-extraction phase 1︰1, and time of 5min. The corresponding per- centage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginine ethyl ester (BAEE) U·mg -1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol·L -1 ceryl-trimethyl-ammonium bromide (CTAB), total extraction percentage of trypsin was 74.18% and specific activ- ity was 3148.3 BAEE U·mg-1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sul- fate-polyacryl amide gel electrophoresis (SDS-PAGE) and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.

Mechanisms of Cytochrome C Extraction by Reverse Micelles

The extraction of cytochrome C was carried out by means of phase transfer technique with three different reverse micellar systems, i.e. , a CTAB micellar solution in n butyl alcohol chloroform(volume ratio 4∶1), an AOT micellar solution in isooctane and a SDSS D 2EHPA micellar solution in isooctane. The extraction mechanisms were studied. The results show that the extraction mechanisms for the same proteins with different types of reverse micellar systems can be distinct. The extraction of cytochrome C with CTAB and SDSS D 2EHPA reverse micellar systems are carried out according to the mechanism of electrostatic interaction. However, in the extraction of cytochrome C with the AOT reverse micellar system, the electrostatic interaction between the protein and the surfactant is not important.

Preparation of Highly Concentrated Silver Nanoparticles in Reverse Micelles of Sucrose Fatty Acid Esters through Solid-Liquid Extraction Method

Silver nanoparticles were synthesized in reverse micelles consisting of sucrose fatty acid esters by dissolving reactant powder in the water pool of reverse micelles through the solid-liquid extraction. Silver nanoparticles having various sizes and shapes were obtained at high concentration. The size of silver nanoparticles was controlled by reaction temperature. Moreover, the size of silver nanoparticles was dependent upon the average esterification degree of sucrose fatty acid esters forming reverse micelles. The wavelength in the peaks, which corresponded upon the localized surface plasmon resonance of resultant silver nanoparticles, was correlated with their sizes.

Size Changes of Reverse Micelles after Extraction of Peanut Protein and Their Forward Extraction Rates

The aim of this study was to detect the size changes of reverse micelles after extraction of peanut protein and their forward extraction rates. Factors that affect the size of reverse micelles and the extraction of peanut protein were also investigated. The size of reverse micelles and the size changes were measured according to the theory of dynamic light scattering under different conditions such as different sodium bis(2-ethylhexyl) sulfosuccinate(AOT) concentrations, p H values, ion concentrations, and salt species.With the increase of AOT surfactant concentrations in a certain range, the size of empty and full reverse micelles increased and the forward extraction rate decreased. The effect of pH on empty reverse micelles was not significant. However, the effect of pH on the full reverse micelle size and forward extraction rate were significant. Its forward extraction rate increased to the maximum39.6% at pH 7.5. The increase of the salt concentration of a buffer solution in a certain range decreased the size of empty and full reverse micelles and reduced the forward extraction rate of peanut protein. Ionic species had important effects on reverse micelles and peanut protein extraction. An increase in the amount of buffer solution enlarged the empty reverse micelle size in 0.03%-0.11%(V/V). However, it did not translate to a larger reverse micelle size. The size of the empty reverse micelles containing K_2SO_2 reached 24.1 nm with a 0.19%(V/V) buffer solution added. The sizes of the full reverse micelles were larger than those of the empty reverse micelles after forward extraction. However, maximum sizes were achieved with the addition of a 0.03%(V/V) buffer solution. The amount of 0.03%(V/V) buffer solution added was appropriate for extracting peanut protein.

Application of Reverse Micelles of Alkyl Glucosides to Synthesis of Silver Nanoparticles

Silver nanoparticles were prepared in reverse micelles of alkyl glucosides by the injection method. The TEM image showed that the obtained silver nanoparticles displayed a wide variety of shapes. The size of silver nanoparticles was strongly dependent upon the kind of reducing agents, and tended to slightly increase with an increase in reaction temperature. The size of silver nanoparticles was hardly dependent upon the molar ratio of water to surfactant and the alkyl chain length of surfactants. Silver nanoparticles having various shapes were obtained at high concentration by the solid-liquid extraction method. The resultant silver colloid could be preserved for at least one month without precipitation.

Phase separation in solvent extraction of cobalt from acidic sulfate solution using synergistic mixture containing dinonylnaphthalene sulfonic acid and 2-ethylhexyl 4-pyridinecarboxylate ester

Phase separation rate is a critical character in determining the usefulness of a solvent extraction system in hydrometallurgy. A survey of the synergistic mixture containing dinonylnaphthalene sulfonic acid (HDNNS) and 2-ethylhexly 4-pyridinecarboxylate ester (4PC) for the extraction of cobalt from acidic single metal sulfate solution was carried out to suggest how the physicochemical properties and the morphology of the reverse micelles in the loaded organic phase affect the phase separation. The results show that effective parameters affecting the phase separation are the viscosity and the excess water uptake of the loaded organic phase. It is obvious that the specific settling rate (SSR) decreases with the apparent increase of these two parameters. The measurement of small angle X-ray scattering (SAXS) proves that the morphology of the reversed micelles in the loaded organic phase changes evidently with the change of the specific settling rate (SSR).

Determination of Teicoplanin A2's Partition Coefficient in Different Liquid-Liquid Extraction Systems

Teicoplanin is one of the macrocyclic glycopeptide antibiotics, which is active against Gram-positive bacteria, and has attracted a lot of attention in the field of chiral separation recently. In this work, the partition coefficients and extraction ratio of teicoplanin in three different solvent systems were studied: conventional extraction, reactive extraction and reverse micelle extraction. With conventional solvent extraction, n-butanol demonstrated high partition coefficient for teicoplanin, but low extraction ratio because of its high solubility in water. Reactive extraction of teicoplanin showed the highest partition coefficient with almost 100% recovery in organic phase when tri-n-octylmethyl ammonium chloride (TOMAC) was used as extractant and pH value was above 5.0. A reverse micelle system, consisted of isooctane +10mmol-L-1 TOMAC +1% n-octanol, also offered high separation factor for teicoplanin. The results are beneficial for the design of teicoplanin separation and purification process.

DTAB反胶束萃取花椒籽中蛋白质和油脂研究

利用十二烷基三甲基溴化铵(dodecyl trimethyl ammonium bromide,DTAB)-异辛烷-正己醇反胶束体系,对花椒籽中的蛋白质和油脂同步萃取进行了探究。结果表明,前萃过程油脂直接溶解于有机相中,蛋白质增溶于反胶束极性核中,蛋白质的前萃率受DTAB浓度、萃取时间、缓冲溶液pH值、氯化钠浓度、料液比的影响。随后,反胶束溶液通过加入等体积含有一定离子强度和pH值的水相构筑WinsorⅡ微乳体系来实现蛋白质转移。其蛋白质后萃率和油脂萃取率受后萃水相的酸度和离子强度等因素的影响。

Reverse micelles extraction ofnattokinase:From model system to real system

Nattokinase is a novel fibrinolytic en- zyme, which is homologous to Subtilisin Carlsberg. In this paper, Subtilisin Carlsberg was taken as a model protein of nattokinase. Effects of pH, ionic strength, concentration of isopropanol on the extraction of Subtilisin Carlsberg with AOT/isooctane reverse mi- celles system were investigated. Further, the process of reverse micelles extraction of nattokinase from fermentation broth was studied. By taking the reverse micelles of AOT/isooctane as extractant to perform a full extraction cycle, it was found that about eighty percent of the total activity of nattokinase in the fer- mentation broth could be recovered and the purifica- tion factor was about 2.5. Homologous protein could be reasonably used as model protein of a target protein.

沙林在CTAB逆胶束介质中的化学发光分析

以 CTAB在氯仿 /环已烷为主体溶剂相中所形成的逆胶束介质 ,基于鲁米诺 - H2 O2 化学发光体系对沙林进行定量分析 ;研究了氯仿与环已烷的不同配比、R值、CTAB浓度、p H、鲁米诺浓度及 H2 O2 浓度对化学发光强度的影响。沙林的检测线性范围为 1× 1 0 -6～ 1× 1 0 -2 mol/L,检出限为 1 .5× 1 0 -7mol/L ,对水样及土样测定的回收率均在 90

湿法磷酸萃取技术发展现状与研究进展

磷酸作为一种重要的化工原料,在化工业中占有极其重要的地位.磷酸的应用主要由磷酸的纯度决定,低纯度磷酸可用于工业和农业领域,而高纯度磷酸则可用于医药、食品和电子等行业.我国磷矿以低品位磷矿为主,生产磷酸主要采用湿法工艺.相比热法生产,湿法工艺更加清洁环保,但产品杂质含量多、种类复杂,故发展磷酸净化技术尤为重要.本文从湿法磷酸纯化技术中的萃取法出发,综述了萃取法的主要研究进展.重点介绍了溶剂萃取法、双水相萃取法、反胶团萃取法、超声协助萃取法和超临界流体萃取法的基本原理和发展趋势.分析了不同萃取方法的优缺点、分离效果和适用条件.突出介绍了溶剂萃取法,梳理了磷酸除杂的主要萃取剂,特别强调了复合萃取剂和新型萃取剂在磷酸纯化方面的显著优势,最后,对磷酸的萃取技术做出了前景展望.

CTAB-己醇-辛烷体系分离纯化醇脱氢酶的反萃研究

本文报道了用反胶束体系萃取醇脱氢酶(ADH)的研究结果。在此萃取体系中,以十六烷基三甲基溴化铵(CTAB)作为表面活性剂,辛烷和己醇作为溶剂和助溶剂。考察了表面活性剂及助溶剂浓度,水相pH值对ADH萃取的影响。详细讨论了离子强度、异丙醇浓度和振荡时间对ADH反萃的影响。确定了最佳反向萃取条件。

超临界CO_2下AOT/CTAB混合反胶团体系萃取乳酸钙中蛋白质的研究

研究了超临界CO2下AOT/CTAB混合反胶团体系萃取乳酸钙中影响产品品质的杂蛋白质。并且分析了萃取压力P、温度T、AOT用量VAOT及AOT/CTAB摩尔混合比nAOT/nCTAB、水量VH2O、pH值等各因素对于该体系中蛋白质的萃取率的影响。通过正交试验得出最佳操作条件:P=25MPa,T=45℃,VAOT=0.15mL.(g乳酸钙)-1,nAOT/nCTAB=300:1,VH2O=1.6mL水.(g乳酸钙)-1,pH=4。在此操作条件下,蛋白质的去除率可达70%。

CTAB混合反胶团萃取工业脂肪酶

研究了CTAB与Tween、含氧有机物形成的混合反胶团对工业脂肪酶进行萃取分离的效果.实验表明,CTAB-Tween85和CTAB?含氧有机物混合反胶团的萃取率高于单一CTAB反胶团;反萃时CTAB-含氧有机物混合反胶团的反萃率与单一CTAB反胶团的反萃率相似,CTAB-Tween60和Tween40混合反胶团的反萃效果优于单一CTAB反胶团.通过测定反萃水相的酶活,发现CTAB?TRPO混合反胶团的效果最好,酶活回收率最高,可以达到70%.

CTAB反微团萃取大豆蛋白

本文考察了CTAB-反微团从水溶液中萃取大豆蛋白质的影响因素,因素包括:萃取时间、离子强度、有机相中表面活性剂(CTAB)浓度、.助溶剂配比、水相pH等,并提出了最佳的萃取条件。最后从反微团微观结构给予解释。

柠檬籽中类柠檬苦素反胶团萃取工艺优化及抑菌活性分析

目的 探究羟丙基甲基纤维素(hydroxypropyl methyl cellulose, HPMC)辅助反胶团萃取柠檬籽中类柠檬苦素的可行性,并对提取物质的抑菌活性进行分析。方法 以初提液中类柠檬苦素含量为响应值,通过正交试验确定初提液制备的最优参数;以Placktett-Burman(P-B)试验筛选影响类柠檬苦素反胶团萃取效率的主要因素,并结合Box-Behnken(B-B)试验确定最优工艺条件;通过测定最小抑菌浓度(minimal inhibitory concentration,MIC)、最小杀菌浓度(minimum bactericidal concentration, MBC)、碱性磷酸酶(alkaline phosphatase, AKP)活性考查类柠檬苦素对青霉菌和绿霉菌的抑菌活性。结果 初提阶段影响类柠檬苦素提取效率的最优条件为提取时间1.5 h、液料比30:1(mL/g)、HPMC添加量0.5%、pH 6.5,该条件下提取率为(3.34±0.02) mg/g。P-B试验结合B-B试验结果表明,类柠檬苦素萃取的最优配方为双十八烷基二甲基氯化铵(dioctadecyl dimethyl ammonium chloride,DODMAC)44.29mmol/L、正己醇13.86%、溴代十四烷3.88%,校正工艺条件下类柠檬苦素萃取率达(87.64±1.12)%。抑菌试验表明,类柠檬苦素对绿霉菌的MIC和MBC分别为1250.00μg/mL、2500.00μg/mL,对青霉菌的MIC和MBC分别为1250.00μg/m L、5000.00μg/mL;通过测定AKP活力发现,当加入抑菌活性物质后,随着药物作用时间延长及药物纯度的提高,AKP活性逐渐增加。结论 通过HPMC辅助反胶团能有效提取柠檬籽中的类柠檬苦素,提取条件温和、提取效率高,该方法具有一定应用前景。采用该方法提取的类柠檬苦素对绿霉菌和青霉菌具有明显的抑制作用,其可通过破坏细胞壁扰乱细胞的正常生理活动,阻碍其正常生长。

MnOOH纳米棒的反胶束法制备及表征

采用水溶液 /CTAB/正丁醇 /庚烷四组分组成的反相胶束体系 ,制备了MnOOH纳米棒 ,并用透射电镜 (TEM)、高分辨透射电镜 (HRTEM)和X射线粉末衍射 (XRD)进行了表征 .结果显示所得MnOOH为单斜晶系 ,直径约为 10nm、长度约为 2 0 0nm .实验表明 ,不同的反应时间所得纳米棒的长度不同 ,但直径可以保持不变 .

反胶束体系中青霉素酰化酶活性及稳定性研究

反胶束体系是一种新型的酶反应介质体系。构建合适的反应体系,使青霉素酰化酶在该体系中保持较高的酶活性,是实现反胶束体系中酶法合成头孢类抗生素的关键所在。本文分别考察了青霉素酰化酶在CTAB/正己醇和CTAB/异辛烷/正己醇两种反胶束体系中不同水含量和pH值对酶活力的影响。结果表明:包埋于CTAB/异辛烷/正己醇反胶束体系中的青霉素酰化酶在W0为14、pH为7.2时表现出较高的酶活性。试验中还考察了不同温度对包埋于CTAB/异辛烷/正己醇反胶束体系中的青霉素酰化酶活力的影响,并与游离酶液进行了比较。该酶在所采用的反胶束体系中的最适水解反应温度为39℃,较游离酶液的最适水解温度升高了2℃。同时酶的热稳定性亦较游离酶液有所提高,表现在CTAB/异辛烷/正己醇反胶束体系中,经50℃保温240 min,酶活保留率为76.0%, 而在同样条件下游离酶液仅为36.5%。