反胶束中单宁酶的光学行为和稳定性

为了测定反胶束系统中单宁酶的光学行为和增溶方式,采用紫外分光光度法和荧光扫描技术对AOT水／异辛烷组成的反胶束体系中单宁酶和水相中单宁酶的光学行为进行研究,同时研究了不同反应体系中单宁酶的稳定性,并对单宁酶在反胶束体系中的增溶方式进行探讨。结果表明：反胶束体系与水相中的单宁酶,其光学行为存在很大差别。反胶束体系有利于单宁酶的稳定,脂肪醇作为反应底物,其碳链的增长有利于单宁酶在反胶束中的稳定性。单宁酶是以嵌入反胶束膜或与反胶束内膜接触的方式增溶的。     

超氧化物歧化酶在反胶束体系中稳定性的研究

目的：考察反胶束体系组成对超氧化物歧化酶(SOD)抗氧化活性的影响。方法：以大豆磷脂氧化产生的氧自由基量为检测指标,考察温度、有机试剂以及表面活性剂浓度对反胶束体系中超氧化物歧化酶抗氧化活性的影响。结果：60℃时,反胶束中超氧化物歧化酶的抑制率由在水溶液中的52．48%升到75．38%,即SOD包封于反胶束中后热稳定性明显提高。表面活性剂可有效阻止有机溶剂对SOD的变性作用,SOD是不具界面活性的酶。结论：超氧化物歧化酶在反胶束体系中稳定性及抗氧化活性增强。     

微乳液凝胶及其固定化脂肪酶研究进展

利用微乳液胶凝现象来固定化酶是90年代初建立起来的酶固定化新技术,该技术为胶束酶学在生物合成与转化领域的应用奠定了基础.就微乳液凝胶及其固定化脂肪酶的制备、性质、微观结构及其潜在应用作了带有知识介绍性质的综述.     

用乙醇消除AOT/异辛烷反胶束体系萃取过程中蛋白质的变性失活

用反胶束技术分离纯化蛋白质,具有高选择性、易于大规模操作等优点,具有良好的工业应用前景。但是离子型表面活性剂形成的反胶束体系萃取蛋白质容易引起蛋白质的变性,这是由于离子型表面活性剂的强电荷作用会导致蛋白质发生变性,从而在两相界面上产生沉淀。这也是离子型反胶束体系用于蛋白质萃取所存在的最大的困难。本文对用AOT/异辛烷反胶束体系从胰酶粗提物中萃取胰蛋白酶进行了研究,通过在反胶束相加入乙醇,解决了反胶束萃取蛋白质时使蛋白质变性失活的问题,并且大大减少了分相的时间。前萃取和反萃取之后的分相时间只需要10分钟左右,简化了实验步骤,优化了实验方法,在工业上的大规模应用成为可能。在本研究中,胰蛋白酶的前萃取率达到90%,反萃取率接近100%。最终得率为88%。纯化后的比活提高了5倍多,从300U/mg左右提高到了1800U/mg。     

用反胶束萃取法提取与分离蛋白质和酶

本文介绍了反胶束萃取的概念、体系、萃取原理及分配影响因素,综述了这一技术在蛋白质和酶的分离与纯化中的应用。     

热力学体系的物理学特性与酶学机制的热力学原理

本文总结讨论了复杂热力学体系的特点与原理.在此基础上,修改了阿累尼乌斯公式,修改后酶学反应速率公式包含蛋白质构象变化的热力学参数.因而可以在理论上分析与确立蛋白质构象变化与酶活性之间的关系.在本理论体系中,酶的调节与酶的作用机制是蛋白质运动的不同体现,二者的基本原理是一致的.可以证明,酶催化过程中活化能的降低来源于酶与底物的结合能.理论表明基因互作现象是生物信息的热力学整合过程.综上所述,蛋白质热力学是分析任何生物功能与过程的基础与原理.     

AOT/异辛烷反胶束萃取纯化胰蛋白酶及酶变性失活问题的解决

用反胶束技术分离纯化蛋白质,具有高选择性、易于大规模操作等优点,具有良好的工业应用前景。但是离子型表面活性剂形成的反胶束体系萃取蛋白质容易引起蛋白质的变性,这是由于离子型表面活性剂的强电荷作用所导致的。对用AOT/异辛烷反胶束体系从胰酶粗提物中萃取胰蛋白酶进行了研究,通过在反胶束相加入乙醇,解决了反胶束萃取蛋白质时蛋白质变性失活的问题。并且由于乙醇的加入大大减少了分相的时间,简化了实验步骤,优化了实验方法,使此技术在工业上的大规模应用成为可能。通过优化各种实验条件,胰蛋白酶的前萃取率达到90%,反萃取率接近100%。最终得率为88%。纯化后的比活提高了5倍多,从300U/mg左右提高到了1800U/mg。     

非离子反胶束中木素过氧化物酶催化性能研究

木素过氧化物酶（LiP）在环己烷/Brij30/水反胶束体系中可体现催化活力,然而在水/醇/TritonX100/环己烷反胶束体系中却没有催化活力。对影响Brij30反胶束中LiP催化活力各主要因素进行了优化并测定了LiP在其中的时间稳定性;结果表明,20℃下,使LiP体现最佳活力的Brij30反胶束介质条件为：ω₀＝8.5,pH＝2.2,［Brij30］＝600mmol/L;在此条件下,LiP的半衰期可达到50h;与水介质相比,酶活力下降了,但稳定性却提高了。直链醇是TritonX100形成反胶束的必要组分,为揭示醇的作用,还考察了戊醇对Brij30 反胶束中LiP催化活力的影响,发现高浓度戊醇对LiP有失活作用。据此推测助表面活性剂醇可能是LiP在环己烷/TritonX100/戊醇/水反胶束中不能体现催化活力的主要原因。     

反胶束中单宁酶催化没食子酸烷基酯的生物合成

为了研究单宁酶在有机相中的催化性能,建立了AOT/异辛烷／水反胶束单宁酶催化没食子酸与脂肪醇酯合成反应体系。结果显示：反胶束单宁酶催化体系可成功催化合成C3-C5脂肪醇与没食子酸的酯合成反应。不同反应体系中由于不同脂肪醇的存在,单宁酶的动力学参数和紫外光谱存在差别。结果表明单宁酶对脂肪醇的专一性不强,根据Vmax/Km比值,丁醇与异丁醇是其最适底物,单宁酶催化没食子酸烷基酯合成的动力学符合米氏方程。反应体系中不同的脂肪醇导致了单宁酶构象的差别。     

辣根过氧化物酶在水相胶束中的动力学

研究了辣根过氧化物酶在三种表面活性剂（ＳＤＳ,ＴｒｉｔｏｎＸ－１００及ＣＴＡＢ）的水相胶束中催化联苯胺聚合反应的动力学。结果表明水相胶束体系有利于反应的进行。辣根过氧化物酶在水相胶束体系中遵循米氏反应,Ｋ_ｍ在ＳＤＳ、ＴｒｉｔｏｎＸ－１００及ＣＴＡＢ三种体系中分别为３．０１４×１０~（－４）ｍｏｌ／Ｌ、１．７２８×１０~（－４）ｍｏｌ／Ｌ和５．６６４×１０~（－５）ｍｏｌ／Ｌ。由于微环境的不同,ＨＲＰ在三种体系中表现出不同的最适反应温度和最适ｐＨ。     

Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles

The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent media, respectively. The influences of the molar ratio of water to surfactant omega0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show "superactivity" in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of omega0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at omega0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.     

Reverse micelles as life-mimicking systems

In this review, we attempt to demonstrate that reverse micelles are simple artificial systems that mimic many life systems from cell division to the creation of an enzyme catalytic mechanism. For a membranous enzyme like placental alkaline phosphatase, the kinetic properties observed in reverse micelles might represent those found under physiological conditions. The reverse micellar system, consisting of a positively charged surfactant, mimics a detoxification enzyme glutathione transferase. We propose a novel island-in-oil-lake reverse micellar model for the glutathione transferase that can account for almost all the catalytic properties of this enzyme. Reverse micelles may provide an excellent model system in investigating the reaction mechanism of other detoxification enzymes.     

Solubilization of enzymes and nucleic acids in hydrocarbon micellar solutions

The present state of the field of biopolymers solubilized in apolar solvents via reverse micelles is reviewed. First, an extensive discussion of the physical and chemical properties of reverse micelles is presented. Particular attention is devoted to the nature of water in the water pools of reverse micelles; to the structure and shape of the micellar aggregates; and to the dynamic properties of the reverse micelles. In the second part of the paper, the mechanism of solubilization of proteins and nucleic acids in hydrocarbon reverse micelles is discussed. Spectroscopic data, mostly circular dichroism and fluorescence, are reviewed in order to clarify the conformational changes which the biopolymers undergo upon their uptake into the micellar environment and determine the location of the biopolymers inside the reverse micelles. Data from neutron scattering, light scattering, ultracentrifugation, and electron microscopy of the protein-containing micelles are reviewed and discussed with the aim of illustrating the structure of the micellar aggregates containing the biopolymer as guest molecules. The activity of enzymes and nucleic acids is discussed, with emphasis on the influence upon the chemical reactivity brought about by the micellar parameters. Finally, a brief review of the applications and potentialities of biopolymer-containing reverse micelles is presented.     

Trypsin-SBTI interaction in reverse micelles. A slow intermicellar exchange-dependent binding

Solubilisate exchange between reverse micelles must take place before any reaction inside reverse micelles occurs if the reactants are confined to the aqueous micellar core. When the interacting species are 2 small molecules or one small molecule and one macromolecule, it has been shown that the exchange is faster than the typical turnover of an enzymatic reaction. The study of the interaction between 2 macromolecules (trypsin and soybean trypsin inhibitor) in reverse micelles carried out in this work reveals that the exchange between these macromolecule-containing reverse micelles slows down by a thousand times and the limiting-step in the exchange, the fusion, by 10(6) times. Both reverse micellar size (omega 0 = [water]/[surfactant]) and temperature affected the rate of the fusion process. A hypothesis for the proposed active role of macromolecules in the exchange process is also given.     

Stability and Enzymatic Studies of Glucose Dehydrogenase from the Archaeon Haloferax mediterranei in reverse micelles

Reverse micelles were used as a cytoplasmic model to study the kinetics of an extreme halophilic enzyme such as the recombinant glucose dehydrogenase from the Archaeon Haloferax mediterranei. This enzyme was solubilized in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as co-surfactant. Glucose dehydrogenase retained its catalytic properties in this organic medium, showing good stability at low water content, even at low salt concentration (125 mM NaCl). The dependence of the enzymatic activity on the molar water surfactant ratio (w₀=[H₂O]/[surfactant]) increased with rising water content. Surprisingly, the activity of this extreme halophilic enzyme did not depend on the salt concentration in reverse micelles. The kinetic of the enzymatic oxidation of β-D-glucose to D-glucono-1,5-lactone using NADP⁺ as coenzyme for the glucose dehydrogenase from Haloferax mediterranei was also studied in the reverse micellar system.     

Reverse micelles as reaction media for lipases

Reversed micelles are at the present time faced as common organic media to perform biocatalysis. They have been associated to the idea of a microreactor where the enzyme can be sheltered and protected from solvent detrimental effects. This simplistic idea led some investigators to ignore some basic understanding, such as the recognition of the enzyme-specific microenvironment and what the enzyme experiences inside the reversed micelle. To date the number of reactions catalyzed by lipases in reversed micelles is large. This review aims to highlight some of the fundamental aspects of the lipase microencapsulation as well as to resume the outstanding progress of the reversed micellar systems. The properties of the micellar microenvironment are reviewed and related to the lipases' performance both in terms of activity and stability. The heterogeneity of reversed micellar systems is discussed in relation to component distribution models and also to enzymatic kinetics. The new trends and the practical aspects where efforts should be centralized in order to spread out the micellar bioreactor technology over industrial processes are also discussed.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30-40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Chirality of reverse micelles

Four chiral analogues of the surfactant Aerosol-OT (AOT) have been synthesized and characterized. All of them form reverse micelles in apolar solvents in the w₀ range 0–30 (w₀ = [water]/[tenside]). Reverse micellar solutions have been investigated by UV absorption and circular dichroism spectroscopies with the aim of clarifying whether the formation of the macromolecular micellar structure induces the appearance of new chromophoric bands or perturbs the existing ones. Methanolic solutions of the surfactants, in which no micellar aggregates are formed, were taken as references. One of the products 1(S),1′(S)-dimethylbisheptylsulphosuccinate sodium salt (MH-AOT) was capable of forming reverse micelles of relatively high water content (w₀ up to 40) and this process was accompanied by a specific increase in the intensity of the circular dichroism band associated with the ester absorbance of the molecule. As no concomitant changes were seen in the UV absorbance spectrum, it was concluded that this observation reflected conformational events occurring within the surfactant rather than chromophoric perturbation. These results are qualitatively similar to those found recently for lecithin reverse micelles which, however, form gels at sufficiently high water contents. The chiroptical properties of these supramolecular aggregates are compared with those of covalent macromolecular systems such as polypeptides.     

Activity and conformational changes of alpha-chymotrypsin in reverse micelles studied by spin labeling.

alpha-Chymotrypsin (CT), spin-labeled at the active site by using an acylating label which constitutes a substrate for this protein, has been investigated in reverse micelles formed by AOT in isooctane. The electron spin resonance spectra provided information on conformation, dynamics and deacylation activity. The dynamics of the label bound to CT appears to be more hindered in reverse micelles than in aqueous solution, probably owing to the effect of the micellar environment on protein conformation. The deacylation rate in reverse micelles does not show the characteristic bell-shaped dependence on water content which is generally found for CT enzymatic activity.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.