表面活性剂和Ca~(++)对大麦根质膜透性的作用

阳离子型表面活性剂(CTMA)以低于CMC的浓度、非离子型表面活性剂(Triton-X-100)以高于CMC的浓度引起大麦离体根K~+及可溶性糖的外流,并有浓度效应。5℃时表面活性剂引起溶质外流,CTMA预处理10 min后,Ca~(++)无抑制作用。Ca~(++)与 Triton-X-100同时处理大麦根促进溶质外流。Mg~(++)、Mn~(++)对CTMA及Triton-X-100引起溶质外流的效应与Ca~(++)的相类似,但不如Ca~(++)有效。     

驱油生物表面活性剂生产菌的筛选与评价

目的:筛选适合油田的生物表面活性剂生产菌。方法:通过发酵培养,研究生物表面活性剂生产菌生长代谢规律;采用正交试验法,优选出其最佳培养条件;通过室内驱油实验评价生物表面活性剂驱油效果。结果:2#菌株最佳培养时间为96小时,最优发酵培养条件为:葡萄糖4.0 g、玉米浆1.6 g、Na2HPO40.1 g、KH2PO40.05 g、MgSO40.05 g、CaCl20.005 g、水100 mL、pH 7.2,培养温度35℃,摇床转速120 r/min,生物表面活性剂驱油提高采收率6.16%。结论:筛选出最优生物表面活性剂产生菌2#,菌株具备产表面活性剂的能力且产物量较高,其生物表面活性剂驱油效果良好。     

一株铜绿假单胞菌及其产生的鼠李糖脂特性研究

研究高效代谢表面活性剂的菌种,并对菌种和表面活性剂的理化性质进行分析。采用菌种16S rDNA序列分析对菌种进行鉴定,采用紫外光谱扫描、色谱柱层析、薄层层析、单糖分析对生物表面活性剂的种类进行鉴定,并对表面活性剂的理化性质进行研究。从油田采出水中筛选出一株高效代谢生物表面活性剂的菌株T-1,经16S rDNA鉴定为铜绿假单胞菌属(Pseudomonas aeruginosa)。该菌种以甘油和大豆油为碳源的培养基培养4 d后,其发酵液表面张力30.216 mN/m,EI24为100%。根据表面活性剂的红外光谱分析并结合薄层分析,可确定T-1样品中含有糖脂类物质,进一步的表面活性剂的单糖分析显示水解终产物为单一的鼠李糖,最终确定其产物为鼠李糖脂,苯酚-硫酸法测定其鼠李糖脂产率为5.2 g/L,从发酵液提取的棕黄色生物表面活性剂粗品,其表观临界胶束浓度为45 mg/L,该鼠李糖脂对环境(耐温、耐酸碱、耐盐)具有较强的适应性。该表面活性剂对恶劣环境具有较强的耐受性,可应用于微生物采油等用途。     

生物表面活性剂对红球菌SY095降解正十六烷及细胞表面性质的影响

目的:研究红球菌SY095产生物表面活性剂对正十六烷的溶解性、微生物降解正十六烷的效率、菌体生长及菌体表面疏水性的影响。方法:测定添加不同生物表面活性剂的降解体系中菌体生物量、细胞表面疏水性、正十六烷含量的变化。结果:生物表面活性剂对疏水性底物正十六烷具有很强的增溶作用,可以显著提高正十六烷的表观溶解度;生物表面活性剂对正十六烷的生物降解具有促进作用,添加量为100 mg/L时,96 h正十六烷去除率达93.32%;生物表面活性剂能明显促进红球菌SY095生长,添加量为300 mg/L时,菌株32 h生物量为未添加生物表面活性剂对照组的2.7倍;生物表面活性剂还能引起红球菌SY095菌体表面疏水性明显增大,添加量为25 mg/L时,菌株对数生长期BATH值达66.94%,高于未添加生物表面活性剂对照组的42.99%。结论:生物表面活性剂可以增加菌体的表面疏水性,促进微生物对正十六烷的生物降解。     

表面活性剂对D-阿拉伯糖醇发酵的影响

为提高D-阿拉伯糖醇的产量,研究不同类型表面活性剂对德巴利汉逊酵母(Debaryomyces hansenii)发酵生产D-阿拉伯糖醇的影响。结果表明:阳离子和阴离子表面活性剂对D-阿拉伯糖醇的生成几乎没有影响,部分非离子表面活性剂对D-阿拉伯糖醇的生产有促进作用,其中Trition X-100的影响最为显著。在不同发酵时间加入不同浓度的Trition X-100均对D-阿拉伯糖醇的生产有促进作用,当发酵24 h添加30 g/LTrition X-100时,D-阿拉伯糖醇的产量达到最高(92.9 g/L),相比于对照增加了27.2%。     

酶法合成表面活性剂

用生物技术来研制表面活性剂是当前国际生物工程领域中发展起来的一个新课题。本文概述了用酶法合成五种类型的表面活性剂,单酰化甘油酯类表面活性剂,糖酯类表面活性剂,氨基酸类表面活性剂,磷酯类表面活性剂及氨基酸糖类表面活性剂。     

表面活性剂对林可霉素发酵生产的影响

在林可霉素发酵过程中,当向培养基中加入表面活性剂十二烷基磺酸钠(SDS)、吐温80(Tween 80)和曲拉通(Triton X-100)时,林可霉素的产量受到较大影响。本研究应用响应面设计法(Response surface design)对表面活性剂的配比进行了优化,得到的优化配比为:十二烷基磺酸钠为31.13 mg/100 mL,吐温80为51.97 mg/100 mL,曲拉通为16.9 mg/100 mL。将该优化配比应用于林可霉素发酵,产量提高了36.67%。     

聚乙烯醇脱氢酶的提取及检测

为解决结合在细胞上的可溶性蛋白聚乙烯醇脱氢酶（PVADH）的检测困难问题,从提取及检测两方面对该酶进行研究,并对检测方法进行改进。结果表明,非离子型表面活性剂Triton X-100对可溶性蛋白PVADH的提取效果优于离子型表面活性剂炕基苯磺酸钠（LAS）和溴化十六烷基吡啶（CPB）,酶活力比LAS和CPB提取后所得酶活力分别提高246．5％和831．3％。而非离子型表面活性剂中,Triton X-100与Tween80相比,所得最高酶活提高了101．4％。Triton X—100浓度和提取时间对测定有明显影响,以1％Triton X-100提取18h为宜,最高比酶活达14．9U／g。在PVADH检测体系中,加入电子受体启动反应比加入酶液与底物启动反应可使酶活性分别提高60．6％和126．5％;酶液与吡咯喹啉醌（PQQ）预先保温对检测该酶活性是十分重要的,可使酶活性提高59．1％.在检测系统中加入的KCN、CaCl2和PQQ的适宜浓度分别为1．ommol／L、0．5mmol／L和2μmol／L,可使测定酶活分别提高37．1％、38．7％和214．0％．     

离子强度、温度及表面活性剂Triton X-100对紫膜质子泵效率的影响

用毫秒级闪光动力学光谱仪研究了离子强度、温度及表面活性剂Triton X-100对紫膜质子泵效率的影响。结果表明:紫膜溶液中适量离子的加入可使其质子泵效率(H~+/M_(412))显著提高(纯水中,H~+/M_(412)—0.56,而在150mM KCl中可达—1.3),高价离子的影响显然远大于低价离子;在5℃—50℃的较大温度范围内,紫膜的质子泵效率变化不大,但当温度升至60℃以上时,紫膜的质子泵效率迅速下降直至为零;非离子表面活性剂Triton X-100的加入对紫膜的质子泵效率影响不大,仅随Triton量的增加而略微下降。以上实验现象,直接或间接地说明了离子在紫膜质子泵功能中的重要作用。就此对离子及脂在紫膜质子泵中的作用机制进行了进一步的探讨。     

表面活性剂对出芽短梗霉多糖生产影响的研究

研究了表面活性剂对出芽短梗霉细胞培养过程中多糖释放的影响。在摇瓶中,比较添加0.05%(w/v)的Tween 80、Tween 60、Tween 40,结果显示几种表面活性剂均能促进细胞释放多糖,其中以Tween 80的效果最佳。在5L发酵罐中,以100g/L玉米粉水解液做碳源的出芽短梗霉细胞培养液中分别添加了表面活性剂Tween 80 0.01%、0.05%、0.1%,其中以添加Tween 800.05%时的效果最好,与不添加表面活性剂相比多糖产量提高25%左右,发酵周期缩短了将近2d。     

Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.     

Phospholipase A2 activity towards phosphatidylcholine in mixed micelles: surface dilution kinetics and the effect of thermotropic phase transitions

Phospholipase A₂ will act on dipalmitoyl phosphatidylcholine as substrate when the phospholipid is part of a mixed micelle with Triton X-100 at a molar ratio of Triton to phospholipid of 2:1 or greater. Kinetic studies at high molar ratios of Triton X-100 to phospholipid are reported and show that the binding of phospholipase A₂ to substrate depends on the total concentration of Triton X-100 and phospholipid, but that the rate of enzymatic catalysis decreases proportionally to the Triton X-100 concentration. These results are interpreted in terms of a model involving surface dilution kinetics. The relationship of this model to that of competitive inhibition is discussed. In addition, the activity of phospholipase A₂ towards dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine at different temperatures is reported, and the results show a direct effect of the thermotropic phase transition of dipalmitoyl phosphatidylcholine on enzymatic activity.     

Phospholipid binding and the activation of group IA secreted phospholipase A2

Equilibrium dialysis was used to study the binding of two nonhydrolyzable, short chain phospholipid analogues to the secreted group IA phospholipase A(2) (PLA(2)), which has been shown to contain several phospholipid binding sites that dramatically affect activity. This study provides new insight into how these activations occur. One analogue contained a phosphorylethanolamine (DiC(6)SNPE) headgroup, while the other contained a phosphorylcholine (DiC(6)SNPC) headgroup. Using phospholipase D, we incorporated tritium into each analogue. No binding of DiC(6)SNPE to PLA(2) was observed under submicellar conditions. Addition of submicellar amounts of Triton X-100 resulted in a linear nonsaturating response to lipid concentration, suggestive of premicellar aggregation of the DiC(6)SNPE with Triton X-100 and PLA(2). Binding of DiC(6)SNPE when presented as Triton X-100 mixed micelles saturated at 0.93 binding sites per PLA(2) with a K(D) of 38 microM. Addition of sphingomyelin, a potent activator of PLA(2) hydrolysis of phosphorylethanolamine containing compounds, resulted in a 13-fold decrease in the K(D), to 2.8 microM. This suggests that changes in the catalytic site binding affinity contribute to "phosphatidylcholine activation". Binding of DiC(6)SNPC with 2.0 mM Triton X-100 showed positive cooperativity (Hill coefficient of 1.7), which saturated at 2.0 binding sites per PLA(2). No binding of either analogue was observed when the catalytic site was alkylated with p-bromophenacyl bromide. Since p-bromophenacyl bromide does not physically block the phosphatidylcholine activator site, this indicates that the two phosphatidylcholine binding sites interact. The binding studies show that DiC(6)SNPC binds cooperatively to two sites on group IA PLA(2), while DiC(6)SNPE binds to only one site.     

Visualization and analysis of lipopolysaccharide distribution in binary phospholipid bilayers

Lipopolysaccharide (LPS) is an endotoxin released from the outer membrane of Gram-negative bacteria during infections. It have been reported that LPS may play a role in the outer membrane of bacteria similar to that of cholesterol in eukaryotic plasma membranes.In this article we compare the effect of introducing LPS or cholesterol in liposomes made of dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine on the solubilization process by Triton X-100. The results show that liposomes containing LPS or cholesterol are more resistant to solubilization by Triton X-100 than the binary phospholipid mixtures at 4 °C.The LPS distribution was analyzed on GUVs of DPPC:DOPC using FITC-LPS. Solid and liquid-crystalline domains were visualized labeling the GUVs with LAURDAN and GP images were acquired using a two-photon microscope. The images show a selective distribution of LPS in gel domains.Our results support the hypothesis that LPS could aggregate and concentrate selectively in biological membranes providing a mechanism to bring together several components of the LPS-sensing machinery.     

Role of phospholipid and protein-protein associations in activation and stabilization of soluble Ca2+-ATPase of sarcoplasmic reticulum

The effect of increasing concentrations of the nonionic detergent Triton X-100 on catalytic activity, stability, phospholipid content, and aggregational state of solubilized Ca2+ ion activated adenosinetriphosphatase (Ca2+-ATPase) of sarcoplasmic reticulum has been investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (w/v) inhibited ATP hydrolysis and p-nitrophenyl phosphate hydrolysis in parallel to the extent of 50% and 95%, respectively. Inactivation of p-nitrophenyl phosphate hydrolysis by preincubation in excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 degrees C was monophasic and first order at all concentrations of Triton X-100. The rate constant for inactivation increased sharply in the range 0.1-0.6% Triton X-100. At higher concentrations, the increase was less marked. Protein-protein associations of the solubilized ATPase were assessed by glutaraldehyde cross-linking and by ultracentrifugation in sucrose gradients. Both methods indicated a decrease in these associations in the 0.1-0.5% range. Cross-linking studies established that above 0.5% Triton X-100 the enzyme is greater than 90% monomeric. The amount of phospholipid associated with the ATPase, recovered from sucrose gradients, decreased from about 50 mol of phospholipid/mol of ATPase at 0.1% Triton X-100 to about 3 mol of phospholipid/mol of ATPase at 0.5% and higher concentrations. Monomeric ATPase and aggregated ATPase isolated from equilibrium mixtures of these components had similar phospholipid/protein ratios. The results indicated that with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations, and loss of phospholipid occur concurrently.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 31P- and 2H-NMR study on lecithins in liquid crystalline polyoxyethylene detergents

Phosphatidylcholines were incorporated into hexagonal liquid cyrstalline mixtures of the non-ionic detergents Triton X-100 and octaethyleneglycoldodecylether with D₂O. It is shown by nuclear magnetic resonance (NMR) that the phospholipids adopt the hexagonal liquid crystalline structure of the detergent host lattice. The anisotropic motion of the phospholipid headgroups seems to be unaffected, whereas the acyl chains are disordered. Increasing phospholipid concentration leads to separation of a lamellar phase. The lamellar structure is also preferred at elevated temperatures. Phosphatidylcholines with saturated acyl chains undergo a transition from the hexagonal liquid crystalline to an ordered lamellar state. The shape of the ³¹P-NMR signals suggests that pure gel phase phospholipid separates out. The headgroup region of this gel phase phospholipid becomes immobilized after a few weeks of storage below the transition temperature as judged from ³¹P-NMR. At the same time ²H-NMR exhibits a new signal from D₆₂O undergoing slow isotropic motion. This behavior bears resemblance to the formation of a coagel in fatty acid-water systems.     

The isolation and structure of membrane lipid rafts from rat brain

The action of detergents in the isolation of detergent-resistant membrane fractions from rat brain is reported. Triton X-100 treatment of whole rat brain homogenate at 4 degrees C produced detergent-resistant membranes with a density of 1.07g/ml compared with Brij96 where the density of the membrane was only 1.05g/ml. The DRM fractions isolated using Triton X-100 are considerably heavier than those isolated from homogenates treated with Brij96. The major polar lipid composition of DRMs derived from Brij96 treated homogenates have a higher proportion of aminophospholipids compared with choline phospholipids than Triton X-100 derived DRMs; this may indicate that DRMs from Brij96 treated homogenates are more closely related to the parent membrane in lipid composition. Solubilization by Triton X-100 at higher temperatures resulted in the appearance of a second detergent-resistant membrane fraction distinctly lighter in density than the membrane recovered at density 1.07g/ml. Analysis of phospholipid composition of the brain homogenate during detergent treatment for up to 30min at 37 degrees C showed a decreasing proportion of sphingomyelin. Treatment of homogenates at 37 degrees C appears to activate phospholipases/sphingomyelinases that may alter the lipid content of isolated DRMs. The presence of K+/Mg2+ with Brij96 treatment results in DRM fractions with significantly thicker bilayers and of larger vesicle diameter than DRMs isolated from either Triton X-100 or Brij96 treated homogenates in the absence of cations.     

Regulation of Plasma Membrane beta-Glucan Synthase from Red Beet Root by Phospholipids : Reactivation of Triton X-100 Extracted Glucan Synthase by Phospholipids

Extraction of red beet root plasma membranes with the detergent Triton X-100 at a level of 2.0% (weight/volume) resulted in the depletion of over 90% of total membrane phospholipid and the reduction of glucan synthase activity by 80 to 90%. Reconstitution of the delipidated Triton X-100, 100,000g fraction in the presence of phospholipids restored glucan synthase activity. The most effective phospholipid was phosphatidyl-ethanolamine, which restored 110 to 144% of the original activity at 0.5% (weight/volume). Glucan synthase in the phospholipid-reactivated Triton X-100-treated fraction was enriched 9-fold in specific activity relative to microsomal membranes but was unstable in digitonin. These results support the hypothesis that glucan synthase activity is regulated by its phospholipid environment.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.     

Studies on mixed micelles of triton X–100 and phosphatidylcholine using nuclear magnetic resonance techniques

The addition of the nonionic detergent Triton X-100 to aqueous phosphatidyl-choline dispersions converts the bilayer structures to mixed micellar structures containing Triton X-100. High-resolution nuclear magnetic resonance spectroscopy at 220 MHz was used to follow this conversion, and the general spectral characteristics of the mixed micelles are presented. The results are discussed in terms of the precise change in structure which occurs as Triton is mixed with the phospholipid bilayers, and it is concluded that, above a molar ratio of about 2:1 Triton to phospholipid, most or all of the phospholipid is in mixed micelles. The relevance of these results to the study of enzymes which require substrate in the form of micelles is discussed.