Adsorption and activity of Thermomyces lanuginosus lipase on hydrophobic and hydrophilic surfaces measured with dual polarization interferometry (DPI) and confocal microscopy

The adsorption and activity of Thermomyces lanuginosus lipase (TLL) was measured with dual polarization interferometry (DPI) and confocal microscopy at a hydrophilic and hydrophobic surface. In the adsorption isotherms, it was evident that TLL both had higher affinity for the hydrophobic surface and adsorbed to a higher adsorbed amount (1.90 mg/m²) compared to the hydrophilic surface (1.40–1.50 mg/m²). The thickness of the adsorbed layer was constant (3.5 nm) on both surfaces at an adsorbed amount >1.0 mg/m², but decreased on the hydrophilic surface at lower surface coverage, which might be explained by partially unfolding of the TLL structure. However, a linear dependence of the refractive index of the adsorbed layer on adsorbed amount of TLL on C18 surfaces indicated that the structure of TLL was similar at low and high surface coverage. The activity of adsorbed TLL was measured towards carboxyfluorescein diacetate (CFDA) in solution, which upon lipase activity formed a fluorescent product. The surface fluorescence intensity increase was measured in a confocal microscope as a function of time after lipase adsorption. It was evident that TLL was more active on the hydrophilic surface, which suggested that a larger fraction of adsorbed TLL molecules were oriented with the active site facing the solution compared to the hydrophobic surface. Moreover, most of the activity remained when the TLL surface coverage decreased. Earlier reports on TLL surface mobility on the same surfaces have found that the lateral diffusion was highest on hydrophilic surfaces and at low surface coverage of TLL. Hence, a high lateral mobility might lead to a longer exposure time of the active site towards solution, thereby increasing the activity against a water-soluble substrate.     

Adsorption and mobility of a lipase at a hydrophobic surface in the presence of surfactants

With the aim of being able to manipulate the processes involved in interfacial catalysis, we have studied the effects of a mixture of nonionic/anionic surfactants, C12E6/LAS (1:2 mol %), on the adsorption and surface mobility of a lipase obtained from Thermomyces lanuginosus (TLL). Surface plasmon resonance (SPR) and ellipsometry were used to analyze the competitive adsorption process between surfactants and TLL onto hydrophobic model surfaces intended to mimic an oily substrate for the lipase. We obtained the surface diffusion coefficient of a fluorescently labeled TLL variant on silica silanized with octadecyltrichlorosilane (OTS) by fluorescence recovery after photobleaching (FRAP) on a confocal laser scanning microscope. By means of ellipsometry we calibrated the fluorescence intensity with the surface density of the lipase. The TLL diffusion was measured at different surface densities of the enzyme and at two time intervals after coadsorption with different concentrations of C12E6/LAS. The surfactant concentrations were chosen to represent concentrations below the critical micelle concentration (CMC), in the CMC region, and above the CMC. The apparent TLL surface diffusion was extrapolated to infinite surface dilution, D0. We found that the presence of surfactants strongly modulated the surface mobility of TLL: with D(0) = 0.8 x 10(-11) cm2/s without surfactants and D0 = 13.1 x 10(-11) cm2/s with surfactants above the CMC. The increase in lipase mobility on passing the CMC was also accompanied by a 2-fold increase in the mobile fraction of TLL. SPR analysis revealed that surface bound TLL was displaced by C12E6/LAS in a concentration-dependent manner, suggesting that the observed increase in surface mobility imparts bulk-mediated diffusion and so-called rebinding of TLL to the surface. Our combined results on lipase/surfactant competitive adsorption and lipase surface mobility show how surfactants may play an important role in regulating interfacial catalysis from physiological digestion to technical applications such as detergency.     

Lipase surface diffusion studied by fluorescence recovery after photobleaching

We have analyzed surface diffusion properties of a variant of Thermomyces lanuginosa lipase (TLL) on hydrophilic silica and silica methylated with dichlorodimethylsilane (DDS) or octadecyltrichlorosilane (OTS). For this study a novel method for analysis of diffusion on solid surfaces was developed. The method is based on fluorescence recovery after photobleaching using confocal microscopy. When a rectangular area of the sample was photobleached, fluorescence recovery could be analyzed as one-dimensional diffusion, resulting in simplified mathematical expressions for fitting the data. The method was initially tested by measuring bovine serum albumin diffusion on glass, which led to a diffusion coefficient in good correspondence to earlier reports. For the analysis of TLL diffusion, ellipsometry data of TLL adsorption were used to calibrate fluorescence intensity to surface density of lipase, enabling measurements of the diffusion coefficient at different surface densities. The average diffusion coefficient was calculated in two time intervals after adsorption. Mobile fraction and diffusion coefficient were lowest on the OTS surface, when extrapolated to infinite surface dilution. Moreover, the diffusion rate decreased with time on the hydrophobic surfaces. Our observations can be explained by the surface dependence on the distribution of orientations and conformations of adsorbed TLL, where the transition from the closed to the catalytically active open and more hydrophobic structure is important.     

Tracking single lipase molecules on a trimyristin substrate surface using quantum dots

The mobility of single lipase molecules has been analyzed using single molecule tracking on a trimyristin substrate surface. This was achieved by conjugating lipases to quantum dots and imaging on spin-coated trimyristin surfaces by means of confocal laser scanning microscopy. Image series of single lipase molecules were collected, and the diffusion coefficient was quantified by analyzing the mean square displacement of the calculated trajectories. During no-flow conditions, the lipase diffusion coefficient was (8.0+/-5.0)x10(-10) cm2/s. The trajectories had a "bead on a string" appearance, with the lipase molecule restricted in certain regions of the surface and then migrating to another region where the restricted diffusion continued. This gave rise to clusters in the trajectories. When a flow was applied to the system, the total distance and average step length between the clusters increased, but the restricted diffusion in the cluster regions was unaffected. This can be explained by the lipase operating in two different modes on the surface. In the cluster regions, the lipase is likely oriented with the active site toward the surface and hydrolyzes the substrate. Between these regions, a diffusion process is proposed where the lipase is in contact with the surface but affected by the external flow.     

A comparison between dual polarization interferometry (DPI) and surface plasmon resonance (SPR) for protein adsorption studies

This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30–1.35 mg/m² at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.     

Single-step purification of different lipases from Staphylococcus warneri

Three different lipases from the extract crude of Staphylococcus warneri have been purified by specific lipase–lipase interactions using different lipases (TLL, RML, PFL, BTL2) covalently attached to a solid support as adsorption matrix. BTL2 immobilized on glyoxyl-DTT adsorbed selectivity only a 30 kDa lipase from the crude, which was desorbed by adding 0.1% triton X-100. Using glyoxyl-PFL as matrix, two new lipases (28 and 40 kDa) were adsorbed, and completely pure 40 kDa lipase was obtained after desorption using 0.01% triton, whereas 28 kDa lipase was desorbed after the incubation of the lipase matrix with 3% detergent. When using other matrixes as glyoxyl-TLL or glyoxyl-RML, different lipases were adsorbed. This methodology could be a very efficient and useful method to purify several lipases from crude extracts from different sources.     

Protein-surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS)

The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 microM C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.     

Lipid mobility controls the diffusion of small biopolymer adsorbates

Recent studies on the diffusion of adsorbed polymers such as DNA on supported lipid bilayers have suggested that such strongly adsorbed polymers can be treated similarly to a polymer "in" a 2D fluid, but this conjecture has not been experimentally verified. To test this hypothesis and also to gain a better understanding of polymer dynamics in two dimensions, we designed an experimental protocol-the lateral transport of a short, single-stranded DNA oligonucleotide adsorbed on a supported cationic lipid bilayer. Fluorescence recovery after photobleaching (FRAP) analysis reveals that the diffusivity of the adsorbed DNA quantitatively tracks that of the underlying lipid, even though the bilayer mobility changes by 2 orders of magnitude with changes in temperature. Interestingly, our results for short, extended, adsorbed biopolymers quantitatively track those for globular proteins in lipid bilayers. We thus conclude that short macromolecules that are strongly adsorbed on lipid bilayers can be treated similarly to macromolecules in the bilayer.     

SPONTANEOUS RECOVERY OF FLUORESCENCE BY PHOTOBLEACHED SURFACE-ADSORBED PROTEINS

Abstract-Fluorescence photobleaching of a carboxyfiuorescein-labeled protein (erythrocyte cytoskel-etal protein 4.1) immobilized on bare glass is found to be spontaneously reversible, provided that the sample is deoxygenated. After a short (hundredths of seconds) photobleaching laser flash, the subsequent fluorescence excited by a dim probe beam partly recovers on a long (tenths of second) time scale, even in the absence of chemical exchange or diffusion processes. Neither the fraction of the fluorescence that bleaches reversibly nor its recovery rate is a strong function of fluorophore surface concentration. At a fixed surface concentration, the reversibly photobleached fraction and its recovery rate decreases with increasing duration or intensity of the bleaching flash. On the other hand, nondeoxygenated air-equilibrated samples exhibit almost total irreversible bleaching on this time scale. Quantitative fluorescence microscopy experiments occasionally require deoxygenation to avoid photochemical crosslinking or photobleaching or to enhance the triplet state population. The observations presented here indicate that fluorescence recovery after photobleaching (FRAP) experiments performed under deoxygenated conditions for measuring diffusion or chemical kinetics should be interpreted with caution: fluorescence recoveries may be due to intrinsic photochemical processes rather than fluorophore mobility. The recovery effect appears too slow to be ascribed simply to a relaxation of a triplet state; other possible explanations are offered.     

Application of ion mobility spectrometry in study of surfactants adsorbed on different dish surfaces

Sodium lauryl sulfate (SLS) and sodium lauryl ether sulfate (SLES) are commonly used in many dishwashing liquids. These chemicals are adsorbed on the dish surface during the washing process and then transferred to food or drink in the cooking process. In this work, the adsorption of SLS and SLES on different dish surfaces in aqueous solution was studied. Stainless steel, copper, aluminum, Pyrex, Teflon and arcopal china ware were used in this study. The adsorbed chemical remained on the surface after rinsing was measured by thermal desorption using an ion mobility spectrometer as the detector. Although arcopal china ware showed the maximum amount of adsorption, and Pyrex and stainless steel dishes showed the minimum amount of residual chemical, the results showed that the amount of adsorbed chemicals on dish surfaces is less than 428 ng.cm^?2, which is well below the health risk dosage. The released SLS and SLES from dish surfaces into cold or hot water were also measured and compared for different dishes.     

Reversible Photobleaching of Fluorescein Conjugates in Air-Saturated Viscous Solutions: Singlet and Triplet State Quenching by Tryptophan

Abstract— Fluorescence recovery after photobleaching (FRAP) measurements on air-saturated aqueous solutions of fluorescein made viscous with glycerol or sucrose revealed a rapid component of fluorescence recovery with exponential time constants of 30-120 μs at viscosities of 15-300 cP. The rapid recovery process was not related to fluorophore translational diffusion and was insensitive to fluorophore concentration and the additive used to increase solution viscosity. At constant viscosity, the rate of reversible photobleaching recovery increased 2.5-fold in an O₂- vs N₂-saturated solution. The relative efficiency of reversible-to-irreversible photobleaching decreased with increasing photobleaching time and/or beam intensity. Reversible photobleaching was also detected for conjugates of fluorescein with dextrans and proteins in viscous media. In screening triplet state quenchers that might influence the reversible recovery, it was found that tryptophan enhanced the rate of reversible photobleaching recovery (two-fold increase at 8 m M ) and quenched the fluorescein singlet state (Stern-Volmer constant, 12 M ⁻¹). Analysis of fluorescein lifetimes and photobleaching parameters for a series of fluorescein-labeled proteins with different numbers of tryptophans were also carried out. The results provide evidence for an oxygen-dependent, reversible photobleaching mechanism for the fluorescein chromophore involving triplet state relaxation. The identification of reversible fluorescein photobleaching has important implications for FRAP measurements of rapid solute diffusion in biological systems.     

Surface modification of poly(oligoethylene oxide methacrylate) for resisting protein adsorption

We prepare poly(2-methoxyethyl-, 2-(2-methoxyethoxy)ethyl-, 2-[2-(2-methoxyethoxy)ethoxy]ethyl methacrylate) (p(nEOMA), n=1, 2, and 3) brushed surfaces with varying the polymer density by surface initiated polymerization. The amount of bovine serum albumin (BSA) adsorbed on the surfaces is investigated. The mobility of the polymer chain in the polymer/water interfaces and the structure of adsorbed water on the surfaces are characterized by Electron Spin Resonance (ESR) and transmission-Fourier transform infrared (FT-IR) spectroscopy, respectively. This work reports the relationship between these surface properties and albumin adsorption. As a result, the surface having both a high molecular mobility and bulk-like water found to be very effective in preventing albumin adsorption.     

In situ formation and characterization of poly(ethylene glycol)-supported lipid bilayers on gold surfaces

Inclusion of a polymer cushion between a lipid bilayer membrane and a solid surface has been suggested as a means to provide a soft, deformable layer that will allow for transmembrane protein insertion and mobility. In this study, mobile, tethered lipid bilayers were formed on a poly(ethylene glycol) (PEG) support via a two-step adsorption process. The PEG films were prepared by coadsorbing a heterofunctional, telechelic PEG lipopolymer (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-(pyridyldithio)propionate]) (DSPE-PEG-PDP) and a nonlipid functionalized PEG-PDP from an ethanol/water mixture, as described in a previous paper (Munro, J. C.; Frank, C. W. Langmuir 2004, 20, 3339-3349). Then a two-step lipid adsorption strategy was used. First, lipids were adsorbed onto the PEG support from a hexane solution. Second, vesicles were adsorbed and fused on the surface to create a bilayer in an aqueous environment. Fluorescence recovery after photobleaching experiments show that this process results in mobile bilayers with diffusion coefficients on the order of 2 microm2/s. The mobility of the bilayers is decreased slightly by increasing the density of tethered lipids. The formation of bilayers, and not multilayer structures, is also confirmed by surface plasmon resonance, which was used to determine in situ film thickness, and by fluorimetry, which was used to determine quantitatively the fluorescence intensity for each 18 by 18 mm sample. Unfortunately, fluorescence microscopy also shows that there are large defects on the samples, which limits the utility of this system.     

镀金和碳纳米管修饰金电极上吸附态葡萄糖氧化酶比活性的EQCM研究

采用石英晶体微天平(EQCM)技术监测了裸金电极、镀金和碳纳米管修饰金电极上葡萄糖氧化酶(GOD)的吸附过程. 通过EQCM测量吸附固定的GOD质量, 并实时检测酶反应产物H2O2的氧化电量, 求算了各表面上吸附态GOD的比活性(ESAi). 结果表明, 各表面上均可吸附一定的GOD, 且吸附态GOD均有一定的酶活性; 修饰CNTs可增大酶吸附量和酶电极对葡萄糖的响应电流, 但ESAi随CNTs修饰量的增大而降低; Au电极上电镀金后, 酶吸附量和酶电极对葡萄糖的响应电流亦增大, 但ESAi与裸金电极上的基本一致.     

Parallel pore and surface diffusion of levulinic acid in basic polymeric adsorbents

The equilibrium and kinetics of levulinic acid (LA) adsorption on two basic polymeric adsorbents, 335 (highly porous gel) and D315 (macroreticular), were investigated. Experimental adsorption rates in batch stirred vessels under a variety of operating conditions were described successfully by the parallel pore and surface diffusion model taking into account external mass transfer and nonlinear Toth isotherm. The film-pore diffusion model was matched with the rate data and the resulting apparent pore diffusivities were strongly concentration-dependent and approached to a constant value for 335 adsorbent. Thus, the constant value was taken as the accurate pore diffusivity, while the pore diffusivity in D315 was estimated from the particle porosity. The surface diffusivities decreased with increasing initial bulk concentration for both adsorbents. The inverse concentration dependence was correlated reasonably well to the change of isosteric heat of adsorption as amount adsorbed.     

Influence of PEG architecture on protein adsorption and conformation

Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers with various grafting ratios were adsorbed to niobium pentoxide-coated silicon wafers and characterized before and after protein adsorption using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Three proteins of different sizes, myoglobin (16 kD), albumin (67 kD), and fibrinogen (340 kD), were studied. XPS was used to quantify the amount of protein adsorbed to the bare and PEGylated surfaces. ToF-SIMS and principal component analysis (PCA) were used to study protein conformational changes on these surfaces. The smallest protein, myoglobin, generally adsorbed in higher numbers than the much larger fibrinogen. Protein adsorption was lowest on the surfaces with the highest PEG chain surface density and increased as the PEG layer density decreased. The highest adsorption was found on lysine-coated and bare niobium surfaces. ToF-SIMS and PCA data evaluation provided further information on the degree of protein denaturation, which, for a particular protein, were found to decrease with increasing PEG surface density and increase with decreasing protein size.     

Adsorption of water vapor on ZnO: Effects of annealing and grinding

TG, DTA, and DTGA study of water vapor adsorption on ZnO showed that the water vapor was adsorbed reversibly at 500°C (0.12 mg/g ZnO). Irreversible desorption of water vapor was also found at approximately 270°C. Both the specific surface area and the amount of water vapor adsorbed reversibly decreased with increasing annealing temperature above 500°C. When as-received ZnO was ground, the amount of water vapor adsorbed reversibly decreased sharply before any significant change took place in the specific surface area. At longer grinding time, the specific surface area increased but the amount of water vapor adsorbed reversibly increased only slightly with the grinding time. When the specimen was ground after annealing, the reversible adsorption of water vapor was not affected by the thermal history before grinding.     

分形表面上CO氧化反应的蒙特卡罗模拟

众所周知,人们在理想完好的单晶表面上的动力学研究已取得了很大的成功[1],然而对许多实际催化剂来说,传统催化理论和实验之间不符的情况仍然俯拾皆是．现在人们对造成这种情况的原因已经有了更加深入的认识：大量的实验事实表明,在表面催化反应体系中,不但催化剂表面具有复杂的分形结构,而且催化剂表面上的活性中心分布也具有复杂的分形特性[2]．“分形”是指那些具有分数维数的几何对象．这些对象往往是不规则的,不能用通常的欧氏几何来描述[3]．将分形引入催化科学中最早的是Pfeifer和Avnir等[4]人,至今已有近二十年的历史,现…     

Lateral mobility of polyelectrolyte chains in multilayers

In this work, the lateral mobility of polyelectrolyte multilayers was investigated by means of the fluorescence recovery after photobleaching (FRAP) technique, with special attention to the effect of relevant parameters during and after preparation. Different polyelectrolytes with respect to charge density, stiffness, and hydrophilicity were compared. From the experimental results emerged that the density of charged sites along the polymer is the most important parameter controlling the formation of polymer complexes. At higher charge density, more complexes are formed, and the diffusion coefficient decreases. It was observed that the intrinsic backbone stiffness reduces the interpenetration of polyelectrolyte layers and the formation of complexes promoting the lateral mobility. In addition, the lateral mobility increases with increasing ionic strength and with decreasing hydration shell of the added anion in the polyelectrolyte solution. The effect of heating or annealing in electrolyte solution after preparation was also investigated along with the embedding of the probing layer at controlled distances to the multilayer surface.     

Spreading of proteins and its effect on adsorption and desorption kinetics

The kinetics of adsorption of lysozyme and alpha-lactalbumin from aqueous solution on silica and hydrophobized silica has been studied. The initial rate of adsorption of lysozyme at the hydrophilic surface is comparable with the limiting flux. For lysozyme at the hydrophobic surface and alpha-lactalbumin on both surfaces, the rate of adsorption is lower than the limiting flux, but the adsorption proceeds cooperatively, as manifested by an increase in the adsorption rate after the first protein molecules are adsorbed. At the hydrophilic surface, adsorption saturation (reflected in a steady-state value of the adsorbed amount) of both proteins strongly depends on the rate of adsorption, but for the hydrophobic surface no such dependency is observed. It points to structural relaxation ("spreading") of the adsorbed protein molecules, which occurs at the hydrophobic surface faster than at the hydrophilic one. For lysozyme, desorption has been studied as well. It is found that the desorbable fraction decreases after longer residence time of the protein at the interface.