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  • 1.
    Sonesson, Andreas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces2007Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    This thesis aimed to give more insight in the dynamics of enzymes at interfaces. The adsorption and mobility of adsorbed proteins can e.g. give a better understanding of structure-function properties of interfacially active enzymes. Studied enzyme was the lipase from Thermomyces lanuginosus (TLL).

    Adsorption of TLL to surfaces of different hydrophobicity was studied by Dual Polarization Interferometry (DPI), Surface Plasmon Resonance (SPR) and ellipsometry. It was found that TLL had highest affinity and adsorbed to largest adsorbed amount on a hydrophobic, C18 terminated surface. Moreover, activity studies of adsorbed TLL suggested that a larger fraction of the lipases were orientated with the active site facing the surface on hydrophobic surfaces.

    Mobility of adsorbed enzymes was studied by means of Fluorescence Recovery After Photobleaching (FRAP) with Confocal Laser Scanning Microscopy (CLSM). CLSM was also used as a tool to image the role of TLL in the detergency of lipids from single cotton fibers. The TLL surface mobility was measured on model surfaces of different hydrophobicity. The rate of TLL surface diffusion was strongly dependent on the surface density of lipase, which was explained by sterical hindrance and intermolecular repulsion. The diffusion was both lowest and decreased as a function of time after adsorption on the most hydrophobic surface. This was thought to be due to a larger fraction of adsorbed TLL oriented with the active site towards the hydrophobic surface and that this fraction increased as a function of time.

    The presence of surfactants affected the TLL mobility on hydrophobic surfaces. The diffusion increased more than tenfold when TLL was coadsorbed with C12E6/LAS above the critical micellar concentration (cmc) of the surfactant. This was thought to be due to a surfactant induced desorption-rebinding mechanism of TLL. Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS) supported this theory and was implemented as a technique to quantify kinetic processes of protein-surfactant interactions at surfaces.

    The surface mobility of TLL was higher on a trimyristin substrate surface compared to the model hydrophobic surface. Single particle tracing of lipases could be performed by conjugation of TLL to Quantum Dots (QDs). The microscopic behavior of QD-lipases on trimyristin suggested that the enzyme operated in two different modes on the surface, which gave the trajectories of single lipase molecules a “bead on a string” appearance.

  • 2.
    Sonesson, Andreas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Lipase diffusion on solid surfaces2005Licentiate thesis, comprehensive summary (Other scientific)
  • 3.
    Sonesson, Andreas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hassler, Kai
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Elofsson, Ulla
    YKI, Institute for Surface Chemistry, Stockholm.
    Callisen, Thomas H.
    Novozymes A/S, Bagsvaerd.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Protein-surfactant interactions at hydrophobic interfaces studied with Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS)2008In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 317, no 2, p. 449-457Article in journal (Refereed)
    Abstract [en]

    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 C 18 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 mu M 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 C 18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

  • 4.
    Sonesson, Andreas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Callisen, Thomas H.
    Novozymes A/S, Bagsvaerd.
    Elofsson, Ulla
    YKI, Institute for Surface Chemistry, Stockholm.
    Mobility of Thermomyces lanuginosus lipase on a trimyristin substrate surface2007In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 23, no 5, p. 2706-2713Article in journal (Refereed)
    Abstract [en]

    We have studied the mobility of active and inactive Thermomyces lanuginosus lipase (TLL) on a spin-coated trimyristin substrate surface using fluorescence recovery after photobleaching (FRAP) in a confocal microscopy setup. By photobleaching a circular spot of fluorescently labeled TLL adsorbed on a smooth trimyristin surface, both the diffusion coefficient D and the mobile fraction f could be quantified. FRAP was performed on surfaces with different surface density of lipase and as a function of time after adsorption. The data showed that the mobility of TLL was significantly higher on the trimyristin substrate surfaces compared to our previous studies on hydrophobic model surfaces. For both lipase variants, the diffusion decreased to similar rates at high relative surface density of lipase, suggesting that crowding effects are dominant with higher adsorbed amount of lipase. However, the diffusion coefficient at extrapolated infinite surface dilution, D-0, was higher for the active TLL compared to the inactive (D-0 = 17.9 x 10(-11) cm(2)/s vs D-0 = 4.1 x 10(-11) cm(2)/s, data for the first time interval after adsorption). Moreover, the diffusion decreased with time after adsorption, most evident for the active TLL. We explain the results by product inhibition, i.e., that the accumulation of negatively charged fatty acid products decreased the diffusion rate of active lipases with time. This was supported by sequential adsorption experiments, where the adsorbed amount under flow conditions was studied as a function of time after adsorption. A second injection of lipase led to a significantly lower increase in adsorbed amount when the trimyristin surface was pretreated with active TLL compared to pretreatment of inactive TLL.

  • 5.
    Sonesson, Andreas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Callisen, Thomas H.
    Novozymes A/S, Bagsvaerd.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Elofsson, Ulla
    YKI, Institute for Surface Chemistry, Stockholm.
    A comparison between Dual Polarization Interferometry (DPI) and Surface Plasmon Resonance (SPR) for protein adsorption studies2007In: Colloids and Surfaces B: Biointerfaces, ISSN 0927-7765, E-ISSN 1873-4367, Vol. 54, no 2, p. 236-240Article in journal (Refereed)
    Abstract [en]

    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/m(2) 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.

  • 6.
    Sonesson, Andreas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Callisen, Thomas H,
    Novozymes A/S, Bagsvaerd.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Elofsson, Ulla
    YKI, Institute for Surface Chemistry, Stockholm.
    Adsorption and activity of Thermomyces lanuginosus lipase on hydrophobic and hydrophilic surfaces measured with dual polarization interferometry (DPI) and confocal microscopy2008In: Colloids and Surfaces B: Biointerfaces, ISSN 0927-7765, E-ISSN 1873-4367, Vol. 61, no 2, p. 208-215Article in journal (Refereed)
    Abstract [en]

    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/m(2)) compared to the hydrophilic surface (1.40-1.50 mg/m(2)). The thickness of the adsorbed layer was constant (similar to 3.5 nm) on both surfaces at an adsorbed amount > 1.0 mg/m(2), 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.

  • 7.
    Sonesson, Andreas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Callisen, Thomas H.
    Novozymes A/S, Bagsvaerd.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Elofsson, Ulla
    YKI, Institute for Surface Chemistry, Stockholm.
    Lipase surface diffusion studied by Fluorescence Recovery After Photobleaching2005In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, no 25, p. 11949-11956Article in journal (Refereed)
    Abstract [en]

    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.

  • 8.
    Sonesson, Andreas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Callisen, Thomas H.
    Novozymes A/S, Bagsvaerd.
    Elofsson, Ulla
    YKI, Institute for Surface Chemistry, Stockholm.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Imaging the detergency of single cotton fibers with confocal microscopy: the effect of surfactants and lipases2007In: Journal of Surfactants and Detergents (JSD), ISSN 1097-3958, E-ISSN 1558-9293, Vol. 10, no 4, p. 211-218Article in journal (Refereed)
    Abstract [en]

    Detergency mechanisms of lipids from single cotton fibers were visualized by means of confocal laser scanning microscopy (CLSM). Fibers were soiled with different types of lipids: olive oil, lard and tri-C-10, and subsequently stained with the fluorescent probe Nile Red. A surfactant composition of 300 M C12E6/LAS (1:2 mol%) was used to mimic the surfactants used in a common washing solution. It was evident from the captured image series that the different kinds of soiling were removed by different mechanisms by the surfactants, depending on the fluidity of the lipid. Roll-up was the main mechanism when removing olive oil, whereas emulsification (necking) and/or solubilization were observed in the removal of lard and tri-C-10. Only 20-25% of the olive oil remained after treatment with surfactants, which was much less compared to the solid fats where roughly 50% remained at end of treatment. The effect of adding lipases to the detergent formulation was clearly seen, both by an apparently higher rate of removal of olive oil but also using double injection when removing lard. A first injection of only surfactants removed a certain part of the lard as emulsion droplets stuck onto the fiber. A second injection of both lipases and surfactants was able to remove some of the preformed emulsion particles and reduce the overall remaining lard content on the cotton fiber.

  • 9.
    Sonesson, Andreas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Elofsson, Ulla
    YKI, Institute for Surface Chemistry, Stockholm.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Callisen, Thomas H.
    Novozymes A/S, Bagsvaerd.
    Adsorption and mobility of a lipase at a hydrophobic surface in the presence of surfactants2006In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 22, no 13, p. 5810-1817Article in journal (Refereed)
    Abstract [en]

    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, D-0. We found that the presence of surfactants strongly modulated the surface mobility of TLL: with D-0 = 0.8 x 10(-11) cm(2)/s without surfactants and D-0 = 13.1 x 10(-11) cm(2)/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.

  • 10.
    Sonesson, Andreas
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Elofsson, Ulla M.
    YKI, Institute for Surface Chemistry, Department of Cell Physics, Stockholm.
    Callisen, Thomas H.
    Novozymes A/S, Bagsvaerd.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Tracking single lipase molecules on a trimyristin substrate surface using quantum dots2007In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 23, no 16, p. 8352-8356Article in journal (Refereed)
    Abstract [en]

    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) x 10(-10) cm(2)/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.

  • 11.
    Sonesson, Andreas W.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    A FRAP-based method to study protein surface diffusionIn: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126Article in journal (Other academic)
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