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Adsorption and mobility of a lipase at a hydrophobic surface in the presence of surfactants
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
YKI, Institute for Surface Chemistry, Stockholm.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0003-0578-4003
Novozymes A/S, Bagsvaerd.
2006 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 22, no 13, 5810-1817 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2006. Vol. 22, no 13, 5810-1817 p.
Keyword [en]
Adsorption; Hydrophobicity; Mathematical models; Physiology; Surface active agents; Surface tension; Interfacial catalysis; Oily substrate; Surface plasmon resonance (SPR; Thermomyces lanuginosus; Enzymes; immobilized enzyme; surfactant; triacylglycerol lipase; adsorption; article; Ascomycetes; chemical structure; chemistry; enzymology; fluorescence recovery after photobleaching; hydrophobicity; surface plasmon resonance; surface property; surface tension; Adsorption; Ascomycota; Enzymes, Immobilized; Fluorescence Recovery After Photobleaching; Hydrophobicity; Lipase; Models, Molecular; Surface Plasmon Resonance; Surface Properties; Surface Tension; Surface-Active Agents
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-6930DOI: 10.1021/la0531244ISI: 000238217000048Scopus ID: 2-s2.0-33745760732OAI: oai:DiVA.org:kth-6930DiVA: diva2:11781
Note
QC 20100818Available from: 2007-03-27 Created: 2007-03-27 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces
Open this publication in new window or tab >>Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces
2007 (English)Doctoral 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.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. 57 p.
Series
Trita-FYS, ISSN 0280-316X ; 2007:20
Keyword
biophysics, surface chemistry, diffusion, enzymes, lipases, adsorption, mobility
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-4319 (URN)978-91-7178-604-3 (ISBN)
Public defence
2007-04-23, FB53, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100818Available from: 2007-03-27 Created: 2007-03-27 Last updated: 2010-08-18Bibliographically approved
2. Lipase diffusion on solid surfaces
Open this publication in new window or tab >>Lipase diffusion on solid surfaces
2005 (English)Licentiate thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 2005. vii, 38 p.
Series
Trita-FYS, ISSN 0280-316X ; 2005:28
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-381 (URN)91-7178-073-4 (ISBN)
Presentation
2005-05-30, Sal FA31, AlbaNova, Roslagstullsbacken 21, Stockhlm, 10:00
Supervisors
Note
QC 20101221Available from: 2005-08-09 Created: 2005-08-09 Last updated: 2010-12-21Bibliographically approved

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