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Adsorption and activity of Thermomyces lanuginosus lipase on hydrophobic and hydrophilic surfaces measured with dual polarization interferometry (DPI) and confocal microscopy
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
Novozymes A/S, Bagsvaerd.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0003-0578-4003
YKI, Institute for Surface Chemistry, Stockholm.
2008 (English)In: Colloids and Surfaces B: Biointerfaces, ISSN 0927-7765, E-ISSN 1873-4367, Vol. 61, no 2, 208-215 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2008. Vol. 61, no 2, 208-215 p.
Keyword [en]
Activity; Confocal microscopy; Dual polarization interferometry; Protein adsorption; Thermomyces lanuginosus lipase; Adsorption; Confocal microscopy; Hydrophilicity; Hydrophobicity; Interferometry; Proteins; fungal enzyme; silicon dioxide; triacylglycerol lipase; water; adsorption; article; confocal laser microscopy; dual polarization interferometry; enzyme activity; enzyme substrate; fungus; hydrophilicity; hydrophobicity; instrumentation; interferometry; measurement; microscope; nonhuman; priority journal; Thermomyces lanuginosus; Ascomycota; Carbon; Fluoresceins; Hydrolysis; Hydrophobicity; Interferometry; Lipase; Microscopy, Confocal; Refractometry; Silicon; Time Factors
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-6928DOI: 10.1016/j.colsurfb.2007.08.005ISI: 000252919500012PubMedID: 17890066Scopus ID: 2-s2.0-37549038279OAI: oai:DiVA.org:kth-6928DiVA: diva2:11779
Note
QC 20100818. Uppdaterad från Submitted till Published 20100818.Available 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

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