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Tracking single lipase molecules on a trimyristin substrate surface using quantum dots
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
YKI, Institute for Surface Chemistry, Department of Cell Physics, Stockholm.
Novozymes A/S, Bagsvaerd.
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.ORCID-id: 0000-0003-0578-4003
2007 (engelsk)Inngår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 23, nr 16, s. 8352-8356Artikkel i tidsskrift (Fagfellevurdert) Published
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.

sted, utgiver, år, opplag, sider
2007. Vol. 23, nr 16, s. 8352-8356
Emneord [en]
humicola-lanuginosa lipase, interfacial activation, biological detection, enzyme-kinetics, diffusion, membrane, receptors, dynamics, mobility, domains
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-16817DOI: 10.1021/la700918rISI: 000248229900014PubMedID: 17616159Scopus ID: 2-s2.0-34547824727OAI: oai:DiVA.org:kth-16817DiVA, id: diva2:334860
Merknad
QC 20100818Tilgjengelig fra: 2010-08-05 Laget: 2010-08-05 Sist oppdatert: 2017-12-12bibliografisk kontrollert
Inngår i avhandling
1. Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces
Åpne denne publikasjonen i ny fane eller vindu >>Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces
2007 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2007. s. 57
Serie
Trita-FYS, ISSN 0280-316X ; 2007:20
Emneord
biophysics, surface chemistry, diffusion, enzymes, lipases, adsorption, mobility
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-4319 (URN)978-91-7178-604-3 (ISBN)
Disputas
2007-04-23, FB53, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Veileder
Merknad
QC 20100818Tilgjengelig fra: 2007-03-27 Laget: 2007-03-27 Sist oppdatert: 2010-08-18bibliografisk kontrollert

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