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Mobility of Thermomyces lanuginosus lipase on a trimyristin substrate surface
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0003-0578-4003
Novozymes A/S, Bagsvaerd.
YKI, Institute for Surface Chemistry, Stockholm.
2007 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 23, no 5, 2706-2713 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2007. Vol. 23, no 5, 2706-2713 p.
Keyword [en]
Flow conditions; Surface density; Thermomyces lanuginosus lipase; Adsorption; Confocal microscopy; Diffusion; Hydrophobicity; Photobleaching; Substrates; Enzymes; triacylglycerol; triacylglycerol lipase; trimyristin; unclassified drug; adsorption; article; Ascomycetes; biological model; biophysics; chemistry; confocal microscopy; diffusion; enzymology; fluorescence recovery after photobleaching; methodology; movement (physiology); physical chemistry; statistical model; surface property; time; Adsorption; Ascomycota; Biophysics; Chemistry, Physical; Diffusion; Fluorescence Recovery After Photobleaching; Lipase; Microscopy, Confocal; Models, Biological; Models, Statistical; Movement; Surface Properties; Time Factors; Triglycerides
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-6932DOI: 10.1021/la062003gISI: 000244248700066PubMedID: 17261037Scopus ID: 2-s2.0-33847752732OAI: oai:DiVA.org:kth-6932DiVA: diva2:11783
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

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