Mobility of Thermomyces lanuginosus lipase on a trimyristin substrate surface
2007 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 23, no 5, 2706-2713 p.Article in journal (Refereed) Published
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.
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
IdentifiersURN: urn:nbn:se:kth:diva-6932DOI: 10.1021/la062003gISI: 000244248700066PubMedID: 17261037ScopusID: 2-s2.0-33847752732OAI: oai:DiVA.org:kth-6932DiVA: diva2:11783
QC 201008182007-03-272007-03-272010-08-18Bibliographically approved