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Mobility of Thermomyces lanuginosus lipase on a trimyristin substrate surface
YKI – Ytkemiska institutet.
2007 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 23, no 5, p. 2706-2713Article in journal (Refereed)
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, D0 , was higher for the active TLL compared to the inactive (D0 = 17.9 x10-11 cm2/s vs D0 = 4.1 x10-11 cm2/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, p. 2706-2713
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:ri:diva-27139OAI: oai:DiVA.org:ri-27139DiVA: diva2:1054143
Note
A1838Available from: 2016-12-08 Created: 2016-12-08 Last updated: 2017-11-29Bibliographically approved

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