Mobility of Thermomyces lanuginosus Lipase on a Trimyristin Substrate Surface

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• 作者：Andreas W. Sonesson ; Hjalmar Brismar ; Thomas H. Callisen ; Ulla M. Elofsson
• 刊名：Langmuir
• 出版年：2007
• 出版时间：February 27, 2007
• 年：2007
• 卷：23
• 期：5
• 页码：2706 - 2713
• 全文大小：234K
• 年卷期：v.23,no.5(February 27, 2007)
• ISSN：1520-5827

文摘

We have studied the mobility of active and inactive Thermomyces lanuginosus lipase (TLL) on a spin-coatedtrimyristin 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 thediffusion coefficient D and the mobile fraction f could be quantified. FRAP was performed on surfaces with differentsurface density of lipase and as a function of time after adsorption. The data showed that the mobility of TLL wassignificantly higher on the trimyristin substrate surfaces compared to our previous studies on hydrophobic modelsurfaces. 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 coefficientat extrapolated infinite surface dilution, D₀, was higher for the active TLL compared to the inactive (D₀ = 17.9 ×10^-11 cm²/s vs D₀ = 4.1 × 10^-11 cm²/s, data for the first time interval after adsorption). Moreover, the diffusiondecreased 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 withtime. This was supported by sequential adsorption experiments, where the adsorbed amount under flow conditionswas studied as a function of time after adsorption. A second injection of lipase led to a significantly lower increasein adsorbed amount when the trimyristin surface was pretreated with active TLL compared to pretreatment of inactiveTLL.