Adsorption and activity of Thermomyces lanuginosus lipase on hydrophobic and hydrophilic surfaces measured with dual polarization interferometry (DPI) and confocal microscopy.
The adsorption and activity of Thermomyces lanuginosus lipase (TLL) was measured with dual polarization interferometry (DPI) and confocal microscopy at a hydrophilic and hydrophobic surface. In the adsorption isotherms, it was evident that TLL both had higher affinity for the hydrophobic surface and adsorbed to a higher adsorbed amount (1.90 mg/m(2)) compared to the hydrophilic surface (1.40-1.50mg/m(2)). The thickness of the adsorbed layer was constant (approximately 3.5 nm) on both surfaces at an adsorbed amount >1.0mg/m(2), but decreased on the hydrophilic surface at lower surface coverage, which might be explained by partially unfolding of the TLL structure. However, a linear dependence of the refractive index of the adsorbed layer on adsorbed amount of TLL on C18 surfaces indicated that the structure of TLL was similar at low and high surface coverage. The activity of adsorbed TLL was measured towards carboxyfluorescein diacetate (CFDA) in solution, which upon lipase activity formed a fluorescent product. The surface fluorescence intensity increase was measured in a confocal microscope as a function of time after lipase adsorption. It was evident that TLL was more active on the hydrophilic surface, which suggested that a larger fraction of adsorbed TLL molecules were oriented with the active site facing the solution compared to the hydrophobic surface. Moreover, most of the activity remained when the TLL surface coverage decreased. Earlier reports on TLL surface mobility on the same surfaces have found that the lateral diffusion was highest on hydrophilic surfaces and at low surface coverage of TLL. Hence, a high lateral mobility might lead to a longer exposure time of the active site towards solution, thereby increasing the activity against a water-soluble substrate.
Sonesson
Andreas W
AW
YKI, Institute for Surface Chemistry, Stockholm, Sweden. andreas.sonesson@surfchem.kth.se
Callisen
Thomas H
TH
Brismar
Hjalmar
H
Elofsson
Ulla M
UM
D000327:Adsorption; D001203:Ascomycota / Q000201:enzymology*; D002244:Carbon / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007368:Interferometry / Q000379:methods*; D008049:Lipase / Q000378:metabolism*; D018613:Microscopy, Confocal; D012031:Refractometry; D012825:Silicon / Q000378:metabolism; D013997:Time Factors
D005452:Fluoresceins; D002244:Carbon; C084577:5-carboxyfluorescein diacetate; D008049:Lipase; D012825:Silicon