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Stability and kinetics of interfacial protein films.

机译:界面蛋白膜的稳定性和动力学。

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Surface forces in protein-stabilized thin-liquid films were determined, together with protein adsorption kinetics, interfacial rheology, and interfacial reaction kinetics to explain the stability of protein layers against rupture and the activity of adsorbed enzymes. The results provide approaches for improving biopharmaceutical formulation, biocatalysis, and food technology applications.; Force-distance curves were measured using a thin-film balance interferometer, and are reported as disjoining-pressure isotherms. A novel, microfabricated, film holder was constructed to permit the measurement of the force-distance relationship in strongly adsorbing polyelectrolyte systems such as proteins, at different degrees of interfacial aging. We measured, for the first time, disjoining-pressure isotherms for single, isolated, thin films stabilized by proteins adsorbed at the two air/water interfaces.; Film stability is obtained when electrostatic interactions are screened or proteins are near their isoelectric points. Steric-repulsive interactions then dominate, reflecting protein interfacial structuring within the film. Upon confinement, proteins stack in either bilayers or trilayers. Although globular bovine serum albumin (BSA) retained its native dimensions upon compression, flexible β-casein did not, resulting in more stable films. Aged films develop visible interfacial aggregates and are no longer stabilized by equilibrium thin-film forces.; We characterized the mechanical properties of aged BSA and β-casein layers by measurement of interfacial rheology at the air/water interface, using a novel interfacial rheometer. Because BSA has well-characterized conformers in solution, we were able to establish, for the first time, a connection between protein structure and interfacial rheology. Mechanical stability arises from two contributions: (1) conformational rearrangement following adsorption that leads to formation of an interfacial protein network, and (2) the intrinsic structural stability of the adsorbed protein within the network. The latter is the most important restoring contribution and accounts for adsorbed layers of globular proteins being more elastic than those of flexible proteins.; We developed a kinetic framework to describe enzyme adsorption, activity and stability in two-phase liquid/liquid systems. The model was successfully applied to the enzymatic cleavage of mandelonitrile by Prunus amygdalus hydroxynitrilase adsorbed at the oil/water interface. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.
机译:测定了蛋白质稳定的薄膜中的表面力,以及蛋白质吸附动力学,界面流变学和界面反应动力学,以解释蛋白质层抗破裂的稳定性和吸附的酶的活性。结果提供了改善生物药物制剂,生物催化和食品技术应用的方法。使用薄膜平衡干涉仪测量力-距离曲线,并记录为分离压力等温线。构造了一种新型的,微制造的薄膜固定器,可以在界面老化程度不同的情况下测量强吸附性聚电解质系统(例如蛋白质)中的力-距离关系。我们首次测量了由两个空气/水界面处吸附的蛋白质稳定的单个隔离薄膜的分离压力等温线。当筛选静电相互作用或蛋白质接近其等电点时,可获得薄膜稳定性。然后,排斥-排斥相互作用占主导,反映了膜内的蛋白质界面结构。封闭后,蛋白质堆积成双层或三层。尽管球状牛血清白蛋白(BSA)压缩后仍保持其原始尺寸,但柔性的β-酪蛋白却没有,从而形成了更稳定的薄膜。老化的薄膜会形成可见的界面聚集体,并且不再被平衡的薄膜力所稳定。我们通过使用新型界面流变仪在空气/水界面处测量界面流变学来表征老化的BSA和β-酪蛋白层的机械性能。由于BSA在溶液中具有良好表征的构象异构体,因此我们首次能够在蛋白质结构和界面流变学之间建立联系。机械稳定性来自两方面的贡献:(1)吸附后的构象重排导致形成界面蛋白网络,以及(2)网络中吸附蛋白的固有结构稳定性。后者是最重要的恢复作用,并说明球形蛋白质的吸附层比柔性蛋白质的吸附层更具弹性。我们开发了动力学框架来描述两相液/液系统中酶的吸附,活性和稳定性。该模型已成功地应用于油/水界面吸附的扁桃体羟腈酶对扁桃腈的酶促裂解。最后,我们指出界面张力滞后时间如何为合理的溶剂选择和酶工程学提供强大的工具。

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