Cascão Pereira Luis G, Hickel Andrea, Radke Clayton J, Blanch Harvey W
Department of Chemical Engineering, University of California, Berkeley, CA 94720-1462, USA.
Biotechnol Bioeng. 2002 Jun 20;78(6):595-605. doi: 10.1002/bit.10241.
A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.
开发了一个动力学框架来描述两相液 - 液系统中的酶活性和稳定性。具体而言，该模型应用于杏仁腈水解酶（pa - Hnl）在二异丙醚（DIPE）/水性缓冲液界面（pH = 5.5）吸附时从扁桃腈酶促生产苯甲醛的过程。我们定量描述了我们之前获得的实验动力学结果（希克尔等人，1999年；2001年），并且成功解释了产物形成速率对水相酶浓度的依赖性以及早期观察到的反应速率。多层生长解释了通过酶活性和动态界面张力冲洗实验观察到的早期酶在DIPE/缓冲液界面吸附的可逆性，这些实验用缓冲液替换了水相酶溶液。对吸附在DIPE/缓冲液界面的pa - Hnl异常稳定性的假定解释归因于双层吸附机制。在第一层中，从天然状态缓慢的构象变化导致不可逆的附着和催化活性的部分丧失。在第二层中，pa - Hnl可逆吸附且催化活性无损失。测得的催化活性是第一层失活动力学和每层吸附动力学的综合作用。对于吸附在DIPE/缓冲液界面的pa - Hnl的特定情况，这种综合作用在几个小时内几乎保持恒定，导致催化活性没有明显损失。我们提出的动力学模型可以扩展到其他界面活性酶和其他有机溶剂。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提供一个强大工具。
