利用擴散−碰撞理論建構一量測酵素反應活化能之新穎方法

Abstract

酵素催化時的活化能不論在藥物釋放的研究上或生物醫學的應用上都扮演了相當重要的角色。大家常會利用阿瑞尼士(Arrhenius)方程式藉由改變溫度量測活化能，但卻忽略了酵素本身對於溫度的變化是很靈敏的。因此，我們利用擴散−碰撞理論建構一套嶄新的方法量測酵素的活化能。在本篇研究中，我們將脂肪分解酵素固定在不同粒徑大小的奈米金粒子上進行催化反應，探討遮蔽係數改變帶來的影響；遮蔽效應會產生一個接觸的立體角影響脂肪分解酵素−奈米金粒子和受質(pNPP)的親和力。利用此方法我們測得的活化能為4.902 kJ/mol (37□C)，其結果與阿瑞尼士方程式測得之活化能不相同。另外，我們藉由加入甘油改變黏度的方法，在葡萄糖氧化酵素的反應溶液中來量測活化能；不同的黏度限制了葡萄糖氧化酵素催化反應中所有反應物的擴散能力，其活化能在20□C、30□C和40□C下分別為25.02 kJ/mol、26.30 kJ/mol和26.83 kJ/mol。這些結果都顯示出，在不同溫度下的活化能並不相等，亦可進一步斷定利用阿瑞尼士方程式量測活化能的方法並不適用於酵素催化反應。最後，我們有足夠的證據證明我們所使用的方法於藥物釋放和生物醫學的發展有更成功的應用。

Activation energy (Ea) plays an important role as a factor of enzyme functions and has been extensively utilized in the developments of drug delivery and biomedical applications. So far, Arrhenius equation is the most common way to evaluate the Ea by changing the temperature of reaction. However, it is ignored that errors occurs on applications of enzyme catalysis while temperature changes due to the ultrasensitivity to temperature for enzymes. In this study, a novel way based on the diffusion−collision theory was put forward to quantify the Ea of enzymatic reaction. Lipase from Candida rugosa (CRL) immobilization onto various sizes of gold nanoparticles (AuNPs) and addition with glycerol in the reaction buffer of Glucose oxidase (GOx) kinetics to change the shielding factor and viscosity respectively are our two strategies. We get the Ea of CRL−AuNP is about 4.902 kJ/mol (310 K) according to the method that shielding factor yields a solid−angle to affect the affinity between CRL−AuNP and pNPP, and this result is dissimilar with the value from Arrhenius equation. On the other hand, the Ea of GOx are about 25.02 kJ/mol, 26.30 kJ/mol, and 26.83 kJ/mol at 20□C, 30□C, and 40□C, respectively because various viscosities limit the diffusibility of reactants. It is obviously that the Ea is indeed different under different temperature. Based on the above results, we may present a conclusion that the Arrhenius equation is an inappropriate method for evaluating the Ea of enzymatic reactions. Furthermore, we have sufficient evidences to support that our methods were successfully applied in the development of drug delivery and biomedical applications.