Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden.
Lab Chip. 2013 May 7;13(9):1754-61. doi: 10.1039/c3lc41398e.
Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine and industrial bioprocess technology. We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzyme substrate inhibitor systems in a single experiment utilizing an integrated droplet picoinjector for bioanalysis. This device enables the scanning of multiple fluorescently-barcoded inhibitor concentrations and substrate conditions in a single, highly time-resolved experiment yielding the Michaelis constant (Km), the turnover number (kcat) and the enzyme inhibitor dissociation constants (ki, ki'). Using this device we determine Km and kcat for β-galactosidase and the fluorogenic substrate Resorufin β-D-galactopyranoside (RBG) to be 442 μM and 1070 s(-1), respectively. Furthermore, we examine the inhibitory effects of isopropyl-β-D-thiogalactopyranoside (IPTG) on β-galactosidase. This system has a number of potential applications, for example it could be used to screen inhibitors to pharmaceutically relevant enzymes and to characterize engineered enzyme variants for biofuels production, in both cases acquiring detailed information about the enzyme catalysis and enzyme inhibitor interaction at high throughput and low cost.
酶动力学和抑制作用对于包括药理学、医学和工业生物工艺学在内的广泛学科都非常重要。我们提出了一种新颖的基于微滴的设备，用于在单个实验中广泛表征酶-底物-抑制剂系统的反应动力学，该实验利用集成的微滴皮升注射器进行生物分析。该设备能够在单个高度时间分辨的实验中扫描多个荧光标记的抑制剂浓度和底物条件，从而得出米氏常数（Km）、转换数（kcat）和酶抑制剂解离常数（ki，ki'）。使用该设备，我们确定β-半乳糖苷酶和荧光底物 Resorufin β-D-galactopyranoside（RBG）的 Km 和 kcat 分别为 442 μM 和 1070 s(-1)。此外，我们还研究了异丙基-β-D-硫代半乳糖吡喃糖苷（IPTG）对β-半乳糖苷酶的抑制作用。该系统具有许多潜在的应用，例如可用于筛选与药物相关的酶的抑制剂，以及用于生物燃料生产的工程酶变体的表征，在这两种情况下，都可以以高通量和低成本获取有关酶催化和酶抑制剂相互作用的详细信息。
