Single-Shot Characterization of Enzymatic Reaction Constants Km and kcat by an Acoustic-Driven, Bubble-Based Fast Micromixer

• Published in: Analytical chemistry (Washington) Vol. 84; no. 17; pp. 7495 - 7501
• Main Authors: YULIANG XIE, AHMED, Daniel, IAN LAPSLEY, Michael, STEVEN LIN, Sz-Chin, AHSAN NAWAZ, Ahmad, LIN WANG, TONY JUN HUANG
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 04.09.2012
• Subjects: Acoustics; Analytical chemistry; beta-Galactosidase - metabolism; Chemistry; Dimethylpolysiloxanes - chemistry; Escherichia coli - enzymology; Exact sciences and technology; Galactosides - metabolism; Kinetics; Microfluidic Analytical Techniques; Oxazines - metabolism; Enzyme; Reaction product; Concentration; Reaction rate; Glycosylases; Characterization; Design; Substrate; Transducer; Reporter gene; Chemical reaction; Glycosidases; High speed; Hydrolases; Yield; Kinetics; β-Galactosidase; Enzymatic reaction
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac301590y

In this work we present an acoustofluidic approach for rapid, single-shot characterization of enzymatic reaction constants K(m) and k(cat). The acoustofluidic design involves a bubble anchored in a horseshoe structure which can be stimulated by a piezoelectric transducer to generate vortices in the fluid. The enzyme and substrate can thus be mixed rapidly, within 100 ms, by the vortices to yield the product. Enzymatic reaction constants K(m) and k(cat) can then be obtained from the reaction rate curves for different concentrations of substrate while holding the enzyme concentration constant. We studied the enzymatic reaction for β-galactosidase and its substrate (resorufin-β-D-galactopyranoside) and found K(m) and k(cat) to be 333 ± 130 μM and 64 ± 8 s(-1), respectively, which are in agreement with published data. Our approach is valuable for studying the kinetics of high-speed enzymatic reactions and other chemical reactions.