Kinetic Profiling of Homogeneous and Heterogeneous Biocatalysts in Continuous Flow by Online Mass Spectrometry.

ABSTRACT

Enzyme kinetics is normally assessed by performing individual kinetic measurements using batch-type reactors (test tubes, microtiter plates), in which enzymes are mixed with different substrates. Some drawbacks of conventional methods are the large amounts of experimental materials, long analysis times, and limitations of spectrophotometry. Therefore, we have developed a method for facile determination of enzyme kinetics using online flow-based mass spectrometry. A concentration ramp of substrate or product was created by dynamically adjusting flow rates of pumps delivering stock solution of substrate and diluent. Precise kinetic measurements were performed by reaction product quantification and initial rate calculation. In the presence of ascending substrate concentrations, the rate of a target enzyme (penicillinase)-catalyzed hydrolysis was varied. By measuring the reaction product continuously, Michaelis constants (KM) could be calculated. The enzyme kinetic measurements for hydrolysis of penicillins were conducted based on this simple, rapid, and low sample consumption online flow device. In the homogeneous reaction, the KM values for amoxicillin, ampicillin, penicillin G, and penicillin V were 254.9 ± 14.5, 29.2 ± 0.3, 2.6 ± 0.1, and 5.4 ± 0.1 µM, respectively. In the heterogeneous reaction, the KM values for amoxicillin, ampicillin, penicillin G, and penicillin V were 408.9 ± 75.1, 114.4 ± 8.0, 21.8 ± 0.7, and 83.3 ± 4.8 µM, respectively. Apart from enzyme assay, the showcased method for the generation of temporal concentration ramps can be utilized to perform rapid quantity calibrations for mass spectrometric analyses.