Measurements of kinetic parameters in a microfluidic reactor.
Continuous flow microfluidic reactors that use immobilized components of enzymatic reactions present special challenges in interpretation of kinetic data. This study evaluates the difference between mass-transfer effects and reduced efficiencies of an enzyme reaction. The kinetic properties of immobilized alkaline phosphatase (AP) were measured by the dephosphorylation of 6,8-difluoro-4-methylumbelliferyl/phosphate to a fluorescent 6,8-difluoro-4-methylumbelliferone. A glass microfluidic chip with an in-channel weir was created for the capture of solid silica microbeads functionalized with enzyme. The input substrate concentrations and flow rates across the bed were varied to probe the flow-dependent transport and kinetic properties of the reaction in the microreactor bed. Unlike previous reactors, substrate was titrated directly over the fixed enzyme bed by controlling the air pressure over the chip reservoirs. The reactor explored substrate conversions from near zero to 100%. The average bed porosity, residence time, and bed resistance were measured with dye pulses. A simple criterion was derived to evaluate the importance of flow-dependent mass-transfer resistances when using microreactors for calculating kinetic rate constants. In the absence of mass-transfer resistances, the Michaelis-Menten kinetic parameters are shown to be flow independent and are appropriately predicted using low substrate conversion data. A comparison of the kinetic parameters with those obtained using solution-phase enzymatic reactions shows a significant decrease in enzyme activity in the immobilized conformation. The immobilized Km of AP is approximately 6 times greater while the kcat is reduced by approximately 28 times. Contradictions found in literature on the evaluation of Michaelis-Menten kinetic parameters for immobilized enzymes in microfluidic reactors are addressed. When product molecules occupy a significant number of enzymatic sites or modify the enzyme activity, the assumed Michaelis-Menten mechanism can no longer be valid. Under these conditions, the calculations of "apparent" kinetic rate constants, based on Michaelis-Menten kinetics, can superficially show a dependence on flow rate conditions even in the absence of mass-transfer resistances. High substrate conversions are shown to depend on flow rate. A kinetic model based on known mechanisms of the alkaline phosphatase enzyme reaction is tested to predict the measurements for high substrate conversion. The study provides a basis for appropriate use of mass-transfer and reaction arguments in successful application of enzymatic microreactors.
Kerby
Matthew B
MB
Biochemical Engineering Laboratory, Division of Engineering, Brown University, Providence, Rhode Island 02912, USA.
Legge
Robert S
RS
Tripathi
Anubhav
A
D000327:Adsorption; D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D007202:Indicators and Reagents; D007700:Kinetics; D044085:Microfluidics; D008863:Microspheres; D010316:Particle Size; D010766:Phosphorylation; D016062:Porosity; D012822:Silicon Dioxide / Q000737:chemistry; D013499:Surface Properties
D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D006923:Hymecromone; D012822:Silicon Dioxide; D000469:Alkaline Phosphatase