Abstract
Monitoring and control of pH is essential for the control of reaction conditions and reaction progress for any biocatalytic or biotechnological process. Microfluidic enzymatic reactors are increasingly proposed for process development, however typically lack instrumentation, such as pH monitoring. We present a microfluidic side-entry reactor (μSER) and demonstrate for the first time real-time pH monitoring of the progression of an enzymatic reaction in a microfluidic reactor as a first step towards achieving pH control. Two different types of optical pH sensors were integrated at several positions in the reactor channel which enabled pH monitoring between pH 3.5 and pH 8.5, thus a broader range than typically reported. The sensors withstood the thermal bonding temperatures typical of microfluidic device fabrication. Additionally, fluidic inputs along the reaction channel were implemented to adjust the pH of the reaction. Time-course profiles of pH were recorded for a transketolase and a penicillin G acylase catalyzed reaction. Without pH adjustment, the former showed a pH increase of 1 pH unit and the latter a pH decrease of about 2.5 pH units. With pH adjustment, the pH drop of the penicillin G acylase catalyzed reaction was significantly attenuated, the reaction condition kept at a pH suitable for the operation of the enzyme, and the product yield increased. This contribution represents a further step towards fully instrumented and controlled microfluidic reactors for biocatalytic process development.
Highlights
The effects of pH on the activity and stability of enzymes, ionization and stability of substrates, products and otherAbbreviations: 6-amino penicillanic acid (6-APA), 6-amino benzyl penicillanic acid; ERY, L-erythrulose; GA, glycol aldehyde; GC, gas chromatography; HPA, hydroxyl pyruvate; HPLC, high performance liquid chromatography; ion-sensitive field effect transistors (ISFETs), ion-sensitive field effect transistor; PG, penicillin G; PGA, penicillin G acylase; TFA, trifluoroacetic acid; TK, transketolase; μSER, microfluidic side-entry reactor components in the reaction mixture are fundamental to enzymatic reactions and belong to the historic foundations of biochemistry [1]
Two enzyme-catalyzed reactions were studied in this research: (A) The transketolase (TK) catalyzed synthesis of L-erythrulose (ERY). (B) The penicillin G acylase (PGA) catalyzed formation of 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PA)
We developed a novel microfluidic side-entry reactor with integrated optical pH sensors
Summary
The effects of pH on the activity and stability of enzymes, ionization and stability of substrates, products and other. The pH can be monitored and controlled using pH electrodes in combination with feedback control systems that add acid or base to the reaction mixture (e.g. pH-stat titration) [2]. When down-scaling to reactors with operating volumes of millilitres and smaller, the integration of microelectrodes or any needle-type (1 of 12) 1600475. Two enzyme-catalyzed reactions were studied in this research: (A) The transketolase (TK) catalyzed synthesis of L-erythrulose (ERY). ThDP is thiamine pyrophosphate or thiamine diphosphate
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