A Compartmented Flow Microreactor System for Automated Optimization of Bioprocesses Applying Immobilized Enzymes

Abstract

In the course of their development, industrial biocatalysis processes have to be optimized in small-scale, e. g., within microfluidic bioreactors. Recently, we introduced a novel microfluidic reactor device, which can handle defined reaction compartments of up to 250 μL in combination with magnetic micro carriers. By transferring the magnetic carriers between subsequent compartments of differing compositions, small scale synthesis, and bioanalytical assays can be conducted. In the current work, this device is modified and extended to broaden its application range to the screening and optimization of bioprocesses applying immobilized enzymes. Besides scaling the maximum compartment volume up to 3 mL, a temperature control module, as well as a focused infrared spot were integrated. By adjusting the pump rate, compartment volumes can be accurately dosed with an error <5% and adjusted to the requested temperature within less than a minute. For demonstration of bioprocess parameter optimization within such compartments, the influence of pH, temperature, substrate concentration, and enzyme carrier loading was automatically screened for the case of transferring UDP-Gal onto N-acetylglucosamine linked to a tert-butyloxycarbonyl protected amino group using immobilized β1,4-galactosyltransferase-1. In addition, multiple recycling of the enzyme carriers and the use of increased compartment volumes also allows the simple production of preparative amounts of reaction products.

Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

“…The MEB were separated from the storage buffer (as described before), resuspended in 100 μL of a preheated reaction buffer compartment and pumped to the temperature control module (TCM). Details of the IR‐unit for preheating and the TCM are discussed by Heinzler et al., describing the reactor system. Compound composition for the activity assays of the analyzed enzymes are listed in Table S1 (supporting information).…”

“…A sophisticated example is demonstrated in this work by handling enzymes immobilized on magnetic particles in an automated compartmented flow microreactor system (CFMS). The principle of the CFMS was described before illustrating the system well suitable for automated optimization of bioprocesses applying single immobilized enzymes, in this case, immobilized β1,4‐galactosyltransferase. The reaction progress can be monitored online with spectroscopic methods and the temperature can be controlled precisely.…”

“…The principle of the CFMS was described before [11] illustrating the system well suitable for automated optimization of bioprocesses applying single immobilized enzymes, in this case, immobilized β1,4-galactosyltransferase. A sophisticated example is demonstrated in this work by handling enzymes immobilized on magnetic particles in an automated compartmented flow microreactor system (CFMS).…”

Repetitive Synthesis of High‐Molecular‐Weight Hyaluronic Acid with Immobilized Enzyme Cascades

“…[28][29][30][31] His-Tag-based immobilization is often combined with magnetic nanoparticles or beads. [27,[32][33][34] Magnetic carriers offer a time-saving inexpensive way to separate enzymes from the reaction just by applying a magnetic field. [33] Therefore, easy reusability of enzymes is enabled.…”

Self‐Immobilizing Oxidoreductases for Flow Biocatalysis in Miniaturized Packed‐Bed Reactors

“…However, in the light of these recent developments, it is also necessary to consider established protein tags, such as the hexahistidine (His)‐tag, which is widely applied in affinity purification of recombinant proteins. Despite several drawbacks associated with His‐tag immobilization, such as a relatively low affinity ( K d ∼ 3 μM) and specificity , as well as potentially adverse effects on the activity of enzymes containing divalent metal ions , this approach has been successfully applied to one‐step purification and immobilization to generate enzymatic cascade reactions in microchips , on microbeads , or on polymer‐coated controlled porous glass beads (EziG TM ) . Other advantages of the His‐tag include its short six‐amino acid sequence that can be introduced conveniently in a single polymerase chain reaction (PCR) step and the variety of well‐established and commercially available equipment for purification and analysis.…”