Process intensification for O2‐dependent enzymatic transformations in continuous single‐phase pressurized flow

Abstract Oxidative O2‐dependent biotransformations are promising for chemical synthesis, but their development to an efficiency required in fine chemical manufacturing has proven difficult. General problem for process engineering of these systems is that thermodynamic and kinetic limitations on supplying O2 to the enzymatic reaction combine to create a complex bottleneck on conversion efficiency. We show here that continuous‐flow microreactor technology offers a comprehensive solution. It does so by expanding the process window to the medium pressure range (here, ≤34 bar) and thus enables biotransformations to be conducted in a single liquid phase at boosted concentrations of the dissolved O2 (here, up to 43 mM). We take reactions of glucose oxidase and d‐amino acid oxidase as exemplary cases to demonstrate that the pressurized microreactor presents a powerful engineering tool uniquely apt to overcome restrictions inherent to the individual O2‐dependent transformation considered. Using soluble enzymes in liquid flow, we show reaction rate enhancement (up to six‐fold) due to the effect of elevated O2 concentrations on the oxidase kinetics. When additional catalase was used to recycle dissolved O2 from the H2O2 released in the oxidase reaction, product formation was doubled compared to the O2 supplied, in the absence of transfer from a gas phase. A packed‐bed reactor containing oxidase and catalase coimmobilized on porous beads was implemented to demonstrate catalyst recyclability and operational stability during continuous high‐pressure conversion. Product concentrations of up to 80 mM were obtained at low residence times (1–4 min). Up to 360 reactor cycles were performed at constant product release and near‐theoretical utilization of the O2 supplied. Therefore, we show that the pressurized microreactor is practical embodiment of a general reaction‐engineering concept for process intensification in enzymatic conversions requiring O2 as the cosubstrate.


| INTRODUCTION
Advanced process technologies for chemical production are increasingly built on process intensification and continuous processing as the central pillars of their development (Adamo et al., 2016;Clomburg, Crumbley, & Gonzalez, 2017;Hessel, Kralisch, Kockmann, Noël, & Wang, 2013;Wiles & Watts, 2014). In this context, biocatalysis is promising to enable cleaner, safer and more energyefficient process technologies (Sheldon & Pereira, 2017;Sheldon & Woodley, 2018). Oxidative transformations represent an area of the chemical production in which biocatalysis is expected to have a profound impact (Dong et al., 2018;. A strong oxidant (e.g., O 2 ) is often required in these transformations, so running them safely and with high chemical selectivity is a difficult problem Hone, Roberge, & Kappe, 2017).
However, the biocatalysis happens in water and supplying O 2 to an aqueous environment faces several well-known restrictions. In fact, the main parameters of reaction efficiency (product concentration, space-time yield (STY), and catalyst turnover) all depend on, and are often severely limited by, how effectively O 2 is made available within the liquid phase . In addition, it is paramount that the reactor design and the preparation of the enzyme used (e.g., immobilized enzyme and whole cell) both are brought in good accordance with the requirements of O 2 supply to the continuous biotransformation envisaged (Dong et al., 2018; (Karande, Schmid, & Buehler, 2016;Toftgaard Pedersen et al., 2015;. These STYs are significantly smaller than the maximum OTR of 100-200 mM/h obtainable in conventional reactors for gas-liquid contacting Toftgaard Pedersen et al., 2015).

| Immobilization
GOX and BlCAT were coimmobilized on Sep-PEI and Rel-PEI based on ionic adsorption of the enzymes. A reported protocol was used with slight modifications described in the Supporting Information Methods S1). DAAO and BpCAT were coimmobilized on Relsulfonate. Previously reported procedure (Bolivar, Schelch, et al., 2016) was used. The DAAO was immobilized before the BpCAT. Of note, the enzyme immobilization involved affinity-like ionic adsorption via the Z basic2 module. This confers high selectivity to the enzyme immobilization directly from the cell extract and also ensures enzyme-surface interaction in a defined molecular orientation via Z basic2 (Bolivar, Schelch, et al., 2016;Wiesbauer et al., 2011). The immobilization was monitored by enzyme activity measurement, both in solution and directly on the carrier. The total activity immobilized, E imm (U/g_carrier), was calculated from the activity balance in solution. E obs (U/g_carrier) is the directly measured activity of the enzyme immobilized on the carrier. An E obs lower than E imm is explainable by effect of the immobilization on the intrinsic enzyme activity, diffusional effects or both.

| Pressurized flow reactor design and set-up
The reactor in Figure

| Pressurized flow reactor operation
All reactions were performed at 24 ± 1°C using 50 mM potassium phosphate buffer (GOX, pH 7.0; DAAO, pH 8.0). The system pressure F I G U R E 1 The flowchart of the high-pressure reactor operated with soluble enzymes is shown. The system comprised the reactor coil, a mass-flow controller for gas delivery, two pumps controlling liquid inflow, two flow-through pressure sensors at the inlet and the outlet of the reactor unit, and a backpressure regulator. The reactor components were made of stainless steel. Observation windows made from Teflon tubes were included as indicated [Color figure can be viewed at wileyonlinelibrary.com]

| Reaction kinetic analysis
The reaction kinetics of the soluble oxidases were described using Equation (2) which is the rate equation for a Ping-Pong two-substrate enzyme mechanism. Equation (2) is known to apply to the kinetics of GOX and DAAO.
In Equation (2) 2). Therefore, F L and F G were adjusted to form a single liquid phase (Supporting Information Figure S1) whose flow rate corresponded to an average residence time (τ res ) of 1 min. (1-4 min), therefore, the assumption of plug flow was justified.
Operating the flow reactor in the experiments described below  Figure   S2). Increase of τ res to 2 or 4 min hardly affected the concentration of the product released (Supporting Information Figure S2). We
Boundaries in terms of STY and catalyst productivity were theoretically discussed in a seminal study by Dencic and coworkers . Using a tube-in-tube reactor for hydroxylation of 2-hydroxybiphenyl, Tomaszewski and coworkers (Tomaszewski, Schmid, T A B L E 1 Summary of the performance metrics of the single-phase pressurized reactor operated with free enzymes  (Bolivar, Schelch, et al., 2016).
Besides issues of enzyme stability caused by a significant level of H 2 O 2 present at steady state under such conditions, keeping the balance between the enzyme activities present as to prevent O 2 gas formation seems challenging. Anyway, in the study of Chapman et al. (2018), the maximum STY was 8 mM/min, which is below the standard OTR limit using gasification with pure O 2 (Figure 4c), the TON was~2 × 10 3 .

| Implementation of pressurized packed-bed reactor
When performing enzymatic transformations in flow, it is customary to use the enzyme in a form suitable for continuous processing with enzyme recycling (Karande et al., 2016;Tamborini et al., 2018).
Enzyme immobilization on a solid support is most commonly used to that end. Despite significant advances in flow reactor applications (Karande et al., 2016;Tamborini et al., 2018), study of the intensification of O 2 -dependent conversions using immobilized enzymes is lacking. In Figure 5 we show the pressurized flow reactor for use with immobilized enzymes. The overall reactor design reflects the idea of expanding the current boundaries of reactor performance in terms of V (10 mM/min; . Our choice of carrier material for enzyme immobilization took into account specifically that the pressure drop In contrast, when the reactor was operated at the same conditions (i.e., F L and F G ) as in Figure 7b, but at atmospheric pressure, the [P] released was decreased to less than 10% of the [P] released under the pressurized conditions (data not shown). Figure 7a also shows that the increase of the substrate concentration improved  Figure S8), indicated a massive limitation of the observable V by diffusion. Therefore, the E obs was expected to be reduced to ≤10% of the actual E imm under these conditions (Doran, 2013). In addition, one can calculate, that for substrate to reach the center of the carrier particle when V is 700 mM/min, the particle radius would have to be lower than 20 µm (Supporting Information Figure S8). This however is not a practical particle size. A more detailed study of diffusional limitations in DAAO immobilizates at high-pressure reaction conditions was left for consideration in future research. In any event, further strategies of reaction intensification with immobilized enzymes are of high interest (Bolivar, Valikhani, & Nidetzky, 2018). They have significant potential to create synergy with the "high-pressure flow approach" developed in this study.
Stable operation of the pressurized flow reactor for 360 reactor cycles is shown in Figure 7b. Enzyme elution was not observed under the conditions used. This is worth emphasizing because both DAAO and [P] (80 mM). The pressurized reactor is unique in avoiding trade-off between these process efficiency parameters which it is difficult to manage even in the currently most advanced reactors requiring OTR at ambient pressure (Dencic, Hassel, et al., 2012;Karande et al., 2016;Kashid et al., 2011). The Note. DAAO: D-amino acid oxidase; GOX: glucose oxidase; STY: space-time yield; TOF: turnover frequency. a TOF was calculated from the the product concentration (mM), F tot and the molar amount of enzyme used. The amount of enzyme was calculated from the E imm , the mass of carrier used, the specific activity of the free enzyme, and the molecular mass of the monomer (GOX: 80.0 kDa; DAAO: 46.3 kDa). Note that TON= TOF × time of reactor operation (here, 6 hr). b The catalyst productivity was calculated from the mass concentration of product, F tot and the mass amount of enzyme used. The mass amount of enzyme used was calculated from the E imm , the mass of carrier used and the specific activity of enzyme. Results are shown for a τ res of 1 min.