Introduction

Nowadays, enzymes are broadly used as highly selective and greener biocatalysts with respect to other traditional chemical catalysts that can be toxic and/or result into more waste production [1, 2]. However, the full implementation of biocatalytic reactions on large scale is still a challenging task [3]. Enzymes may suffer from (i) low stability under harsh conditions such as presence of organic co-solvents, extreme temperatures (i.e. mesophilic enzymes operate at 20–45ºC), or extreme pH (i.e. highest activity of most enzymes remains between pH 6 and 8), (ii) substrate/product inhibition, (iii) dependency on costly cofactors (i.e. price for NADPH can be > 1,000 €/g), [4] and (iv) low productivity rate, among others. Several technologies have emerged with the aim to alleviate those drawbacks and bring biocatalysis closer to industrial set-ups [3]. A clear step to boost the potential of biocatalysis is the incorporation of enzymatic reactions into continuous flow reactors, which is generally known as Flow Biocatalysis [5]. The transition from batch to flow offers numerous advantages. For instance, flow set ups offer a higher surface-volume ratio; increased automation of the systems, reducing the risks associated with hazardous intermediates; better experimental reproducibility, lower production of waste; etc. Specifically, flow biocatalysis offers the great advantage to be able to continuously synthesize a product of interest with high control of the reaction parameters (i.e. substrate concentration, reaction rate) and a straightforward coupling of downstream processes (i.e. product purification, reuse of co-solvents). Such a set-up is highly desired to develop more cost-effective processes for chemical manufacturing, while contributing to greener synthetic routes.

The rise of flow biocatalysis is easily recognizable when looking at the steady increase of scientific articles, conference presentations, and lectures over the last years. In fact, scientific publications containing the keyword ‘Flow Biocatalysis’ started to increase intensely in 2008 according to the database Scopus (Fig. 1). Just in the last five years almost 400 publications came out, including entire reviews focusing on this topic [5,6,7,8,9,10,11,12,13]. Flow biocatalytic approaches are also gaining attention in industry, especially in pharmaceutical synthesis [14,15,16,17]. Yet, while enzymatic reactions in continuous flow are far from being extensively exploited in industrial processes, the growing interest in biocatalytic transformations is clear, as it has been recently shown by the survey carried out by Gallou, Gröger, and Lipshutz to different chemical industries around the world [18]. Herein, we will discuss the current and future directions for flow biocatalysis and its role in the leverage of enzymatic transformations towards more efficient and sustainable chemistry.

Fig. 1
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Scientific publications including the keywords ‘Flow biocatalysis’ reported yearly during the last four decades. The database Scopus has been used as data source

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Current landscape of enzymatic reactions in continuous flow

Enzyme immobilization

Enzyme immobilization as the linkage of enzyme molecules to a solid support is typically associated with the integration of biocatalytic reactions into flow systems [19]. Not only, enzyme immobilization can stabilize the protein structure under the required operational conditions, but also facilitates the reusability of the biocatalyst through easy separation of the heterogenous biocatalyst (enzyme-support entity) from the reaction medium that continuously flows through the reactor. This is one of the key concepts that enhances the sustainability and efficiency of biocatalytic reactions in flow. Notwithstanding, the impact of the costs and waste production on to the final productivity must be carefully evaluated when using immobilized enzymes. In order to evaluate the suitability of a heterogeneous biocatalyst for its implementation into a flow reactor, Bolivar and López-Gallego have summarized the most fundamental aspects that should be considered [20]. Surface-protein engineering, material engineering, and reactor design are the three pillars which must be optimized to achieve a balance between the activity and stability of the biocatalyst. Therefore, the field of flow biocatalysis advances parallelly to the field of enzyme immobilization.

The immobilization of enzymes on solid supports has indeed come a long way during the last 50 years. The first examples of biocatalyst immobilization featured the adsorption of only one enzyme, typically a hydrolase, to a solid particle [21]. Nowadays, the co-immobilization of several enzymes working synergistically on an enzymatic cascade is possible, even tuning each enzyme immobilization through a different binding chemistry [22,23,24]. Furthermore, the reversible co-immobilization of cofactors needed for the biocatalytic reactions have been achieved, allowing the development of self-sufficient heterogenous biocatalysts that contribute to reduce the costs of the synthetic processes [25]. Regarding the support onto which the enzyme is attached, different techniques are available: anchoring to microparticles, entrapment into hydrogels, or carrier-free immobilization (such as crosslinking of enzymes) [19, 26]. Noteworthy, the immobilization of biocatalysts from whole cells or cell extracts can be also performed through all those techniques [27,28,29].

In the industrial landscape, the increasing interest on stabilization of enzymes via immobilization has encouraged the establishment of companies specifically focused on the preparation of immobilized biocatalysts [30].

Reactor design

Transferring enzymatic reactions from batch to flow mode requires the adaptation of the reactor as well. Several types of reactors which were already used in flow chemistry have been adopted for the integration of enzymatic reactions in continuous flow (Fig. 2). The most user-friendly configuration are likely continuous stirred tank reactors (CSTRs) as their set-up is very similar to a stirred batch reactor [31,32,33]. The operation of CSTRs in continuous mode involves an inlet to feed the substrate solution to the reactor vessel and the outlet line to recover the product within the reaction mixture. This is a good option for newcomers to the field of flow biocatalysis, but the use of immobilized enzymes in CSTRs must be carefully considered because the mechanical stirring can damage the heterogeneous biocatalysts by shredding the support. As an alternative, membrane reactors (MRs) offer the opportunity to work with free biocatalysts due to the use of a membrane that can separate the biocatalyst from the product molecules which are much smaller [34], although MR can be also operated with immobilized biocatalysts.

The most promising and widely used reactors for flow biocatalysts at present are packed-bed reactors (PBRs) [22, 35,36,37,38]. These reactors are column-shaped and contain the heterogenous biocatalyst occupying around half of the reactor volume. Due to the high surface/volume ratio, the reactants flowing through the PBR can be transformed to the corresponding products accomplishing high conversion rates. Unfortunately, PBRs also suffer limitations such as the inability to process solid reactants or slurries.

In order to screen and optimize the reaction conditions, microreactors are an attractive solution that permit to perform different reactions simultaneously while reducing the reagent consumption and waste generation [39, 40]. The microfluidic devices also allow the study of immobilized enzymes as those are usually attached to the reactor wall. The last trend on (micro)reactor design for flow biocatalysis are 3D-printed reactors which allow the tailor-made design of the reactor in consideration of the reaction challenges [41, 42].

Fig. 2
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Schematic representation of different types of flow reactors

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Downstream processing

One of the biggest advantages of performing enzymatic reactions under flow conditions is the in-line coupling of downstream processing. After the enzymatic reaction is completed, the separation of the product from the unreacted substrates, by-products, or co-solvents used during the reaction requires special attention. In-line downstream processing contributes to reducing the waste production as well as simplifying the multi-step synthetic reactions. Recently, Kara and co-workers have reviewed the main downstream techniques used in flow biocatalysis [43]:

  • Liquid-liquid extraction is a simple strategy in which the different physico-chemical properties of the product are harnessed to enable its solubility into a target solvent, thus promoting its purification. This strategy also allows for the separation of unreacted starting materials or cofactors that are water-soluble and can be recycled into the flow system (Fig. 3A) [44,45,46,47,48].

  • Scavenger resins are widely employed to purify a target molecule in a liquid-solid extraction. The process known as “catch and release” strategy allows for the separation of the product (or other target molecules) by attachment to the resin, and then the purified molecule is released into a new liquid phase (Fig. 3B) [35,36,37, 49]. Typically, sorbent materials which are commercially available are packed into a column that is connected downstream to the flow bioreactor. In addition, novel scavenger materials (i.e. lignin) from natural sources have been successfully applied [50].

  • Precipitation and filtration are often employed for an easy product recovery (Fig. 3C) [38]. Different methods are available for the crystallization of the product, not only to allow its downstream purification but also to shift the reaction equilibrium when the crystallization occurs in situ [51].

Noteworthy, in-line analysis by UV or NMR among others can be also coupled downstream the continuous flow reactor [32].

Fig. 3
figure 3

Flow biocatalytic reactors coupled to in-line downstream processes. (A) liquid-liquid-extraction was used to separate the product (ester) in the organic phase [45]. (B) liquid-liquid extraction allowed a first separation of the co-substrates and side product from the intermediate and product. In a second step, a scavenger column was used to isolate the final product (S-alcohol). Moreover, another scavenger column was employed for the separation and recycling of the co-substrates by “catch-and-release” strategies. For example, glucuronic acid was retained onto Ambersep 900 OH which is a strongly basic resin, and could be released by the addition of an acidic solution [35]. (C) Product precipitation was achieved by cooling down the solution exiting the flow stream. Then, in a separate step, filtration and drying were carried out to isolate the final product (vidarabine) [38]. Figure 3A and 3B are reproduced from ref. [45] and ref. [35] with permissions of Springer Nature

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New trends in flow biocatalysis

Multi-enzyme cascades

The current accessibility to numerous and diverse biocatalysts has certainly unveiled the potential of multi-enzyme reactions [52]. In this regard, flow biocatalysis facilitates the integration of different enzymatic steps into a cascade. On one hand, the compartmentalization of each biocatalytic step into single reactors allows to operate each enzyme at its optimal conditions (temperature, pH, co-solvents) [53]. On the other hand, the integration of different biocatalysts into the same flow reactor, either by co-immobilization of enzymes on the same support [25, 54, 55] or by mixing of different immobilized biocatalysts [35, 56, 57], has proven to favor the reaction rate compared to batch reactions. Certainly, what makes flow biocatalysis truly valuable for enzymatic cascades is the elimination of issues related to product inhibition, as the flow itself continuously removes the newly formed product. Despite all the above-mentioned benefits, the intensification of a multi-enzymatic process in flow is still a very complex task that requires optimization efforts for each individual enzyme/reaction [16, 58, 59]. For this reason, the integration of multi-enzyme cascades into industrial set-ups has been barely explored to date, although this field is advancing steadily.

Chemoenzymatic approaches

The combination of chemical and enzymatic steps in synthetic procedures is a very appealing strategy to introduce (flow) biocatalysis at large scale [58]. The integration of telescoped chemoenzymatic approaches in flow systems facilitates the separation of chemo- and biocatalysts, allowing them to perform at their best reaction conditions that might be difficult to achieve, if at all possible, when performed in batch mode (for instance, enzyme incompatibility with a chemical reaction that happens at 150ºC). In-line purification/separation methods are also a great advantage of flow systems to promote the compatibility of chemo- and biocatalytic reactions, as precise control over the product of one catalytic step can be executed before carrying out the next one. As result, reduced reactions times and more reproducible yields can be obtained. Hence, chemo-enzymatic flow reactions are a very active research area in both academia and industry as it can be appreciated in the recent literature [48, 60,61,62,63,64].

Photobiocatalysis in flow

One of the most recent additions to the field of flow biocatalysis are photobiocatalytic reactions [65]. Light can be harnessed either (i) to excite the substrate before the reaction proceeds, or (ii) to bring the biocatalyst to an excited state in which it is active [66, 67]. In photobiocatalytic reactions, the use of whole-cell biocatalyst is more common than free enzymes due to the instability of the latter. Indeed, this is an important parameter when integrating photobiocatalysis in flow reactors. New types of reactors have been designed to adapt photobiocatalytic reactions to flow set-ups, for instance, to avoid the decrease on enzyme stability due to exposure to high temperatures while ensuring the right light penetration into the photobioreactor [68]. Overall, photobiocatalysis has brought new-to-nature reactivities as well as an alternative manner to improve the sustainability of biotransformations (i.e. light-dependent cofactor regeneration). Now, the biggest challenge to overcome is the obtainment of industrially-relevant processes since both the techniques have limitations in terms of instruments/reactors.

Bioinformatics and machine learning

In recent years, bioinformatic tools have played a key role in the development of enzymatic processes, from the optimization of the catalytic activity of the enzyme to the reaction process [69,70,71,72]. Now, artificial intelligence and machine learning strategies are entering the field of continuous flow biocatalysis aiming at further automation of continuous processes [73,74,75]. Nevertheless, not all reactions can be operated in continuous flow and databases are still limited. But the potential of computational tools on the development of more efficient biocatalysts, reaction optimization, and reaction screening can be revolutionary in the near future by reducing the experimental time required.

Conclusions

In this perspective, we have highlighted the main research streams within the field of flow biocatalysis, focusing on the biocatalyst, the reactor, and the downstream processes. The advantages of this key enabling technology have been discussed and summarized in Table 1. Yet, Flow Biocatalysis is a field in development that present certain challenges, but it is rapidly growing to meet the request of process intensification at industrial scale.

Table 1 Summary of the benefits that flow biocatalysis technology has brought to enzymatic reactions, and the current challenges that still must be overcome to completely leverage its potential
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It is worth mentioning that the integration of enzymatic reactions into continuous flow processes is not a one-step procedure but rather a multidisciplinary effort. Therefore, enzymologists, material scientists, and process engineers work in synchrony to develop the most optimal flow biocatalytic processes. Nevertheless, not all the enzymatic reactions can be intensified in continuous flow, and batch mode may offer more advantages in certain scenarios. New technologies such as photocatalysis or computational studies are entering this field and providing valuable tools to tackle the current limitations as well as expanding the opportunities to apply flow biocatalysis. We envision an even more increasing number of works integrating this technology in the next years. Specially, (chemo)enzymatic reactions in continuous flow are expected to be applied in the chemical and pharmaceutical industries.