The rise of biocatalysis in continuous flow

By Andrew Mansfield on October 24th, 2018 in Flow chemistry, The Flow Chemistry Collection

Flow chemistry has application in a diverse range of areas and has the potential to improve on many chemical processes. The increased use of these enabling technologies for research and development applications has increased rapidly over the last 15 years with the development of commercially available apparatus and the increase in academic literature and take-up from industry.

The area of continuous flow biocatalysis is fast becoming a key area of focus for chemists with applications in the production of fine chemicals, drugs, biotherapeutics, and biofuels to name a few. This is reflected in the adoption of flow techniques in modern laboratories and the increased knowledge of these techniques in industry and academia. Continuous flow biocatalysis use is demonstrated in the significantly increased number of publications over the last few years.

The graph below shows the total number of patents and publications in continuous flow biocatalysis since 2000. This graph was constructed using data from SciFinder with the terms ‘‘continuous flow biocatalysis’’. Data was analyzed to January 2018.(¹)

Why perform continuous flow biocatalysis?

In traditional biocatalysis systems batch stirred tank reactors are the most common method and rightly so as this is the most widely available type of reactor. This method, however, has relatively low volumetric productivity and the collision of the enzyme with stirrers and impellers causes degradation and attrition of the enzyme. The use of free enzymes in batch reactions suffers from the limitations of recycling and recovery of the biocatalyst.

Flow chemistry has a lot to offer and shows several advantages over traditional methods. The method has the potential to increase rates of biotransformations due to its increased mass transfer making its use more economical by decreasing reaction times and increasing throughput of material. Immobilization of enzymes allows for better stability, reduces product purification, allows better control of substrate contact time and its recyclability reduces costs and extends their applicability for production.

Whole cells vs. purified proteins

There are two general types of biocatalysis, whole-cell, and purified protein biocatalysis. Whole cell catalysis uses the whole organism such as Escherichia coli (E. coli) for the transformation, while purified protein biocatalysis uses an extracted protein without the cell present.

Whole-cell biocatalysis relies on the substrate entering and exiting the cell for the transformation to take place. There are two approaches to whole-cell catalysis: fermentation and biocatalysis. It is biocatalysis that is of interest to chemists. The advantages of whole cell biocatalysis are that it’s less expensive than using purified proteins and the disadvantages being that the cell membrane limits the penetration of the substrate and product making the reaction slower compared to purified protein,

Purified enzymes, however, have the advantage of being specific in their transformations, but this makes enzymes typically quite specific for substrates. When using purified enzymes, the substrate is only required to diffuse into the active site of the protein and not through the cell membrane. Also, compared with whole cells, the concentration of the desired enzyme is higher compared to the same mass. However, the purification process can be expensive and sometimes these purified proteins can be unstable outside of the cell structure.

While both types of biocatalysis are used in continuous flow systems, the use of immobilized enzymes shows the most advantage for this application.

Immobilizing enzymes

There are a range of methods for the immobilization of enzymes and ideally, they should show the same properties. Most have several, including;

• A large surface area
• Chemical and thermal stability
• Suitable (and enough) functional groups for attachment
• Ease of regeneration
• Insolubility in water
• Rigidity and mechanical strength
• Low cost

The most fundamental immobilization techniques are entrapment, adsorption, covalence, affinity, cross-linking, and encapsulation as shown below.

Adsorption of biocatalysts onto solid supports (or carriers) relies on hydrophobic, salt bridge, van der Waals, and hydrogen bonding interactions between the protein or cell, and the immobilization support. Adsorption is easier to perform and can avoid enzyme denaturation through minimal distortion to the protein. However, immobilization lifetimes and efficiencies can be lower than comparable covalent immobilization.

In the case of covalent binding (covalence), the supporting material is functionalized with an active group (e.g., amine, epoxy, etc.) and the enzymes are covalently bonded to the surface through them. The major benefit of covalent immobilization is the potential for improved catalyst lifetime due to decreased leaching.

Affinity immobilization revolves around enzymes having different affinities for immobilization supports under different conditions.

Entrapment immobilization is achieved by trapping the biocatalysts into a caged network via covalent or non-covalent interactions with an immobilization support. One of the methods is direct immobilization onto the microchannel wall. Another approach utilized enzyme immobilization on a solid support inside the microchannel, e.g., on micro- and nanoparticles, porous polymer monoliths or membranes.

Continuous flow biocatalysis examples

There are a steadily-growing plethora of publications now available for continuous flow biocatalysis; here are some of our favourites.

Bioreduction of β-ketoesters by immobilized microorganisms

In this example, researchers at the Universidade Federal do Rio de Janeiro have demonstrated an interesting alternative on the bioreduction of β-ketoesters by immobilized microorganisms².

The group had previously shown the successful bioreduction of β-ketoesters under batch conditions using K. marxianus and Rhodotorula rubra cells and transferred the conditions a continuous flow methodology. The immobilization of microorganisms by calcium alginate entrapment allows systems of this type to be more robust and readily reused and recycled, a big advantage over using whole cells which cannot be recycled. Using this methodology, the group obtained β-hydroxyesters in excellent yields and high enantiomeric excess (>99%). By varying the β-ketoester structure and immobilized microorganism the absolute stereochemistry could also be controlled.

An enantioselective preparation of O-Acetylcyanohydrin in a three-step telescoped continuous process

Researchers at the Department of Chemistry at the University of Cambridge, IBG-1: Biotechnology (Germany), and INB (Aachen University of Applied Sciences) have demonstrated an enantioselective preparation of O-Acetylcyanohydrin in a three-step telescoped continuous process³. This is particularly interesting as biocatalytic multistep approaches to chiral fine chemicals are still rare.

Candida Antarctica CalB and Arabidopsis thaliana AtHNL were employed in a robust continuous telescoped process, involving an in-situ HCN generation followed by addition to aldehydes. High stereocontrol was observed in the subsequent hydrocyanation reaction. An in-line chemical acetylation enabled stabilization of newly formed cyanohydrins and gave access to a class of O-acetylcyanohydrins with very good conversions and ee values over the three steps (75–99% conversion; 40–98% ee).

This method proved to be advantageous over the batch protocols in terms of reaction time (40 min vs. 345 min) and ease of operation, opening access to reactions which have often been neglected due to safety concerns. The modular components enabled an accurate control of two sequential biotransformations, safe handling of an in-situ generated hazardous gas, and in-line stabilization of products.

Synthesis of Geraniol esters for the food, flavor, and fragrance industries

This example from the Department of Chemical Engineering (Institute of Chemical Technology, Mumbai) has demonstrated the synthesis of Geraniol esters, used in the perfume, flavor, and beverage industries⁴.

The global demand for flavors, perfumes, and fragrances forecasts to grow 3.9% per year, reaching $26.3 billion (USD) in 2020. The importance for the synthesis of short-chain fatty acids, esters, and aroma compounds are gaining economic importance at a commercial level. With an evolving demand for green processes, adoption of biocatalysis has been drastically increased in this area.

In this study, a continuous-flow packed bed reactor of immobilized Candida antarctica lipase B (Novozym 435) was employed. Optimization of process parameters such as biocatalyst screening, and the effect of solvent, mole ratio, temperature and acyl donors were studied. Maximum conversion of ~87% of geranyl propionate was achieved in 15 minute residence time at 70 °C using geraniol and propionic acid with a 1:1 mol ratio. Novozym 435 was found to be the most active and stable biocatalyst among all tested.

Interesting reviews and further reading

There are some nice reviews to look through for some extra reading. All have a range of chemistries and applications which may be useful:

• Enzymatic microreactors in biocatalysis: history, features and future perspectives, Biocatalysis, 2015, 1, 148 – 165
• Industrial Applications of Enzymes: Recent Advances, Techniques, and Outlook, Catalysis, 2018, 8, 238
• Flow Bioreactors as Complementary Tools for Biocatalytic Process Intensification, Trends in Biotechnology, 2017, 36(1), 73 – 88

Conclusion

There is a common misconception that enzymes are unstable and expensive, work only under high dilution, and do not lend themselves well to scalable chemical processing. However, there are ever-increasing numbers of biotransformations being performed in a continuous manner. They offer many different solutions for chemical and biochemical problems with the goal of enhancing selectivity and yield of complex reactions.

References

1. Continuous flow biocatalysis, Joshua Britton, Sudipta Majumdar, and Gregory A. Weiss, Chem. Soc. Rev., 2018, 47, 5891—5918
2. Biocatalyzed Acetins Production under Continuous-Flow Conditions: Valorization of Glycerol Derived from Biodiesel Industry, Rodrigo O. M. A. de Souza et al, J. Flow Chem, 2013, 3(2), 41-45
3. An Orthogonal Biocatalytic Approach for the Safe Generation and Use of HCN in a Multistep Continuous Preparation of Chiral O-Acetylcyanohydrins, Steve V. Let et al, Synlett 2016, 27, 262–266
4. Synthesis of Geraniol Esters in a Continuos-Flow Packed-Bed Reactor of Immobilized Lipase: Optimization of Process Parameters and Kinetic Modelling, Ganapati D. Yadav, Appl Biochem Biotechnol. 2018, 184(2), 630-643

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If you’re interested in performing your biocatalysis – or any other type of chemistry – in continuous flow, just fill in the contact form and one of my colleagues or myself will get back to you.