Biocatalysis is the use of enzymes and other biological molecules to catalyze chemical reactions. It is used in a wide range of applications, including the production of chemicals, pharmaceuticals, and food and beverages.
One of the main advantages of biocatalysis is its ability to selectively catalyze reactions under mild conditions, such as at low temperatures and pH values. This makes it a more environmentally friendly alternative to traditional chemical synthesis methods, which often require harsh conditions and produce waste products.
In addition to its environmental benefits, biocatalysis can also offer economic advantages, such as lower production costs and the ability to synthesize complex molecules that are difficult to produce using other methods. These factors make biocatalysis an attractive option for many industries.
Some specific examples of applications for biocatalysis include the production of enzymes for laundry detergents, the synthesis of pharmaceuticals, and the production of biofuels.
Enzymes display remarkable chemo-, regio-, and stereoselectivity. Once a suitable enzyme has been identified that converts the mandated substrate to the desired product isomer, significantly shorter reaction pathways compared to traditional reaction pathways often become accessible. Notably, protection and deprotection strategies can often be omitted, and difficult isomer separation should be avoided.
The mild conditions and modest temperatures employed in enzymatic reactions, in combination with the highly selective catalysis, typically result in few side reactions.
Associated with the mild conditions are the environmental and toxicity benefits of avoiding pathways that employ metal catalysts and reagents.
Enzymes are divided into seven major classes, which are listed below with a brief description.
EC 1 - Oxidoreductases - These enzymes catalyze oxidation-reduction reactions, transferring electrons from one molecule to another.
EC 2 - Transferases - These enzymes transfer a functional group from one molecule to another.
EC 3 - Hydrolases - These enzymes catalyze the hydrolysis of various bonds, such as those between two molecules or between a molecule and a functional group.
EC 4 - Lyases - These enzymes catalyze the cleavage of various bonds by means other than hydrolysis or oxidation, often forming a double bond or a new ring structure.
EC 5 - Isomerases - These enzymes catalyze the rearrangement of atoms within a molecule to form a structural isomer.
EC 6 - Ligases - These enzymes catalyze the formation of a new chemical bond, often by joining two molecules together.
EC 7 - Translocases - These enzymes catalyze the movement of ions or molecules across membranes or their separation within membranes.
The activity and stability of enzymes are influenced by several factors, including temperature, pH, solvent and buffer conditions, water activity, immobilization, mixing, and reaction parameters. Understanding these factors can help us optimize enzyme-catalyzed reactions and improve their efficiency and effectiveness.
Enzymes are subject to two competing, increasing processes as the temperature is raised. One is the increase in catalytic activity as described by the Arrhenius equation, and the other is the accelerated breakdown of the enzyme. The combined result is a bell-shaped temperature-activity curve, i.e., enzymes have a specific temperature with peak activity flanked by diminishing activity at both higher and lower temperatures.
Enzymes are frequently engineered to shift the peak temperature and temperature range with optimal activity and to make them more resistant to denaturation. The desired shift in temperature can be both to higher and lower temperatures to allow for reactions at elevated temperatures or high activity even at low temperatures. Enzymes can also be engineered to allow for a broadened substrate scope or non-natural substrates and products. Finally, engineering may confer stability in non-conventional media.
Similar to temperature dependence, enzymes show peak activity within a narrow range. When moving away from the optimal pH, the activity is diminished and finally lost. Consequently, pH control is often necessary.
Choice of buffer and buffer strength often provide pH control and stability in aqueous systems and consequently also activity and stability. In non-conventional solvents, process analytical technology (PAT) and titration allow for pH control. Choice of solvent also strongly affects the enzymatic activity.
Enzymes require a small amount of water even when running reactions in non-aqueous media for normal function, less than 1% i typically needed. As a consequence and perhaps counter intuitive, a small amount of water is often added to reactions that should be maintained water-free to shift the equilibrium towards product (i.e. condensation reactions).
Immobilization of free enzymes onto solid materials (enzyme carriers) often provides stabilization from denaturation. It frequently alters the activity to either higher or lower levels. The compatibility with the enzyme carrier material strongly influences the success of the immobilization, and experimentation with several materials may be advisable. Arguably, the most common enzymer carrier materials are synthetic polymers (resins), but inorganic materials such as glass, natural fibers, etc. are also used. The choice is determined by the resulting activity along with cost and longevity.
Inherently connected to the enzyme carrier material is the method of attachment. While some enzymes, notably lipases, may be attached non-covalently by hydrophobic interactions, covalent attachment is often needed. A number of chemistries have been used for the attachment of enzymes to solid materials, and the choice may affect the resulting enzyme activity. The nucleophilic free amines and thiols on the surface of the enzyme are typically reacted with electrophilic reactive groups on the enzyme carrier, such as epoxides and aldehydes for amines and maleimide and haloacetyls for thiols.
Read more in our insight “Introduction to enzyme immobilization”.
Especially for immobilized enzymes, the method of mixing will determine the longevity of the solid material. For batch reactors, the rotating bed reactor offers almost complete protection against the grinding that standard stirred tank reactors suffer from. As a result, extended recycling becomes feasible and convenient.
A rotating bed reactor (RBR) is designed to prevent disintegration of solid phase particles by retaining them inside a rotating cylinder during the reaction span. This minimizes solid phase debris even at high speeds, which would normally occur due to mechanical forces when using stirred tank reactors. Due to these properties of the RBR, reaction rates can be improved by increasing the speed at which an RBR is rotated, without shortening the lifespan of the solid phase particles. This ensures easy in-line monitoring of the process, as well as a more efficient recycling of the solid phase particles, making the RBR a cost and time efficient alternative to conventional methods for immobilized enzyme biocatalysis.
Perhaps more related to the efficiency of the enzymatic reaction than the stability of the enzyme are parameters such as stoichiometry, gas introduction method, and mixing. Proper mixing ensures the necessary mass transfer of substrate to enzyme and is crucial for gas saturation.
A RBR shows important advantages over an stirred tank reactor in this aspect as well. More information can be found here.
Don't miss out on the exciting developments in biocatalysis! With engineered enzymes that are more stable, more active, and capable of working in a wider range of conditions, biocatalysis is becoming increasingly accessible and easy to use. And with a growing range of free and immobilized enzymes available on the market, now is the perfect time to start exploring the benefits of biocatalysis for yourself. Join the biocatalysis revolution today!
Katarzyna Szymańska, Klaudia Odrozek, Aurelia Zniszczoł, Wojciech Pudło, and Andrzej B. Jarzębski Chem. Eng. J., 2017, 315, pp. 18-24.
Time lapse video showing how straightforward it is to use immobilized enzymes in a rotating bed reactor. A substrate giving a yellow coloured product was used to follow the reaction progress of an ester hydrolysis by an immobilized lipase. This substrate is commonly used to screen and characterize lipases.
Roger A. Sheldon and Pedro C. Pereira Chem. Soc. Rev., 2017, 46(10), pp. 2678-2691.
Enzyme screening is an important step in the development of a biocatalytic application. The behaviour of the biocatalyst is often hard to predict, meaning that different combinations of materials and reaction conditions need to be tested.
Philipp Petermeier, Jan Philipp Bittner, Simon Müller, Emil Byström, Selin Kara Green Chemistry, 2022, 24(18), pp. 6889-6899.
When working with an emulsion (and particularly with a heterogeneous catalyst) the mass transfer between the phases is critical. Insufficient mixing leads to lower interfacial area per volume, and in turn to poor mass transfer across the phases.
Hendrik Mallin, Jan Muschiol, Emil Byström, and Uwe T. Bornscheuer ChemCatChem, 2013, 5, pp. 3529-3532.
Whole cell biocatalysis is powerful, but not straightforward. One way of utilizing whole cells is to encapsulate them in a matrix such as alginate to make them easier to separate from a reaction mixture. However, alginate beads are not mechanically stable enough to be packed into columns and are easily destroyed in stirred tank reactors (STR). This makes enzyme recycling ineffective, at the same time as mass transfer limitations may prevail.
Finding the optimal chemistry and solid-phase material for immobilization of enzymes relies heavily on trial and error. The right resin will ensure satisfactory immobilization yield, as well as high activity and stability of the enzyme.
Ariana Causevic, Eimantas Gladkauskas, Kim Olofsson, Patrick Adlercreutz, and Carl Grey Biochem. Eng. J., 2022, 187, 108610.
Anup Ashok and Santhosh Kumar Devarai 3 Biotech, 2019, 9(9), 349.
Carl-Johan Aurell, Staffan Karlsson, Fritiof Pontén, and Søren M. Andersen Org. Process Res. Dev., 2014, 18(9), pp. 1116-1119.
Jochen Wachtmeister, Philip Mennicken, Andreas Hunold, and Dörte Rother ChemCatChem, 2016, 8, pp. 607-614.
Jens Johannsen, Francesca Meyer, Claudia Engelmann, Andreas Liese, Georg Fieg, Paul Bubenheim, and Thomas Waluga AIChE J., 2021, 67(4), e17158.
An interesting paper where the authors mentions ”A further refinement, the rotating bed reactor developed by SpinChem... This technology combines the benefits of an STR and a packed bed and has been scaled up successfully to more than 100 litres scale.” Key learnings from the paper (1) The advantages and limitations of immobilised enzymes in industrial applications (2) The different technical and regulatory requirements using immobilised enzymes (3) The different enzyme immobilisation methods (4) The different reactor technologies for immobilised enzymes and biocatalysis in general (5) Recent advances in enzyme immobilisation for better production economy
In biotransformations, obstacles commonly encountered are product inhibition, product toxicity, and reaction equilibria that prevents complete conversion. Enzyme engineering has made tremendous progress in alleviating these problems. The concept of in situ product removal (ISPR) may still be an attractive alternative or complement. The authors have demonstrated concurrent enzymatic reaction and ISPR, referred to as 'extractive biocatalysis'. For the ISPR, the authors evaluated the use of aqueous micellar two-phase systems (ATPMS) as an extraction medium. For the model reaction, Penicillin G hydrolysis by CalB lipase, the demonstrated process was thus a continuous, heterogeneous extractive biocatalysis with cloud point extraction. An RBR was used during the process development work to determine the Michaelis-Menten kinetics of the CalB immobilized in gel coatings on column packing material. Also, the particles were easily re-used in stability experiments.
Alexandra V. Chatzikonstantinou, Αrchontoula Giannakopoulou, Stamatia Spyrou, Yannis V. Simos, Vassiliki G. Kontogianni, Dimitrios Peschos, Petros Katapodis, Angeliki C. Polydera, and Haralambos Stamatis Environ. Sci. Pollut. Res., 2022, 29, pp. 29624-29637.
S. Lokesha, Y. S. Ravi Kumar, P. S. Sujan Ganapathy, Prashant Gaur, and H. M. Arjun 3 Biotech, 2021, 11, 410.
Ingeborg Heuschkel, Selina Hanisch, Daniel C. Volke, Erik Löfgren, Anna Hoschek, Pablo I. Nikel, Rohan Karande, and Katja Bühler Eng. Life Sci., 2021, 21(3-4), pp. 258-269.
M. Aßmann, A. Stöbener, C. Mügge, S. K. Gaßmeyer, L. Hilterhaus, R. Kourist, A. Liese, and S. Kara React. Chem. Eng., 2017, 2(4), pp. 531-540.
Jochen Wachtmeister and Dörte Rother Curr. Opin. Biotechnol., 2016, 42, pp. 169-177.
Immobilized catalyst recycling using a SpinChem® rotating bed reactor (RBR) and a Mettler-Toledo EasyMax™ 102 Advanced synthesis workstation. The process proved very time efficient as no filtration steps were needed between cycles, or for the samples extracted for analysis during each run. Washing of the resin between runs was fast, simple and robust, without running the risk of material loss. Keywords: Biotransformation, Immobilized enzymes, Mettler-Toledo, Organic molecules, Preserved activity, Quick recycling
Roger A. Sheldon and John M. Woodley Chem. Rev., 2018, 118(2), pp. 801-838.
Poster on a case study of applying the rotating bed reactor for the lipase-mediated stereoselective acetylation of a racemate amine as a model reaction for the manufacturing of pharmaceutical building blocks. The results showed that enzyme recycling and synthesis scale up was easy to achieve with preserved yield, enantioselectivity and catalytic activity. Keywords: Biotransformation, Easy handling, Immobilized enzymes, Quick recycling, Seamless scaleup
Stirred vessels tend to damage soft heterogeneous catalysts, like enzymes immobilized in agarose or alginate beads, with activity loss and tedious workup as consequence. In a fixed bed reactor, these materials are easily compressed by the pressure gradient, leading to a loss of flow rate. Overcoming these challenges opens up the possibility to use biocatalysis as a tool for greener processes and more sustainable manufacturing.
The stable reaction environment in the EasyMax™ 102 Advanced synthesis workstation and the high flow rates through the SpinChem® RBR allowed for quick and convenient screening of different immobilized lipases to find the enzyme most suitable for further reaction optimization. Keywords: Biotransformation, Immobilized enzymes, Mettler-Toledo, Organic molecules, Rapid screening
The SpinChem rotating bed reactor (RBR) has been proven to be a time and labour-efficient tool in the screening of biocatalysts. In this application note, six different immobilized lipases were screened in parallel for the esterification of lauric acid into propyl laurate using the pre-packed MagRBR Lipase screening kit.
Research and development quickly takes new directions, and the requirements on a laboratory may vary with every new project. Limiting yourself to equipment with a narrow scope of conditions and applications may become expensive, since new equipment must be acquired for anything out of scope. With budgets quickly consumed by other projects, the need for new equipment may mean significant delays and a reduced capability to take on emerging opportunities. Many heterogeneous processes (e.g. adsorption or catalysis) are made faster by increasing the solid-to-liquid ratio. Studying scale-up effects can also help to predict full-scale performance. For these reasons it’s wise to invest in equipment that can handle different operating conditions such as liquid volume, solids loading, pH and temperature. The RBR S3 Plus is the most modular rotating bed reactor for laboratory use. Made from two stacked rotating bed reactors, the S3 Plus quickly converts to a single RBR S3 for use with smaller liquid volumes. When used in the dedicated glass reactor system, this yields an operating range of 250 - 1500 mL of liquid and 0 - 140 mL of solids. This application note investigates the effect of solids loading on the reaction rate of two applications: the adsorption of a dye and a biocatalytic esterification reaction. These two reactions are mass-transport limited and relatively fast. In the first case, an RBR S3 and an RBR S3 Plus were filled with 50 mL and 100 mL respectively of the ion-exchange resin Purolite® NRW1160. Methylene blue was dissolved in water, and the solution was decolorized by spinning an RBR at 600 RPM (reaction conditions in the details below). The results were clear; each case followed 1st order kinetics with a rate constant for the RBR S3 Plus that was twice that of the RBR S3. Note that the solid-to-liquid ratio for the RBR S3 Plus was also twice that of the RBR S3. For the enzymatic esterification, the same rotating bed reactors (RBR S3 and RBR S3 Plus) were filled with 40 mL and 80 mL respectively of the biocatalyst Purolite® immo PS. The rotating bed reactors were used in separate reactions in mixtures of lauric acid, 1-propanol and water. Also in this case the reaction rate was proportional to the solid-to-liquid ratio, yielding twice the productivity with the RBR S3 Plus compared to the RBR S3. The conclusion is that with a rotating bed reactor you are making the most out of the solid-phase. Doubling the amount of catalyst or adsorbent will generally double the reaction rate constant, which makes scaling up straightforward. Contact us today to discuss how we can scale your process.
The synthesis of products, such as active pharmaceutical ingredients, often involve multiple steps using heterogeneous catalysts or adsorbents. The need often arises for simultaneous use of multiple solid phases.
baffle (noun) : a device (such as a plate, wall, or screen) to deflect, check, or regulate flow or passage (as of a fluid, light, or sound) Can you use a rotating bed reactor (RBR) in any type of vessel? Absolutely. Would the performance be higher with baffles in the vessel? Definitively. A vortex, which forms due to the rotation of an agitator, is detrimental to the mixing in a reactor vessel. If the agitator is a rotating bed reactor, it also disrupts the flow through the RBR. Baffles are features in the reactor vessel that break the circulating flow pattern, preventing vortex formation and improving overall mixing. The importance of baffles has long been established for stirred tank reactors with agitation by impellers, and baffling is equally important for vessels with an RBR installed. You don't need to take our word for it; customers that have investigated the effect of baffling on their mass-transfer limited reactions have found the same result. The most recent data comes from research at the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB. They started out with the RBR S3 in a smooth glass vessel. After observing a deep vortex, drawn down into the RBR, and the resulting disappointing performance, one simple flat-blade baffle was installed. The performance for the enzymatic reaction was quantified with and without the flat-blade baffle, and the result is presented in the figure below. As seen in the data, installing one simple baffle resulted in a doubled yield at each time point on average. The double-walled glass vessels (V2 and V3) that SpinChem offer are custom-made to fit the RBR S2 and S3 respectively. The vessels have structured inner glass walls that serve as "flower-shaped" baffles, which do not take up the same space as traditional flat-blade baffles. This minimizes the required volume of reaction medium, and maximizes mixing performance. Some non-baffled vessels perform as if they had flow breakers installed, just through the shape of their vessel walls. For instance, any non-round geometry such as rectangular IBC-tanks may provide satisfactory mixing and prevention of a deep vortex forming. On benchtop scale, the round beaker is appealing with its simplicity, but a baffled reaction vessel will yield much better performance. If you have more questions about this contact us.
Subhash Pithani, Staffan Karlsson, Hans Emtenäs, and Christopher T. Öberg Org. Process Des. Dev., 2019, 23(9), pp. 1926-1931.
Shuke Wu and Zhi Li ChemCatChem, 2018, 10(10), pp. 2164-2178.
Biocatalysis offers many benefits in the production of chemicals and active pharmaceutical ingredients. One major challenge has been the deployment of immobilized enzymes in an efficient way on large scale. The rotating bed reactor offers a convenient way to scale a biocatalytic process.
Sometimes you don’t want to pack the entire rotating bed reactor full with your solid-phase material. Fully loading might simply be wasteful, or you may want to experiment with your reaction conditions. But how does the amount of solids in the rotating bed reactor influence the reaction performance? Can you use only 10% of the full capacity?
Teng Ma, Weixi Kong, Yunting Liu, Hao Zhao, Yaping Ouyang, Jing Gao, Liya Zhou, and Yanjun Jiang Appl. Biochem. Biotechnol., 2022, 194, pp. 4999–5016.
Adriana Freites Aguilera, Pontus Lindroos, Jani Rahkila, Mark Martinez Klimov, Pasi Tolvanen, and Tapio Salmi Chem. Eng. Process. Process Intensif., 2022, 174, 108882.
Krzysztof Polaczek, Eliza Kaulina, Ralfs Pomilovskis, Anda Fridrihsone, Mikelis Kirpluks Journal of Polymers and the Environment, Volume 30 (2022), pages 4774–4786
Markus Hobisch, Piera De Santis, Simona Serban, Alessandra Basso, Emil Byström, and Selin Kara Org. Process Res. Dev., 2022, 26(9), pp. 2761-2765.
Tobias Heinks, Nicolai Montua, Michelle Teune, Jan Liedtke, Matthias Höhne, Uwe T. Bornscheuer, and Gabriele Fischer von Mollard Catalysts, 2023, 13(2), 300.
Wilhelm Wikström, Adriana Freites Aguilera, Pasi Tolvanen, Robert Lassfolk, Ananias Medina, Kari Eränen, and Tapio Salmi Ind. Eng. Chem. Res., 2023, 62(23), pp. 9169-9187.
Christopher K. Prier, Karla Camacho Soto, Jacob H. Forstater, Nadine Kuhl, Jeffrey T. Kuethe, Wai Ling Cheung-Lee, Michael J. Di Maso, Claire M. Eberle, Shane T. Grosser, Hsing-I Ho, Erik Hoyt, Anne Maguire, Kevin M. Maloney, Amanda Makarewicz, Jonathan P. McMullen, Jeffrey C. Moore, Grant S. Murphy, Karthik Narsimhan, Weilan Pan, Nelo R. Rivera, Anumita Saha-Shah, David A. Thaisrivongs, Deeptak Verma, Adeya Wyatt, and Daniel Zewge ACS Catal., 2023, 13(12), pp. 7707-7714.
Daria Kowalczykiewicz, Katarzyna Szymańska, Danuta Gillner, and Andrzej B. Jarzębsk Microporous Mesoporous Mater., 2021, 312, 110789.
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