Biocatalysis

Using biocatalysts for chemical manufacturing is a highly developed and applied science, used widely for production and processing of pharmaceuticals and food items. Both enzymes and whole cells can be used to catalyse organic reactions, with the most general example of biocatalysis being the yeast-mediated transformation of sugars into alcohols, acids or gases.

However, the most frequently employed group of biocatalysts is lipases. These enzymes are very versatile, facilitating the hydrolysis of lipids into fatty acids as well as the synthesis of esters and amides. Areas of lipase-catalysed reactions include food processing, synthesis of fine chemicals and production of biofuel. Other regularly used enzymatic biocatalysts are proteases, cellulases and amylases, used for the hydrolysis of proteins, cellulose and starch, respectively

The technique of using enzymes to mediate organic synthesis is widely endorsed due to their high selectivity and specificity, which ensures a more satisfactory yield and purity of the transformed products. Furthermore, by the use of protein engineering, the biocatalysis can be made even more efficient by having the enzyme customized to fit a specific need

Immobilized enzymes

When utilizing isolated enzymes for organic synthesis, a typical approach is to keep the enzymes immobilized on the surface of an insoluble material. This is preferred to using soluble enzymes due to both technical and economic benefits. The immobilization will allow for easier recycling, manipulation and separation of the enzyme, as well as an increase in its thermal and operational stability. Additionally, immobilization of the enzyme will ensure minimized protein contamination during the experimental procedure, and improved control over the overall reaction process.

Rotating bed reactors

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.

Application 1014
Biocatalysis by immobilized enzymes in a rotating bed reactor

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.

Application L1704
Biocatalysis engineering: the big picture

A tutoral review taking a holistic approach to the engineering of biotransformations utilising isolated enzymes. The authors highlighted SpinChem® RBR technology benefits and wrote; “… in addition to protecting the biocatalyst from shear forces caused by the stirrer, it greatly accelerates mass transfer, thus affording substantially higher reaction rates and creating the possibility to use much smaller reactors”.   Keywords: Biotransformation, Immobilized enzymes, Organic molecules, Review, Scientific literature

Application 1023
Biocatalysis in rotating bed reactors – from screening to production

Poster describing the screening of resins with immobilized enzymes for esterification reactions. The screening was performed with prepacked cartridges inside a SpinChem® rotating bed reactor (RBR) and compared to parallel screening with prototype disposable magnetic RBR (MagRBR) in 10 mL volume. Both approaches were very quick, required minimum preparation and no filtration. Up-scaled processes enabled quick enzyme recycling, opening for the possibility of semi-continuous processes with attractive production economy.

Application 1003
Effective phase-transfer between immiscible liquids and an ion exchange resin

Illustrative video showing how a phenolic colourant is deprotonated and extracted from an organic to an aqueous solvent. Using SpinChem® RBR in a flower-baffled reaction vessel created fine emulsion droplets resulting in effective phase-transfer between the two liquids and the solid phase. Keywords: Immiscible liquids, Ion exchange, Organic molecules, Technology

Application L1301
Efficient biocatalysis with immobilized enzymes or encapsulated whole cell microorganism by using the SpinChem reactor system

Hendrik Mallin, Jan Muschiol, Dr. Emil Byström, Prof. Dr. Uwe T. BornscheuerChemCatChem, 5 (2013) 3529-3532   "...the immobilized transaminase was better protected from mechanical forces in the SpinChem device."    Keywords: Alginate, Biotransformation, Encapsulated cells, Immobilized enzymes, Organic molecules, Scientific literature

Application 1002
Efficient synthesis of chiral lactones by encapsulated cells in a rotating bed reactor

Comparison of SpinChem® rotating bed reactor (RBR) with traditional reaction set-ups for a demanding biotransformation. SpinChem® RBR matched or outperformed the other systems and gave a 10 to 25-fold more time-efficient recycling of the encapsulated cells.   Keywords: Alginate, Biotransformation, Encapsulated cells, Gas-distribution, Organic molecules, Quick recycling

Application 1028
Enzyme immobilization screening using magnetic rotating bed reactors

The SpinChem® MagRBR ECR screening kit, pre-packed with Purolite® Lifetech™ resins, was used to screen six different enzyme carrier resins in parallel for the immobilization of lipase CalB. Easy sampling and monitoring of the process, together with effortless handling, established the MagRBR as a time and labour efficient screening device. Keywords: Biotransformation, Easy handling, Immobilized enzymes, Rapid screening

Application L1402
Lipase catalyzed regioselective lactamization as a key step in the synthesis of N-Boc (2R)-1,4-oxazepane-2-carboxylic acid

A synthesis of N-Boc (2R)-1,4-oxazepane-2-carboxylic acid was developed in 39% yield over seven steps starting from methyl (2R)-glycidate. The key step was a lipase-catalyzed regioselective lactamization performed using SpinChem® rotating bed reactor that simplified work up and recycling of the enzyme. Carl-Johan Aurell*, Staffan Karlsson, Fritiof Pontén, and Søren M. Andersen Keywords: Biotransformation, Immobilized enzymes, Organic molecules, Quick recycling, Scientific literature

Application L1602
Modularized biocatalysis: Immobilization of whole cells for preparative applications in microaqueous organic solvents

Jochen Wachtmeister, Philip Mennicken, Andreas Hunold, Dörte RotherChemCatChem, 8 (2016) 607-614   Keywords: Biotransformation, Encapsulated cells, Organic molecules, Scientific literature

Application L1703
Reaction engineering of biocatalytic (S)-naproxen synthesis integrating in-line process monitoring by Raman spectroscopy

A traditional stirred tank reactor setup was compared to a rotating bed reactor (RBR) for the biocatalytic synthesis of the anti-inflammatory drug (S)-naproxen. Both setups performed well during five repetitive bathes giving an enantiomeric excess of 99% and an isolated yield of 92%, but the RBR was easier to handle and the authors concluded that “… the rotating bed reactor concept can be regarded as a promising option for industrial applications”. Keywords: Biotransformation, Immobilized enzymes, Organic molecules, Scientific literature

Application L1606
Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale

Jochen Wachtmeister, Dörte RotherCurrent Opinion in Biotechnology, 42 (2016) 169-177 Keywords: Biotransformation, Encapsulated cells, Organic molecules, Scientific literature

Application 9001
Recycling of Immobilized Enzymes – Simple and Streamlined Biocatalysis

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

Application 1032
Rotating bed reactor for immobilized enzymatic reactions

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

Application 1025
Rotating bed reactor setup for in situ formation, loading and handling of alginate beads and whole cell encapsulation

Video showing the formation of alginate beads under conditions mimicking whole cell encapsulation. The use of a SpinChem® rotating bed reactor (RBR) allowed easy collection, maturing and washing of the alginate beads. With the RBR setup, it was possible to immediately continue with filling of the reaction substrate into the same vessel, thus reducing the number of handling steps and facilitating bead recycling. The beads showed no signs of physical wear after use in the RBR.   Keywords: Alginate, Biotransformation, Easy handling, One-pot multistep, Technology

Application 9002
Screening of Immobilized Enzymes – Fast and Convenient Reaction Optimization

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

Application 1027
Screening of immobilized lipases using magnetic rotating bed reactors

Six different immobilized lipases were screened in parallel for the esterification of lauric acid into propyl laurate using the pre-packed SpinChem® MagRBR Lipase screening kit. The process proved fast and simple, as efficient sampling and monitoring of the process was achieved without filtration steps, by keeping the immobilized catalyst confined inside the MagRBR.   Keywords: Biotransformation, Easy handling, Immobilized enzymes, Organic molecules, Rapid screening

Application 1009
Simultaneous extraction of two dyes selectively onto different resins

Video illustrating how a mixture of red and blue dyes with different chemical properties can be selectively extracted onto different adsorbents within the same run using a rotating bed reactor (RBR). The dyes were separated based on ionic and hydrophobic interactions, respectively.  Keywords: Cleantech, Decolouration, Extraction, Ion exchange, One-pot multistep, Organic molecules, Polymeric resin, Technology

Application L2001
Using Spinchem Rotating Bed Reactor Technology for Immobilized Enzymatic Reactions: A Case Study

Subhash Pithani, Staffan Karlsson, Hans Emtenäs, and Christopher T. ÖbergOrganic Process Research & Development 2019 23 (9), 1926-1931 "By using SpiChem RBR technology, we found that recycling of the immobilized enzyme was easy with preserved enantioselectivity and catalytic activity. The final optimized process was successfully demonstrated on a 1 kg scale with 39% isolated yield and 98.8% enantiomeric purity."     Keywords: Scientific literature

Application L1706
Whole-cell cascade biotransformations for one-pot multistep organic synthesis

This scientific minireview highlights that “one-pot multi-catalysis reactions is a revolutionary tool for multistep synthesis” and that “reaction engineering is indispensable to further advance biotransformation processes”. The authors concluded that “advanced reactor design such as … the SpinChem reactor, could also enhance cascade biotransformation processes”.   Keywords: Biotransformation, Encapsulated cells, One-pot multistep, Review, Scientific literature

Application L2110
Process design of a continuous biotransformation with in situ product removal by cloud point extraction

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.

Application 1040
The importance of baffles in a reactor vessels

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.

Application L2117
Multi‐enzyme cascade reaction in a miniplant two‐phase‐system : Model validation and mathematical optimization

In the American Institute of Chemical Engineers, AIChE Journal, the authors of this paper highlights the use of Rotating Bed Reactor (RBR) with two different immobilized enzymes at the same time in a cascade reaction. In the flow chart above of the miniplant consisting of a continuously stirred tank reactor (CSTR) equipped with an RBR (highlighted in orange) (a), a buffer tank (b), an extractive centrifuge (c) and a fixed bed reactor (d)  In the reaction scheme the complete multi-enzyme cascade is shown with the two enzymes placed in the RBR is highlighted. If you would like to get in contact with us give us a call or fill in the form.

Application L2112
Production of hydroxytyrosol rich extract from Olea europaea leaf with enhanced biological activity using immobilized enzyme reactors

The paper describes work to valorize olive leaves, the major by-product in olive oil production. Aqueous olive leaf extract is rich in oleuropein that was enzymatically hydrolyzed by beta-glucosidase to hydroxytyrosol and other products. Compared to the non-modified extract, the modified one demonstrated 20% higher antioxidant activity, seven-fold higher antibacterial activity, and enhanced cytotoxicity against leiomyosarcoma cells. The beta-glucoside was immobilized in chitosan-coated magnetic beads and used in both stirred tank reactor (STR) and rotating bed reactor (RBR). The immobilized enzyme showed good stability over time and the activity was good in both STR and RBR settings. However, the enzyme beads were damaged mechanically in the STR and could not be re-used. The RBR solved the problem of bead attrition allowing the immobilized enzyme to be re-used and thus giving superior total turnover number to the STR.

Application L2119
Pseudomonas taiwanensis biofilms for continuous conversion of cyclohexanone in drip flow and rotating bed reactors

This article involves the use of a rotating bed reactor for a biocatalytic process involving biofilms. The biofilm of Pseudomonas taiwanensis, with a genetically introduced Baeyer-Villiger monooxygenase (BVMO), was grown directly on 3D-printed polyamide inserts installed in the RBR S3. The biofilm, immobilized on the cartridges, was then used to catalyze the conversion of cyclohexanone. The authors comment that the work opens up the possibility to use biofilms in rotating bed reactors for synthesis via biocatalysis.

L-Asparaginase production in rotating bed reactor from Rhizopus microsporus IBBL-2 using immobilized Ca-alginate beads

The present work reports on the production of extracellular l-asparaginase from Rhizopus microsporus IBBL-2 using submerged fermentation (SmF) process free of glutaminase and urease activities. Scale-up studies involving 200-mL and 1-L rotating bed reactor (RBR) using immobilized beads were done and the results obtained are 20.21 U mL-1 and 19.13 U mL-1, respectively, the increased activity with immobilization accounts for reduced shear on cells due to increased stability as compared to the free-flowing cells.

Application L2102
New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy

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

Production of recombinant choline oxidase and its application in betaine production

The paper describes the author’s successful work to express and use recombinant choline oxidase. The gene for the choline oxidase was isolated from a Gram-positive soil bacterium, cloned into an expression vector, inserted into and overexpressed in a Rosetta expression system. The isolated recombinant choline oxidase was subsequently immobilized onto Ni-Sepharose beads, which were loaded in a rotating bed reactor (RBR). With the immobilized choline oxidase in the RBR, reaction conditions such as pH and temperature were optimized and the enzymatic activity measured for the reaction of choline to glycine betaine via betaine aldehyde.

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