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Biocatalysis in Pickering emulsions

Schmidt, Christine Vera Isabel LU (2023) KBTM01 20222
Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
Abstract
Employing enzymes as catalysts in the production of chiral amines that are both used as building blocks and active pharmaceutical ingredients might be promising due to milder reaction conditions compared to non-biological catalysts, exclusion of heavy metals, high selectivity towards substrates and precision in chirality of the products. Transaminases are attractive for the use in this process. In an industrial setting, to obtain feasible yields, the amount of substrate supplied
must be high. Most of the substrates are impolar organic solvents causing formation of two phases and the respective interphase which most of the transaminases are sensitive towards. This has as a consequence the deactivation of the enzymes in a usual biphasic... (More)
Employing enzymes as catalysts in the production of chiral amines that are both used as building blocks and active pharmaceutical ingredients might be promising due to milder reaction conditions compared to non-biological catalysts, exclusion of heavy metals, high selectivity towards substrates and precision in chirality of the products. Transaminases are attractive for the use in this process. In an industrial setting, to obtain feasible yields, the amount of substrate supplied
must be high. Most of the substrates are impolar organic solvents causing formation of two phases and the respective interphase which most of the transaminases are sensitive towards. This has as a consequence the deactivation of the enzymes in a usual biphasic system.
In this project, a possible solution was investigated, namely the use of Pickering emulsions. The hypothesis was that the Pickering emulsion will protect the enzyme from deactivation.
As biocatalyst, an ω-transaminase was employed, using the deactivated E. coli cells the enzymes were expressed in. Both the cells and the Pickering emulsion gave a colloidal system.
The aim was the development of an assay using spectrophotometry and to investigate the hypothesis with the help of it.
In each assay, a Pickering emulsion, a monophasic, and a biphasic system were compared. This was done by two reactions. The first reaction had the purpose to simulate a longterm exposure of the enzyme to the reactants, and the second reaction was used to determine the activity at the time point the sample was taken. Acetophenone was the reaction product of this second reaction.
It has a characteristic absorbance maximum at 245 nm, so this wavelength was used in the spectrophotometric measurements.
Transferring a sample from the first reaction into a 40X diluted solution containing the reactants for the second reaction inside the quartz cuvette without any processing of the suspension and subsequent measurement of the absorbance every 5 seconds lead to unreliable results. The particles in the cuvette disturbed the beam in a manner that the curves resulting from this measurement became very craggy. This lead to the conclusion that removing particles before absorbance measurement is necessary.
To remove all particles, centrifugation was applied on samples taken from the second reaction at every second minute. After dilution, the absorbance was measured. The results were more reliable, so stop assay was the method of choice in all the following experiments.
The assay was performed in two different scales, namely in 4.5 mL glass vials and in Eppendorf tubes. Here, it could be shown that when using Eppendorf tubes, employing the ones with a volume of 2 mL is necessary.
An outcome of the project is that the geometry of the vials is crucial, so when applying 1000 rpm on 4.5 mL vials, the Pickering emulsion did not stay intact in contrast to applying this shaking speed on 2 mL Eppendorf tubes where the results seem to confirm the hypothesis, but this should be double-checked in future investigations.
As well in future research, a method for sampling at t0 has to be developed, and the shaking speed that has to be applied for good mixing and an intact Pickering emulsion has to be found with respect to the vial used. (Less)
Popular Abstract
The left foot is the mirror image of the right foot. Similar to that the left shoe does not fit on the right foot, the correct mirror image of a pharmaceutical molecule needs to be administered in order to reach the desired effects.
In classical chemical reactions, both mirror images of a molecule are obtained in a 50:50 ratio.
Separating these is both costly and causes high amounts of solvent waste.
Enzymes facilitate chemical reactions in biological systems. They are very selective towards reactants, but also in the precision in terms of which mirror image of the product is formed. On top of this, these reactions are carried out at mild conditions. In industrial production, all these properties are very attractive. The successful... (More)
The left foot is the mirror image of the right foot. Similar to that the left shoe does not fit on the right foot, the correct mirror image of a pharmaceutical molecule needs to be administered in order to reach the desired effects.
In classical chemical reactions, both mirror images of a molecule are obtained in a 50:50 ratio.
Separating these is both costly and causes high amounts of solvent waste.
Enzymes facilitate chemical reactions in biological systems. They are very selective towards reactants, but also in the precision in terms of which mirror image of the product is formed. On top of this, these reactions are carried out at mild conditions. In industrial production, all these properties are very attractive. The successful implementation of enzymes would enable a big economization of steps and solvent use, and thereby a reduction in environmental pollution, usually caused by the energy use that is necessary to reach the high temperatures and pressures used, but also solvents. Economizing energy and solvents is reducing overall process costs. The downside of some enzymes, but especially concerning the ones used in this project, is the susceptibility towards surface contacts which is a problem as the enzymes perform the reaction in an aqueous phase, but the substrate is in the organic, non-polar phase. The presence of this surface will cause enzymatic
deactivation.
Here, the employment of an emulsion was investigated. An emulsion is a mixture of two liquids that are immiscible, for example a well-mixed salad dressing made of oil and vinegar. An oil-vinegar dressing is splitting up into an oil and a vinegar phase again if it is not mixed any longer.
This can be avoided by adding an emulsion stabilizing agent as it is done in mayonnaise with egg-yolk. Here, instead of egg-yolk, starch particles were used. The particles cover the surface of the non-polar liquid’s droplets.
The hypothesis here was that the presence of the emulsion comes with a higher surface and thereby higher rates of substrate leakage into the water phase, but with longer lifetime of the enzymes as the starch particles might form a barrier, meaning surface contact is avoided, and thereby the source of deactivation might be reduced.
In the experiments, it turned out that the form of the vial that was used has a very big impact on the enzyme stability. It could be shown that employing Pickering emulsions might be beneficial, but more research is needed to understand the system better which is crucial for successful application. (Less)
Please use this url to cite or link to this publication:
author
Schmidt, Christine Vera Isabel LU
supervisor
organization
course
KBTM01 20222
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Enzymes, Pickering emulsion, Amine Transaminases, Green Chemistry, Stereoselectivity, Biotechnology
language
English
id
9130800
date added to LUP
2023-06-28 11:38:49
date last changed
2024-06-27 07:57:16
@misc{9130800,
  abstract     = {{Employing enzymes as catalysts in the production of chiral amines that are both used as building blocks and active pharmaceutical ingredients might be promising due to milder reaction conditions compared to non-biological catalysts, exclusion of heavy metals, high selectivity towards substrates and precision in chirality of the products. Transaminases are attractive for the use in this process. In an industrial setting, to obtain feasible yields, the amount of substrate supplied
must be high. Most of the substrates are impolar organic solvents causing formation of two phases and the respective interphase which most of the transaminases are sensitive towards. This has as a consequence the deactivation of the enzymes in a usual biphasic system.
In this project, a possible solution was investigated, namely the use of Pickering emulsions. The hypothesis was that the Pickering emulsion will protect the enzyme from deactivation.
As biocatalyst, an ω-transaminase was employed, using the deactivated E. coli cells the enzymes were expressed in. Both the cells and the Pickering emulsion gave a colloidal system.
The aim was the development of an assay using spectrophotometry and to investigate the hypothesis with the help of it.
In each assay, a Pickering emulsion, a monophasic, and a biphasic system were compared. This was done by two reactions. The first reaction had the purpose to simulate a longterm exposure of the enzyme to the reactants, and the second reaction was used to determine the activity at the time point the sample was taken. Acetophenone was the reaction product of this second reaction.
It has a characteristic absorbance maximum at 245 nm, so this wavelength was used in the spectrophotometric measurements.
Transferring a sample from the first reaction into a 40X diluted solution containing the reactants for the second reaction inside the quartz cuvette without any processing of the suspension and subsequent measurement of the absorbance every 5 seconds lead to unreliable results. The particles in the cuvette disturbed the beam in a manner that the curves resulting from this measurement became very craggy. This lead to the conclusion that removing particles before absorbance measurement is necessary.
To remove all particles, centrifugation was applied on samples taken from the second reaction at every second minute. After dilution, the absorbance was measured. The results were more reliable, so stop assay was the method of choice in all the following experiments.
The assay was performed in two different scales, namely in 4.5 mL glass vials and in Eppendorf tubes. Here, it could be shown that when using Eppendorf tubes, employing the ones with a volume of 2 mL is necessary.
An outcome of the project is that the geometry of the vials is crucial, so when applying 1000 rpm on 4.5 mL vials, the Pickering emulsion did not stay intact in contrast to applying this shaking speed on 2 mL Eppendorf tubes where the results seem to confirm the hypothesis, but this should be double-checked in future investigations.
As well in future research, a method for sampling at t0 has to be developed, and the shaking speed that has to be applied for good mixing and an intact Pickering emulsion has to be found with respect to the vial used.}},
  author       = {{Schmidt, Christine Vera Isabel}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Biocatalysis in Pickering emulsions}},
  year         = {{2023}},
}