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Enzyme catalysis at high hydrostatic pressure
Citation Link: https://doi.org/10.15480/882.13865
Publikationstyp
Doctoral Thesis
Date Issued
2024
Sprache
English
Author(s)
Advisor
Referee
Pörtner, Ralf
Title Granting Institution
Technische Universität Hamburg
Place of Title Granting Institution
Hamburg
Examination Date
2024-11-11
Institute
TORE-DOI
Citation
Technische Universität Hamburg (2024)
In the past, several methods have been developed to improve the application of the biocatalysts, such as immobilizing enzymes on support materials to ensure reusability, improve stability, and maintain their activity. Furthermore, the use of non-aqueous media and genetic engineering has been applied to further enhance the enzyme performance with respect to stability, activity and selectivity. However, the evaluation of additional or synergistic process parameters is still part of research with the aim of enabling the application of enzymes in chemical synthesis. Recently, the application and effect of high hydrostatic pressure (HHP) to enzymatic catalyzed reactions is becoming increasingly important. This thesis explored the synergistic potential of combining common and innovative methods to improve enzyme performance with superior stabilization and enzyme activity, thereby contributing to the development of more efficient biocatalysts.
In order to investigate the effect of HHP on three different enzymes, a reactor concept was designed first. A continuously operated packed bed reactor (PBR) was selected to fulfill the requirement for continuous operation as part of process intensification and to enable quick and easy adjustment of process parameters. An appropriate immobilization method was developed for two selected lipases to ensure their application in a packed bed reactor (PBR) with the highest loading of the enzyme immobilizates possible. In particular, mechanical stability during continuous reactor operation under ambient and high hydrostatic pressure conditions and the leaching of enzymes from the carrier were investigated. After the establishment of the HHP reactor system, the influence of HHP on the enzyme performance including enzyme stability, activity, selectivity and kinetic parameters was investigated representing the core of the thesis.
Three industrially relevant enzymes, Candida rugosa lipase (CRL) and Candida antarctica lipase B (CalB) from enzyme class 3 (EC 3), as well as Ruegeria pomeroyi polyphosphate kinase (PPK) of EC 2 were chosen to investigate the impact of HHP of up to 1200 bar. This investigation aims to determine the intra- and inter-enzyme-class-specific effects of HHP on the enzyme properties.
Particularly, the lipase-catalyzed transesterification reaction was used for a detailed study of the effect of HHP, as it allowed the investigation of both stability and activity changes, as well as changes in selectivity. This was of great interest as regio- as well as enantioselectivity differentiates enzymes from chemical catalysts. Additionally, the investigation included the effect of HHP on the kinetic parameters and characteristics of CRL. The resulting kinetic parameters vmax, KM,vin, KM,PP and Ki,vin were adapted to the experimental data using Michaelis-Menten type kinetics at ambient and high pressure.
HHP was investigated as a synergistic and possible process intensification parameter for the cofactor regeneration process catalyzed by the industrially relevant PPK. The applicability of the enzyme immobilizates and their performance in a continuously operated reactor system were studied and compared with results obtained in a discontinuously operated stirred tank reactor (STR).
Therefore, the objective of this work was to conceptualize and investigate the operability of a continuously operated high pressure reactor system. The influence of HHP on different classes of enzymes was investigated as a complementary process parameter in the conceptualized reactor system.
In order to investigate the effect of HHP on three different enzymes, a reactor concept was designed first. A continuously operated packed bed reactor (PBR) was selected to fulfill the requirement for continuous operation as part of process intensification and to enable quick and easy adjustment of process parameters. An appropriate immobilization method was developed for two selected lipases to ensure their application in a packed bed reactor (PBR) with the highest loading of the enzyme immobilizates possible. In particular, mechanical stability during continuous reactor operation under ambient and high hydrostatic pressure conditions and the leaching of enzymes from the carrier were investigated. After the establishment of the HHP reactor system, the influence of HHP on the enzyme performance including enzyme stability, activity, selectivity and kinetic parameters was investigated representing the core of the thesis.
Three industrially relevant enzymes, Candida rugosa lipase (CRL) and Candida antarctica lipase B (CalB) from enzyme class 3 (EC 3), as well as Ruegeria pomeroyi polyphosphate kinase (PPK) of EC 2 were chosen to investigate the impact of HHP of up to 1200 bar. This investigation aims to determine the intra- and inter-enzyme-class-specific effects of HHP on the enzyme properties.
Particularly, the lipase-catalyzed transesterification reaction was used for a detailed study of the effect of HHP, as it allowed the investigation of both stability and activity changes, as well as changes in selectivity. This was of great interest as regio- as well as enantioselectivity differentiates enzymes from chemical catalysts. Additionally, the investigation included the effect of HHP on the kinetic parameters and characteristics of CRL. The resulting kinetic parameters vmax, KM,vin, KM,PP and Ki,vin were adapted to the experimental data using Michaelis-Menten type kinetics at ambient and high pressure.
HHP was investigated as a synergistic and possible process intensification parameter for the cofactor regeneration process catalyzed by the industrially relevant PPK. The applicability of the enzyme immobilizates and their performance in a continuously operated reactor system were studied and compared with results obtained in a discontinuously operated stirred tank reactor (STR).
Therefore, the objective of this work was to conceptualize and investigate the operability of a continuously operated high pressure reactor system. The influence of HHP on different classes of enzymes was investigated as a complementary process parameter in the conceptualized reactor system.
Subjects
Biocatalysis
High Pressure
Enzymes
DDC Class
660.6: Biotechnology
Funding Organisations
More Funding Information
The thesis with the project number 031B0723 was carried out in close cooperation with the industrial partner GALAB Laboratories GmbH.
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