Using Adsorption Energy Distribution for Parameter Estimation of Competitive Cofactor Coupled Enzyme Reaction

The chemical and biotechnology industries are facing new challenges in the use of renewable resources. The complex nature of these materials requires the use of advanced techniques to understand the kinetics of reactions in this context. This study presents an interdisciplinary approach to analyze cofactor coupled enzymatic two-substrate kinetics and competitive two-substrate kinetics in a fast and efficient manner. By studying the adsorption energy distribution (AED), it is possible to determine the individual parameters of the reaction kinetics. In the case of a single alcohol reaction, the AED is able to identify parameters in agreement with the literature with few experimental data points compared to classical methods. In the case of a competitive reaction, AED analysis can automatically determine the number of competing substrates, whereas traditional nonlinear regression requires prior knowledge of this information for parameter identification

Concept of an Enzymatic Reactive Extraction Centrifuge

Biocatalytic processes often provide an ecological alternative to many chemical processes. However, further improvements in terms of the economic efficiency are required. In order to achieve that, the concept of process integration is a promising option. Applying this within a biocatalytic process, a highly integrated apparatus working as a reactive extraction centrifuge was developed and operated. For this purpose, a commercially available extraction centrifuge was modified to implement a biocatalytic reaction. The novel apparatus was used within a multi-enzyme cascade for the production of a natural flavor and fragrance, namely cinnamic ester. The characterization of the reactive extraction centrifuge and the suitable operation conditions for the inlet streams and the rotational speed for a stable operation were determined. Furthermore, different initial substrate concentrations were applied to prove the reaction. The results provide a successful proof of concept for the novel reactive extraction centrifuge

Process for the preparation of fatty aldehydes from renewable resources

Common methods for the chemical production of fatty aldehydes, widely applied as artificial flavors and fragrances, use fossil starting materials, harsh reaction conditions, and hazardous chemicals. This contribution introduces a novel, environmentally friendly process for the preparation of fatty aldehydes from the natural, renewable resource plant oil. It has successfully been shown that bringing a fatty acid into contact with zerovalent iron leads to the reduction of the fatty acid to the corresponding fatty aldehyde. In this paper, the three-step process is presented and discussed, exemplary for the preparation of dodecylaldehyd from dodecanoate.For the financing of this work the authors thank the Free and Hanseatic City of Hamburg (Förderzeichen LFF-FV43)

A multi-enzyme cascade for the production of High-Value Aromatic Compounds

Cascade reactions are the basis of life in nature and are adapted to research and industry in an increasing manner. The focus of this study is the production of the high-value aromatic ester cinnamyl cinnamate, which can be applied in flavors and fragrances. A three-enzyme cascade was established to realize the synthesis, starting from the corresponding aldehyde with in situ cofactor regeneration in a two-phase system. After characterization of the enzymes, a screening with different organic solvents was carried out, whereby xylene was found to be the most suitable solvent for the second phase. The reaction stability of the formate dehydrogenase (FDH) from Candida boidinii is the limiting step during cofactor regeneration. However, the applied enzyme cascade showed an overall yield of 54%. After successful application on lab scale, the limitation by the FDH was overcome by immobilization of the enzymes and an optimized downstream process, transferring the cascade into a miniplant. The upscaling resulted in an increased yield for the esterification, as well as overall yields of 37%.Cascade reactions are the basis of life in nature and are adapted to research and industry in an increasing manner. The focus of this study is the production of the high-value aromatic ester cinnamyl cinnamate, which can be applied in flavors and fragrances. A three-enzyme cascade was established to realize the synthesis, starting from the corresponding aldehyde with in situ cofactor regeneration in a two-phase system. After characterization of the enzymes, a screening with different organic solvents was carried out, whereby xylene was found to be the most suitable solvent for the second phase. The reaction stability of the formate dehydrogenase (FDH) from Candida boidinii is the limiting step during cofactor regeneration. However, the applied enzyme cascade showed an overall yield of 54%. After successful application on lab scale, the limitation by the FDH was overcome by immobilization of the enzymes and an optimized downstream process, transferring the cascade into a miniplant. The upscaling resulted in an increased yield for the esterification, as well as overall yields of 37%.Deutsche Forschungsgemeinschaft: BU3409/1-1 and WA 3957/1-

Evaluation of process integration for the intensification of a biotechnological process

Process intensification is a well-known and established method to improve ecological and economic efficiency of large-scale chemical processes. Process intensification in form of integrating an in-situ product or intermediate removal can also improve processes. In this study the effects of integrating an intermediate separation in form of an extraction centrifuge and a product separation in form of a pre-purification into a multi-enzyme cascade as well as the potential for further process integration in form of a reactive extraction centrifuge are analyzed for a biotechnological process. In order to evaluate the application of process integration, different process configurations are compared regarding to the efficiency using a model-based approach. Although the highly integrated process leads to lower space-time yields in some cases, an operating window can be identified in which the space-time yield is significantly higher compared to a sequential process. In order to distinguish both cases and thus choose a suitable operating point simulative studies are required. Moreover, a potential for further integration can be identified for the reference process studied in this work

Enzyme immobilization on synthesized nanoporous silica particles and their application in a bi-enzymatic reaction

The application of enzymes presents a great advantage regarding highly selective reactions; however, it involves also challenges due to their sensitivity. Immobilization offers one strategy to overcome those challenges enabling enzyme stabilization, as well as retention. In the present study, covalent attachment on hydrophilic amino-functionalized carriers is found to be the most promising immobilization method for the investigated reaction system. To achieve this, a novel method for preparation of silica particles with subsequent amino-functionalization is developed to prepare spherical carriers for enzyme immobilization, whereby high porosities are obtained based on polymerization. With these particles, immobilization of an alcohol dehydrogenase and a formate dehydrogenase is realized with residual activities of 70 and 80 % after 12 consecutive batches, respectively. The two immobilized enzymes are used in the reduction of cinnamyl aldehyde with in situ cofactor regeneration, obtaining a conversion of 100 % and up to 10-fold higher turnover numbers compared to the free enzyme.Supported by Deutsche Forschungsgemeinschaft (DFG, BU 3409/1-1 and DFG, WA 3957/1-1