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%.

The sufficient provision of oxygen is mandatory for enzymatic oxidations in aqueous solution, however, in process optimization this still is a bottleneck that cannot be overcome with the established methods of macrobubble aeration. Providing higher mass transfer performance through microbubble aerators, inefficient aeration can be overcome or improved. Investigating the mass transport performance in a model protein solution, the microbubble aeration results in higher k L a values related to the applied airstream in comparison with macrobubble aeration. Comparing the aerators at identical k L a of 160 and 60 1/h, the microbubble aeration is resulting in 25 and 44 times enhanced gas utility compared with aeration with macrobubbles. To prove the feasibility of microbubbles in biocatalysis, the productivity of a glucose oxidase catalyzed biotransformation is compared with macrobubble aeration as well as the gas-saving potential. In contrast to the expectation that the same productivities are achieved at identically applied k L a, microbubble aeration increased the gluconic acid productivity by 32% and resulted in 41.6 times higher oxygen utilization. The observed advantages of microbubble aeration are based on the large volume-specific interfacial area combined with a prolonged residence time, which results in a high mass transfer performance, less enzyme deactivation by foam formation, and reduced gas consumption. This makes microbubble aerators favorable for application in biocatalysis.

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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 aminofunctionalization 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. afterwards for immobilization without stirring for 22 h at room temperature. Afterwards the resins were filtered and washed buffer.

Carbon capture technology can be set up in combination with biocatalysis to utilize the bound CO2 as substrate in the Kolbe‐Schmitt like enzymatic reaction. The exemplary whole cell biotransformation of catechol to 2,3‐dihydroxybenzoic acid in a triethanolamine‐mediated multiphase system shows increased equilibrium conversion. Apart from the beneficial thermodynamics, the inherent fluid properties of triethanolamine is enabling easy application of CO2 fine bubbles as highly efficient gassing method to minimize the CO2 demand and CO2 emissions.

Biotechnological application of multiple enzymes in different phases for target compounds synthesis poses a significant challenge for industrial process development. At the same time, a growing demand for natural flavors and fragrances opens up possibilities for novel biotechnological processes to replace current chemical synthesis routes, with additional advantages such as avoiding harsh reaction conditions and toxic chemicals, and less by-products in the system. Within complex biotechnological processes, the key for unfolding their industrial application potential in bioprocess engineering lies in their mathematical modeling. In this contribution, a multi-enzyme cascade reaction in a two-phase system implemented in a miniplant-scale reactor setup is mathematically modeled for the example of the flavoring agent cinnamyl cinnamate. Using our validated model and a mathematical optimization tool based on a genetic algorithm, optimization runs are performed to demonstrate the potential of computer-aided process development for complex biotechnological processes.

BACKGROUND Spray congealing is investigated as a new innovative immobilization technique for biocatalysts. The immobilization is realized by atomization of a mixture of a melted carrier and the enzyme, which is re‐solidifyed due to temperature decrease. This method needs neither organic solvents, nor difficult downstream processing after preparation. For the encapsulation of different matrices, long chain alcohols and glyceryl derivatives, with melting points below 86 °C and environmentally friendly properties, are chosen and tested for their suitability for enzyme encapsulation. RESULTS The immobilization of a laccase (EC 1.10.3.2, Novozym 51003, from Myceliophthora thermophila) was investigated and the produced microparticles were characterized. The obtained particles showed a mean particle size around 40 μm and a spherical surface with different structures depending on the used matrix. Spray congealing with the laccase reached yields of 80% and residual activities up to 56%. Furthermore, four consecutive reactions were realized with cetyl alcohol as carrier and an activity of 20% was retained. CONCLUSION Cetyl alcohol was found to be the most appropriate carrier for the immobilization of the investigated laccase, due to easy handling and high yields. In comparison with the other tested carriers, the best retained activities were obtained and recycling of the biocatalyst was realized. © 2017 Society of Chemical Industry

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