The topic of integrating enzymatic reactions with in situ product removal is addressed. Different integrated reactive separations structured accordingly to the corresponding unit operations, i.e., reactive distillation, reactive chromatography, reactive crystallization, and extractive biocatalysis, are discussed. Special attention is given to their realization with homogenous and heterogeneous biocatalysis. Various enzyme immobilization techniques are presented and distinct strategies for installation of heterogeneous catalysts into process equipment are discussed.
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.
In this work, a continuous, heterogeneous extractive biocatalysis was realized with penicillin G hydrolysis as a model reaction. Therefore, commercially available structured packing CY from Sulzer were coated with enzyme‐containing gels and their catalytic activity was examined. Thereby, the potential of aqueous micellar two‐phase systems (ATPMS) as an extraction medium was evaluated. Triton X‐114 was used as model surfactant. It was found that the separation efficiency of the packing is not affected by the coating and ATPMS allows for a good separation yield. With these results, different process concepts for the continuous, heterogeneous extractive biocatalysis were compared. Overall, a countercurrent process with side inlet (YPAAside inlet = 78.1% ± 1.3%) has shown a great potential for a selective product separation and thus, higher yields in comparison to a monophasic batch operation (YPAAitalicbatch,italicmonophasic = 51.9% ± 0.6%) as well as a biphasic batch operation (YPAAitalicbatch,italicbiphasic = 67.6% ± 1.0%, wTriton X‐114 = 5%) were achieved.
The integration of an in situ extraction
into biocatalytic processes
is often limited by the toxicity of organic solvents. Therefore, it
is desirable to use water-based extraction systems (for example, aqueous
micellar two-phase systems). They can be used, for instance, for the
extraction of valuable products from microalgae cultures. Recently,
the nonionic surfactant ROKAnol NL5 was identified as a suitable
surfactant for this purpose, since it forms an upper micellar phase,
enabling an easy separation of whole-cell biocatalysts. However, its
application at temperatures below 45 °C is limited by unstable
phase boundaries, whereas the maximal temperature to ensure the vitality
of the most microalgae cultures is ∼40 °C. To overcome
this problem, the addition of long-chain alcohols to the surfactant–water
mixture during extraction is suggested in this work. Using 1-hexanol,
a continuous extraction process with the model solute trans-cinnamic acid at 40 °C in a stirred column could be realized.
The results of a new suggested water/ROKAnol NL5/1-hexanol
system at 40 °C (extraction yield, Y
cont = 97.67% ± 0.14%; enrichment factor, log10
T
CA = 2.42 ± 0.03; number of theoretical
stages, N
theo = 4.45 ± 0.16) are
comparable to those of the water/ROKAnol NL5 system at 45 °C
(Y
cont = 99.26% ± 0.24%, log10
T
CA = 2.60 ± 0.10, N
theo = 5.88 ± 0.67), ensuring, however,
no damage of microalgae.
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