Formulation of Enzyme-based Biomaterials for Flow-biocatalysis

Introduction
In flow biocatalysis, the concept of interconnected, continuously perfused reaction chambers with
integrated biocatalysts is used to improve reaction control and the efficiency of chemical syntheses. To
align such concepts for industrial use, an efficient and stable immobilization of enzymes and enzyme
cascades into biocatalytically active materials is essential. For this purpose, we have developed enzyme-
based biomaterials that can self-assemble into all-enzyme hydrogels (AEHs).[1] Such AEH-materials are
created by fusing genetically encoding coupling systems (SpyCatcher or SpyTag) to the enzyme of
interest.
Objectives
To show the broad applicability of the AEH-technology a variety of enzymes from different classes,
different multimerization states as well as cascades should be formulated into highly stable materials.
Results
By fusing enzymes, such as a phenolic acid decarboxylase (PAD),[2] or an alcohol dehydrogenase (ADH)
and a glucose 1-dehydrogenase (GDH),[1] or a xylose reductase (XR) and a galactitol dehydrogenase
(GalDH),[3] with the SpyCatcher (SC) or the SpyTag (ST), the site-specific mediated enzyme
... mehr
conjugation allowed the formulation of very stable and highly active biomaterials which consist almost
exclusively of enzymes in contrast to other carrier-based immobilization techniques. AEH based on the
bienzymatic ADH and GDH cascade can also be formulated directly inside cells[4] and recently we
further enhanced the storage and process stability by formulating the material as a foam.[3] The novel
AEH-materials were extensively characterized by a number of methods such as dynamic light scattering
(DLS), scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray tomography,
fluorescence microscopy, laser scanning microscopy (LSM), IR spectroscopy and thermogravimetry.
Moreover, the gels showed excellent stereoselectivity, stable conversion rates, high space-time yields
(STY) and cofactor retention for more than four days in continuous flow experiments.[5-6]
Conclusion
AEHs, especially in their novel formulation as foam, are a promising material with high application
potential for sustainable and environmentally friendly industrial biocatalysis, compatible with a wide
range of enzyme classes. Continuous improvement through innovative formulations will further increase
the stability and expand the application possibilities.
References
1 T. Peschke et al., C. M. Niemeyer, Angew. Chem. Int. Ed. 2018, 57, 17028- 17032.
2 E. Mittmann et al., K. S. Rabe, Micromachines 2019, 10, 795.
3 J. S. Hertel et al., C. M. Niemeyer, Adv. Mater. 2023, 35, e2303952.
4 P. Bitterwolf et al., C. M. Niemeyer, Chem. Eur. J. 2022, 28, e202202157.
5 P. Bitterwolf et al., C. M. Niemeyer, Micromachines 2019, 10, 783.
6 T. Peschke et al., C. M. Niemeyer, Catalysts 2019, 9, 164