Biocatalytic Foams from Microdroplet‐Formulated Self‐Assembling Enzymes

Hertel, Julian S.; Bitterwolf, Patrick; Kröll, Sandra; Winterhalter, Astrid; Weber, Annika J.; Grösche, Maximilian; Walkowsky, Laurenz B.; Heißler, Stefan; Schwotzer, Matthias; Wöll, Christof; van de Kamp, Thomas; Zuber, Marcus; Baumbach, Tilo; Rabe, Kersten S.; Niemeyer, Christof M.

doi:10.1002/adma.202303952

Cited by 6 publications

(2 citation statements)

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“…This could also serve as inspiration for further improvement of other types of cascade systems, such as hybrid enzyme-metal catalysts [70] or micro-and macroporous enzyme materials. [71][72][73][74]…”

Section: Discussionmentioning

confidence: 99%

Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

Kröll,

Burgahn,

Rabe

et al. 2023

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Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion‐reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano‐ and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot‐based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It is shown that the microscale compartment exerts a strong influence on the productivity of the cascade, whereas the nanoscale arrangement of enzymes has no influence but can be modulated by the insertion of a diffusion barrier.

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Section: Discussionmentioning

confidence: 99%

Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

Kröll,

Burgahn,

Rabe

et al. 2023

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“…Super‐resolution microscopy can be exploited to increase the spatial resolution of analysis, yet the sample preparation is highly complex and the enzyme labeling sometimes is not compatible with the reaction conditions (i.e., DNA‐PAINT) [24] . As advantage,, SP analysis can be performed with heterogeneous biocatalysts immobilized on industrially relevant carriers [25] (porous microparticles [26] and, membranes, [27] monoliths, [28] foams, [28] etc.) under process‐relevant conditions (i.e., high substrate concentrations) [29] .…”

Section: Sp/sm Assays To Profoundly Characterize Heterogeneous Biocat...mentioning

confidence: 99%

Single‐Particle and Single‐Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts

Diamanti,

López‐Gallego

2024

Angewandte Chemie

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Heterogeneous biocatalysis is highly relevant in biotechnology as it offers several benefits and practical uses. To leverage the full potential of heterogeneous biocatalysts, the establishment of well‐crafted protocols, and a deeper comprehension of enzyme immobilization on solid substrates are essential. These endeavors seek to optimize immobilized biocatalysts, ensuring maximal enzyme performance within confined spaces. To this aim, multidimensional characterization of heterogeneous biocatalysts is demanded. In this context, spectroscopic and microscopic methodologies conducted at different space and temporal scales can inform about the intraparticle enzyme kinetics, the enzyme spatial distribution, and mass transport issues. In this minireview, we identify enzyme immobilization, enzyme activity, and enzyme inactivation as the three fundamental processes for which advanced characterization tools unveil fundamental information. Recent advances in operando characterization of immobilized enzymes at the single‐particle (SP) and single‐molecule (SM) levels reveal valuable information on their functional properties unlocking the full potential of heterogeneous biocatalysis toward biotechnological applications.

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Flow‐Induced Microfluidic Assembly for Advanced Biocatalysis Materials

Lemke,

Schneider,

Kunz

et al. 2024

Adv Funct Materials

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Exploring the potential of microfluidic systems, this study presents a groundbreaking approach harnessing energy in microfluidic flows within a purpose‐built microreactor, enabling precise deposition of functional biomaterials. Upon optimizing reactor dimensions and integrating it into a microfluidic system, sequentially flow‐induced deposition of DNA hydrogels and transformation into DNA‐protein hybrid materials with SpyTag/SpyCatcher technology is investigated. However, limited functionalization rates restrict its viability for targeted biocatalytic processes. Therefore, the direct deposition of a phenolic acid decarboxylase is investigated, which is efficiently deposited but shows limited biocatalytic performance due to shear‐induced denaturation. This challenge is overcome by a two‐step immobilization process, resulting in microfluidic bioreactors demonstrating initial high space‐time yields of up to 7000 g L−1 d−1, but whose process stability proves unsatisfactory. However, by exploiting the principle of flow‐induced deposition to immobilize recombinant E. coli cells as functional living materials overexpressing biocatalytically relevant enzymes, bioreactors are produced that show equally high space‐time yields in continuous whole‐cell catalysis which remain constant over periods of up to 10 days. The insights gained offer optimization strategies for advanced functional materials and innovative reactor systems holding promise for applications in fundamental materials science, biosensing, and scalable production of microreactors for biocatalysis and bioremediation.

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Biocatalytic Foams from Microdroplet‐Formulated Self‐Assembling Enzymes

Cited by 6 publications

References 50 publications

Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

Single‐Particle and Single‐Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts

Flow‐Induced Microfluidic Assembly for Advanced Biocatalysis Materials

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