Carrier-Free Enzyme Immobilization for High-Density Catalytic Architectures in Flow Biocatalysis

Abstract

Carrier-free enzyme immobilization has become a central design concept for constructing high-density catalytic architectures in flow biocatalysis. This Perspective discusses the evolution of such materials from early cross-linked enzyme aggregates and crystals to biologically derived inclusion bodies, all-enzyme hydrogels (AEHs), and genetically encoded protein crystals. These carrier-free systems achieve high enzyme loadings, enhanced stability, and improved cofactor retention, thereby enabling continuous processing under mild conditions. Among them, AEHs currently provide the most versatile experimental platform, combining high catalytic density with tunable porosity and mechanical flexibility that favor mass transport and integration into microstructured reactors. Genetically encoded crystalline assemblies complement this development through molecular precision and robustness. The article further highlights emerging trends in automation and data-driven reactor control, including the use of inline analytics for autonomous optimization, as well as the growing importance of standardized data management following the FAIR (findable, accessible, interoperable, reusable) principles. Together, these advances outline a path toward precision biocatalysis, where material design, reactor engineering, and digital control converge to enable sustainable and reproducible enzyme-driven manufacturing.

1. From Sustainable Biocatalysis to Continuous Flow: Reimagining Enzyme Immobilization for High-Density Catalytic Architectures

Industrial biocatalysis is widely regarded as a cornerstone of the sustainable chemical industry, providing environmentally benign routes to chemicals, materials, and fuels that traditionally rely on petrochemical synthesis. − The proven advantages of enzymatic catalysis, including mild reaction conditions, high chemo-, regio-, and stereoselectivity, and the possibility of tailoring enzymes by protein engineering, have enabled the green synthesis of a wide range of value-added products such as pharmaceuticals and fine chemicals. − These developments underpin the role of white biotechnology as a transformative pillar of the sustainable economy, advancing the use of renewable feedstocks and environmentally compatible production routes. ,

To translate laboratory success into robust industrial practice, challenges such as catalyst stability, reusability, and process intensification must be overcome. Continuous-flow biocatalysis has emerged as an attractive strategy to meet these requirements. − In this context, flow biocatalysis operates within the general framework of heterogeneous catalysis, where the active species are immobilized and spatially confined, enabling continuous operation and catalyst reuse. Building on the principles of flow chemistry, continuous systems provide constant operation, efficient mass and heat transfer, and accurate control of reaction parameters, which together result in higher reproducibility and scalability. , Moreover, the modular nature of flow reactors facilitates the integration of sequential reaction steps within fluidically coupled compartments, enabling the realization of multienzyme cascades under defined conditions. ,−

A key requirement for implementing continuous biocatalytic processes is the immobilization of enzymes, for which a broad range of methodologies has been developed over the past decades. Beyond classical covalent attachment to solid supports, well-established strategies also include enzyme entrapment or encapsulation within hydrogels, silica, metal–organic frameworks, or polymeric matrices. − In general, immobilization enhances operational stability, simplifies catalyst recovery, and enables spatial organization of biocatalytic functions within micro- or mesostructured reactors. However, conventional carrier-based approaches often suffer from reduced catalytic activity, mass-transfer limitations, or low enzyme loading capacity, which are particularly detrimental in flow systems where high catalyst density and short diffusion distances are essential. ,−

Nature provides powerful design principles for efficient multienzyme catalysis. Within living cells, catalytic cascades proceed in confined compartments such as organelles, metabolons, or enzyme complexes, which ensure high local substrate concentrations and rapid transfer of reactive intermediates while suppressing undesired side reactions. − By contrast, cell-free systems often fail to reproduce this level of organization, as enzymes are typically dispersed in dilute solution or loosely attached to inert carriers. This limitation has motivated the development of catalytic architectures that mimic the efficiency of biological compartmentalization under cell-free conditions.

Recent advances in carrier-free enzyme immobilization address these challenges by creating self-supporting biocatalytic materials with intrinsically high catalytic-site density. Such materials combine the stability benefits of immobilization with minimal diffusion barriers and high volumetric activity, offering significant advantages for continuous-flow operation. The present article discusses emerging strategies for constructing and applying these high-density, carrier-free biocatalytic architectures and outlines how they can enable the next generation of efficient and sustainable flow biocatalysis processes.

2. When Proximity Becomes Performance: Mechanistic Foundations of High-Density Catalysis

Biological systems achieve exceptional catalytic efficiency through spatial organization. Enzymes within cells are not randomly distributed but confined to structured micro- and nanodomains such as organelles, metabolons, and multienzyme complexes, which control metabolite flux and maintain locally high substrate concentrations. − In addition to classical membranes, nature employs membrane-less compartments or biomolecular condensates that dynamically assemble and disassemble to regulate metabolism. These condensates concentrate catalysts and substrates, fine-tune physicochemical microenvironments, and modulate conformational equilibria of enzymes. , The dense phase of such condensates can enhance catalytic turnover by stabilizing active conformations or creating favorable pH microgradients, although diffusion retardation or crowding effects may counteract these benefits. Collectively, these biological systems illustrate how high local enzyme densities and confined environments can promote efficient catalysis through optimized mass transport and environmental control.

Translating these natural design principles into cell-free systems motivates the development of high-density catalytic architectures. Yet, it is crucial to recognize that simple enzyme proximity does not automatically produce the benefits observed in nature. Proximity does not equal substrate channeling. Substrate channeling, or “facilitated transport,” refers to the direct diffusion of an intermediate from one active site to the next without entering the bulk solution, as found in natural multienzyme complexes such as tryptophan synthase (Figure A). − It can arise from physical tunnels, electrostatic steering, or swinging domains that covalently transfer intermediates. , Artificial cascades, however, typically consist of independently evolved enzymes without such structural coupling. Theoretical analyses showed that for freely diffusing intermediates, enzyme proximity enhances reaction rates only transiently (Figure B). The first enzyme (blue) converts the substrate (S) into the intermediate (I), which can either diffuse into the bulk solution or directly reach the second enzyme (green). Accordingly, the overall production rate k _total can be described as the sum of a direct enzyme-to-enzyme flux (k _direct) and a contribution arising from intermediates that diffuse through the surrounding bulk phase (k _bulk) (Figure C). Theoretical modeling demonstrated that, once steady state is reached, diffusion into and out of the bulk phase dominates overall turnover, while direct transfer between enzymes contributes only marginally. −

1.

Mechanistic origins of high-density catalytic performance. (A) Substrate channeling in tryptophan synthase. The indole intermediate generated by the α-subunit (blue) is transferred directly through a protein tunnel to the β-subunit (green) without entering the bulk solution. Adapted with permission from ref . Copyright 2017 Wiley-VCH. (B) Diffusion model illustrating intermediate flux in a two-enzyme cascade. The intermediate I formed by the first enzyme (blue) can either diffuse into and back from the bulk (dashed green arrow) or reach the second enzyme (green) directly (dashed blue arrow), with associated rates k­(bulk) and k­(direct). (C) Time-dependent contribution of the two pathways to product formation. After a short transient phase, k­(bulk) dominates over k­(direct), at t_eq, both streams contribute equally to the product formation at the second enzyme, underscoring that mere proximity does not enhance steady-state cascade throughput. (D) Immobilized enzymes create local diffusion layers that retain intermediates (purple), forming effective microscale or ″virtual″ compartments that reduce escape into the bulk and enhance overall turnover. (B, C) Adapted with permission from ref , Copyright 2017 Wiley-VCH, and ref , Copyright 2013 Elsevier.

Experimental studies corroborate these findings. DNA-origami-based enzyme assemblies revealed that initial rate enhancements vanish after a short transient phase as diffusive equilibration is established (Figure C). − Comparable results were obtained for scaffolds composed of metal–organic frameworks or protein aggregates, where catalytic rates showed no consistent dependence on enzyme spacing. These observations highlight that enhanced cascade throughput arises not from direct substrate channeling between adjacent enzymes but from compartmentalization effects that confine intermediates and substrates near catalytic sites.

Consequently, the concept of compartmentalization, rather than simple proximity, provides the correct framework to explain the enhanced performance of multienzyme assemblies. Diffusion layers surrounding immobilized enzymes or porous matrices can act as microscale compartments (Figure D) that retain intermediates and sustain elevated local concentrations. Weak substrate–surface interactions can further create “virtual compartments” that prevent loss of intermediates into the bulk phase and accelerate overall turnover. The activity increases frequently observed for coimmobilized enzyme cascades − can be rationalized by high local enzyme concentrations that create confined diffusion zones rather than by direct substrate tunneling. Modeling confirms that sustained enhancement arises from the collective behavior of large enzyme populations providing multiple reaction sites for diffusing intermediates, not from pairwise proximity effects. −

At a conceptual level, the functional benefit of metabolic compartmentalization relies on the establishment of controlled microenvironments and regulated boundaries rather than on compartment formation per se. Consequently, compartment-like effects are size- and transport-dependent and do not scale indefinitely. Theoretical analyses predict critical or optimal compartment dimensions, beyond which catalytic productivity plateaus or approaches bulk-like behavior as diffusion paths increase and effective enzyme packing density becomes less favorable. Minimal kinetic models of compartmentalized enzyme cascades further identify boundary transport, in particular diffusional resistance and intermediate leakage, as key determinants of whether compartmentalization outperforms free-solution catalysis, revealing threshold regimes where the advantage vanishes. Importantly, these predictions are supported by experimental work: peroxisomal reconstitution studies demonstrate upper functional limits, where enlarged organelles retain the full enzymatic machinery but fail to sustain efficient coupled reactions due to scaling-dependent effects such as altered enzyme density and cofactor balance.

An analogous principle applies to cofactor-dependent enzymatic reactions, where the local retention and recycling of expensive cofactors such as NAD­(P)⁺/NAD­(P)H are essential for efficient operation in continuous systems. In self-sufficient heterogeneous biocatalysts, cofactors are coimmobilized with their partner enzymes within porous or polymeric matrices, enabling in situ regeneration and minimizing diffusive loss. Recent studies have demonstrated diverse strategies for achieving this goal, including ionic and reversible covalent cofactor attachment, engineered enzyme–particle hybrids, and additive-manufactured hydrogels that encapsulate both catalysts and cofactors to ensure long-term stability under flow. − These approaches illustrate that dense biocatalytic environments can simultaneously promote substrate turnover and cofactor confinement, a prerequisite for developing self-sufficient catalytic architectures.

This concept culminates in carrier-free biocatalytic materials that integrate catalytic and cofactor-regenerating functions within a single dense phase. A representative example involves self-assembling all-enzyme hydrogels, which illustrate the potential of such systems to achieve high activity and exceptional cofactor retention under continuous operation. By eliminating noncatalytic carrier mass, these materials fully exploit the available reactor volume and thereby maximize volumetric catalytic density and productivity, which cannot be achieved with conventional supports. Such carrier-free high-density architectures mark the transition from immobilized enzyme systems to a new generation of compact, self-sufficient biocatalytic materials that will be discussed in detail in the following section.

3. Strategies for Carrier-Free Enzyme Immobilization

Building on the advantages of dense catalytic architectures, carrier-free immobilization transforms enzymes themselves into self-supporting catalytic materials. By eliminating inert support mass, these systems make full use of the reactor volume and enhance volumetric productivity while preserving high catalytic efficiency. Current strategies to realize such materials follow three main concepts. Cross-linked enzyme assemblies convert soluble enzymes into mechanically stable, reusable solids through chemical cross-linking. Catalytically active inclusion bodies exploit nature’s intrinsic tendency for protein aggregation, yielding functional enzyme particles directly from cellular expression systems. Finally, self-assembling all-enzyme hydrogels integrate catalytic and cofactor-regenerating enzymes into continuous, porous networks that combine high enzyme density with structural cohesion. Together, these approaches exemplify how enzymes can be transformed from dispersed molecular catalysts into compact, structurally defined catalytic matter, paving the way for efficient and sustainable flow bioprocesses.

3.1. Cross-Linked Enzyme Assemblies: From Crystals to Aggregates

Cross-linked enzyme assemblies (CLEs) represent one of the earliest and most established routes to carrier-free immobilization. In these systems, soluble enzymes are first brought into the solid phase by either crystallization or nonspecific aggregation, producing enzyme crystals or amorphous aggregates, respectively (Figure A). Chemical cross-linking with bifunctional reagents such as glutaraldehyde then stabilizes these precipitates into insoluble, catalytically active materials that contain no inert support, yielding cross-linked enzyme crystals (CLECs) or cross-linked enzyme aggregates (CLEAs) (Figure B). ,, Their enzyme packing density can approach theoretical maxima. For a 30 kDa globular protein of approximately 2 nm radius, concentrations up to 25 mM, corresponding to roughly 700 mg mL^–1, have been estimated. ,

2.

Schematic overview of carrier-free immobilization routes based on cross-linked enzyme assemblies. (A) Soluble enzymes can be converted into the solid state either by crystallization, forming enzyme crystals (EC), or by nonspecific precipitation, forming enzyme aggregates (EA). Subsequent treatment of these precursors with bifunctional cross-linkers (purple) yields cross-linked enzyme crystals (CLECs) or cross-linked enzyme aggregates (CLEAs), respectively. (B) Chemical cross-linking with reagents such as glutaraldehyde (purple) generates covalent bridges between lysine residues, stabilizing the crystalline or amorphous protein matrices. (C) Microscopic analysis of halohydrin dehalogenase CLECs. Reproduced from ref . Available under a CC-BY 4.0 license. Copyright 2023 Staar et al. (D) SEM image of magnetic combi-CLEAs. Reproduced with permission from ref . Copyright 2018 Elsevier. (E) Coprecipitation and cross-linking of two or more enzymes produces multienzyme assemblies (combi-CLEAs) that support cascade transformations within a single particle.

CLECs were first reported in the 1960s for carboxypeptidase A and have since been applied to a range of enzymes. In crystalline form, enzymes largely retain their native structure and therefore display enhanced resistance to pH fluctuations, elevated temperatures, and organic solvents. CLECs can be produced at preparative scale and offer excellent mechanical robustness, as demonstrated for engineered halohydrin dehalogenase and for lipases in industrially relevant reactor formats. , Their preparation, however, requires high enzyme purity and reproducible crystallization conditions to afford stable enzyme crystals (Figure C). Since only a single enzyme species crystallizes at a time, CLECs are not readily suited to multienzyme cascade systems.

CLEAs provide a more accessible alternative because they rely on precipitation by salts or organic solvents rather than crystallization (Figure A). This simplicity enables the use of partially purified or even crude enzyme preparations and has made CLEAs attractive for scale-up. CLEAs typically exhibit high stability toward temperature, pH, and organic solvents and effectively prevent enzyme leaching. Challenges remain, including potential activity loss due to nonspecific cross-linking and mechanical fragility during handling. These limitations can be mitigated by additives such as aminated feeder molecules for controlled cross-linking, surfactants or ionic polymers to tune porosity, and even magnetic nanoparticles for improved recovery or process integration (Figure D).

Despite their stability and high enzyme loading, applications of CLECs and CLEAs in continuous-flow biocatalysis remain relatively scarce. − Several factors contribute to this limitation, including restricted mechanical robustness, broad particle-size distributions, and a pronounced tendency of CLEAs and CLECs to compact or clog within packed-bed or microfluidic reactors. , These challenges have motivated the development of compartmentalized cross-linked enzyme nano aggregates (c-CLEnAs), in which enzymes are confined within bowl-shaped polymeric nanocompartments prior to cross-linking. The resulting nanoscale aggregates exhibit uniform size, improved mechanical resistance, and reduced clogging propensity, enabling their use in flow reactors with a variety of enzyme systems including lipases, esterases, and oxidoreductase cascades. ,

Multienzyme variants, known as combi-CLEAs, extend the concept to cascade catalysis (Figure E). Coprecipitation of glucose oxidase and horseradish peroxidase, for example, produced CLEAs with enhanced performance and reduced sensitivity to catalase degradation of the hydrogen peroxide intermediate. Similar strategies have been applied to cofactor-regeneration systems for chiral alcohol synthesis.

Overall, cross-linked enzyme assemblies offer a direct route to dense, carrier-free catalytic solids with substantial improvements in stability and solvent tolerance. The evolution from amorphous CLEAs to structurally more ordered frameworks highlights how chemical cross-linking can impose mechanical cohesion and expand functional capability. While CLECs and CLEAs remain important benchmarks for achieving high enzyme loading, their underlying principles increasingly intersect with broader themes in biologically inspired catalyst organization. This trajectory creates a natural conceptual bridge to the intracellularly formed aggregates and genetically programmed crystalline materials discussed in the following sections.

3.2. Catalytically Active Inclusion Bodies: Nature-Inspired Functional Aggregates

Catalytically active inclusion bodies (CatIBs) provide a biologically inspired route to carrier-free enzyme immobilization. Inclusion bodies naturally arise during recombinant protein overexpression when polypeptides partially misfold and self-assemble into dense, insoluble aggregates inside the host cell. Although originally assumed to be inactive, inclusion bodies retaining significant catalytic activity were reported as early as 1989, revealing that enzymatic function can persist within these highly concentrated protein deposits. In contrast to in vitro strategies such as CLEAs or CLECs, CatIBs form directly in vivo, which eliminates the need for precipitation reagents, cross-linkers, or carrier materials. After cell lysis and centrifugation, CatIBs can be isolated through simple washing steps, making them attractive for scalable and low-waste preparation of heterogeneous biocatalysts. Their composition is almost entirely protein, resulting in very high volumetric enzyme densities and minimal noncatalytic mass. CatIBs can display excellent mechanical and operational stability in selected systems, but such robustness is not intrinsic and varies strongly with the enzyme, tag, and aggregation environment. In some cases, additional stabilization steps such as postexpression cross-linking are required to ensure sufficient process resistance under elevated temperature, pH extremes, or organic cosolvents.

CatIB formation can be systematically guided through genetic design. Fusion of the target enzyme to aggregation-inducing tags, typically connected via short peptide linkers, promotes controlled clustering while preserving catalytic competence (Figure A). Over the past decade, extensive work has developed growing libraries of tags and linkers and clarified their influence on activity, morphology, and stability (Figure B). ,− Because optimal combinations are difficult to predict, high-throughput and automated workflows have been introduced to screen CatIB variants and establish structure–property relationships. ,

3.

Design and morphology of catalytically active inclusion bodies (CatIBs). (A) Structural model of a typical fusion protein construct to induce CatIB formation, consisting of an aggregation-inducing TdoT coiled-coil domain, a flexible linker, and the target enzyme. (B) Scanning electron microscopy image of the resulting CatIBs. (A, B) Reproduced with permission from ref . Copyright 2016 Wiley-VCH.

Recent studies have extended CatIBs to cascade catalysis. Coexpression of multiple enzymes can yield mixed CatIBs that perform sequential reactions inside a single dense protein aggregate. Such assemblies have been applied to oxidoreductase and hydrolase cascades and can be combined with intracellular cofactor-regeneration pathways to support cofactor-dependent transformations. , Structural analyses indicate that CatIBs contain native-like secondary structure embedded in partially amorphous regions, creating porous microenvironments that allow substrate diffusion while immobilizing the catalytic centers.

Despite these advantages, the use of CatIBs in continuous flow remains nontrivial. Several studies have reported diffusion limitations due to restricted access to active sites or internal buffer retention, which can impede substrate transport and lower apparent activity. , Their nonporous and compressible nature can also generate high backpressure in packed-bed reactors, limiting applicability in plug-flow configurations. , Nevertheless, successful flow applications exist. Alcohol dehydrogenase CatIBs enabled 120 h of continuous asymmetric ketone reduction with integrated NADP­(H) regeneration when copacked with silica to prevent washout, achieving 92% yield with 98% ee corresponding to a STY of 3.55 g L^–1 h^–1 and a cofactor turnover (TON) of 374 mol/mol. Additional examples include PfBAL CatIBs in an enzyme membrane reactor and lipoxygenase CatIBs in a continuous-flow microreactor. , Due to their small particle size and associated handling challenges, CatIBs are still predominantly employed in repeated-batch operation, ,, sometimes supported by magnetization strategies to facilitate catalyst recovery.

Overall, CatIBs demonstrate how intracellular protein self-assembly can be harnessed to generate robust, carrier-free enzyme materials with minimal downstream processing and good compatibility with continuous operation. Their success highlights the potential of biologically programmed catalytic architectures and motivates the development of next-generation carrier-free systems with greater structural control. Compared with CLEAs, which require in vitro precipitation and often struggle with particle heterogeneity, CatIBs offer genetically programmable formation, cleaner preparation, and a broader set of demonstrated flow applications. This contrast positions CatIBs as the more practical aggregate-based platform for flow biocatalysis today, while underscoring the need for future systems that combine their ease of production with improved mass-transport control.

3.3. Protein Crystal-Based Catalytic Materials: Genetically Encoded and Supramolecularly Designed Assemblies

Protein-based self-assembly offers a second major route to carrier-free immobilization that builds on principles of biological organization rather than chemical cross-linking. These materials range from genetically encoded crystalline scaffolds that form directly in living cells to rationally designed supramolecular protein frameworks assembled in vitro. This progression mirrors the increasing structural order observed in natural protein organization and provides access to highly defined, mechanically robust architectures that complement the biologically derived aggregates discussed in the previous section. In what follows, we first examine in vivo–formed crystalline systems such as Cry3Aa, Cry1Ab, and CipA-based assemblies, which already find application in continuous flow. We then highlight synthetic supramolecular protein crystals that demonstrate how coordination chemistry and computational design can program protein materials with atomic-level precision.

3.3.1. Genetically Encoded Protein Crystals and Aggregates

Porous protein crystals have recently emerged as versatile scaffolds for enzyme immobilization. Foundational studies demonstrated that ordered, solvent-rich lattices can host guest enzymes through metal-affinity interactions, physical entrapment, or lattice-specific adsorption strategies, while retaining activity across multiple assay formats. A broader survey of large-pore crystal architectures further underscored the diversity of available lattice topologies and functionalization strategies, and highlighted their potential in catalysis, delivery, and templating applications.

The crystal-forming protein Cry3Aa from Bacillus thuringiensis provides a genetically programmable scaffold for carrier-free enzyme immobilization. During recombinant expression, Cry3Aa assembles into ordered micron-scale crystals with uniform nanopores that can encapsulate heterologous enzymes (Figure A). Passive entrapment of a Proteus mirabilis lipase demonstrated the basic feasibility of this strategy, yielding hybrid crystals with high enzyme loading, enhanced thermostability and proteolytic resistance, and excellent recyclability for biodiesel synthesis.

4.

(A) Self-assembly of Cry3Aa proteins (red) into ordered micron-scale crystals with internal nanopores that can passively entrap target enzymes (blue). (B) Structural model of a CipA–enzyme fusion showing the CipA domain (red) connected to the enzyme (blue) via a linker (green). Adapted from ref . Copyright 2024 Elsevier. (C) Supramolecular assembly of monomeric RIDC3 proteins into 1D helical tubes and 2D arrays mediated by Zn²⁺ coordination. Amino acids engineered to promote dimerization are shown in cyan, and residues involved in metal binding are highlighted in magenta. Reproduced from ref . Copyright 2014 Proceedings of the National Academy of Sciences.

A key advance addressed one of the major limitations of continuous biocatalysis: the loss of costly cofactors. Surface modification of Cry3Aa crystals with polyethylenimine (PEI) introduced cationic sites for NAD­(H) binding, enabling simultaneous immobilization of enzymes and cofactors within the same ordered lattice. Cofactor-dependent dehydrogenases such as formate dehydrogenase, leucine dehydrogenase, and alcohol dehydrogenase were genetically fused to Cry3Aa, cocrystallized in vivo, and subsequently embedded into agarose beads to form packed-bed flow reactors. These systems supported continuous production of L-tert-leucine and ethyl-(R)-3-hydroxybutyrate for up to 30 days, achieving enzyme turnover numbers above 10⁴ and NADH turnover numbers exceeding 7000, highlighting the ability of Cry-based crystals to retain and recycle expensive cofactors under flow.

To expand the platform to larger enzymes and enable multistep cascades, the homologous crystallizing protein Cry1Ab was introduced, allowing immobilization of proteins up to approximately 80 kDa. Coimmobilization of human arginase I and ornithine decarboxylase afforded a two-enzyme cascade for putrescine synthesis from l-arginine, delivering repeated near-quantitative conversions. The product could subsequently be polymerized to nylon-4,6, illustrating how genetically encoded crystalline scaffolds can bridge biocatalysis with downstream materials chemistry.

Self-assembling aggregates based on the CipA cohesin domain provide a related strategy. Fusion of carbonyl reductase to CipA (Figure B) induced the formation of compact enzyme aggregates that retained NADH and supported continuous reduction of prochiral ketones with 97% yield at substrate concentrations up to 2 M. The corresponding space–time yield (STY) of 1717 g L^–1 d^–1 and cofactor turnover number of 4815 highlight the catalytic potential of genetically programmed, carrier-free aggregates.

Building on these developments, mechanoresponsive Cry3Aa crystals were generated by coating the crystal surface with PEI to modulate flexibility and redox behavior. Incorporation of formate dehydrogenase for NADH regeneration and leucine dehydrogenase for amination yielded a dual-enzyme catalyst that operated continuously for 21 days. Notably, the improved mechanical resilience of these hybrid crystals resulted in a 10-fold increase in NADH turnover number compared with earlier Cry3Aa configurations, demonstrating how crystal–polymer hybrids can combine structural robustness with long-term cofactor recycling.

3.3.2. Designed Supramolecular Protein Crystals

Beyond genetically driven self-assembly, recent work has introduced synthetic supramolecular strategies for constructing ordered protein frameworks with precisely controlled architecture. A major contributor to this field is the Tezcan group, which pioneered the use of metal-coordination motifs to program protein–protein interactions with defined geometry and stoichiometry. By engineering metal-binding sites into homooligomeric enzymes, they generated one-, two-, and three-dimensional crystalline assemblies whose size, anisotropy, and order were tunable through parameters such as metal concentration and pH (Figure C).

Follow-up studies demonstrated the functional breadth of such designed protein crystals. Examples include redox-active supramolecular assemblies with emergent electron-transfer properties, spatially patterned and porous protein crystals with multifunctional material characteristics, and dissipative self-assembling systems in which enzymatic turnover drives nonequilibrium structural remodeling. Although these materials have not yet been integrated into continuous-flow biocatalysis, they illustrate how principles from inorganic chemistry, supramolecular design, and computational modeling can be merged to produce enzyme-based materials with unprecedented structural and functional precision.

Together, genetically encoded crystals and supramolecular protein frameworks demonstrate how ordered protein matter can be engineered to combine high stability, programmable architecture, and intrinsic cofactor retention. Their rigid lattices, however, inevitably constrain mass transport and limit how efficiently these materials can occupy reactor volume. These considerations have motivated the development of more adaptable protein-based architectures in which catalytic function and material structure are fully integrated. This shift leads naturally to all-enzyme hydrogels, discussed in the next section, which offer highly porous, reactor-filling, and mechanically tunable protein networks that achieve some of the highest volumetric productivities reported for carrier-free biocatalysts under continuous-flow conditions.

3.4. All-Enzyme Hydrogels: Self-Assembled Catalytic Networks

Following the ordered and largely rigid protein crystals described above, all-enzyme hydrogels (AEHs) represent a more adaptable form of carrier-free protein matter in which catalytic and structural roles fully merge. AEHs combine molecular precision with macroscopic functionality by turning enzymes themselves into the structural framework of a material. These hydrogels form through spontaneous self-assembly of complementary protein building blocks that link catalytic and cofactor-regenerating enzymes into a continuous network. Unlike conventional immobilization, AEHs contain no inert carrier, and their three-dimensional protein matrix serves simultaneously as catalyst and scaffold, providing high enzyme density, short diffusion paths, and efficient cofactor retention.

The first generation of AEHs was based on covalent conjugation via SpyTag/SpyCatcher (ST/SC) domains, which link enzymes through spontaneous isopeptide bond formation under physiological conditions. In these systems, tetrameric alcohol dehydrogenase (LbADH) and glucose-1-dehydrogenase (BsGDH) self-assembled into porous, catalytically active monoliths that filled microfluidic reactor channels and maintained near-quantitative conversions in the stereoselective reduction of prochiral ketones (Figure A). The covalent protein network retained the NADP­(H) cofactor within the gel and enabled total turnover numbers exceeding 10⁴, representing a record value for cofactor utilization and establishing AEHs as a benchmark for carrier-free, cofactor-retaining biocatalysts.

5.

All-enzyme hydrogels (AEHs) as structurally integrated, carrier-free catalytic materials. (A) Covalent AEH formation via SpyTag/SpyCatcher coupling. The two homotetrameric fusion enzymes, BsGDH-ST and SC-LbADH (light and dark blue, respectively), spontaneously assemble into a protein-only hydrogel through formation of covalent isopeptide bonds. Shown are schematic representations of both building blocks, together with a photograph and a representative SEM image of the resulting hydrogel (scale bar 300 nm). The porous network supports continuous stereoselective ketone reduction. Adapted with permission from ref . Copyright 2018 Wiley-VCH. (B) Generation of monodisperse AEH foams by microfluidic air-in-water droplet formation. Fresh droplets solidify upon drying into open-pore, honeycomb-like catalytic foams, as shown by representative optical micrographs. (C) Continuous-flow performance of AEH foams evaluated at different flow rates (color-coded). Drying markedly improves mechanical robustness, allowing the foams to sustain high conversions over prolonged operation and outperform undried materials. (B, C) Adapted from ref . Available under a CC-BY 4.0 license. Copyright 2023 Hertel et al.

Subsequent work broadened the applicability of the approach to include monomeric enzymes by tuning the number and positioning of ST/SC tags, yielding hydrogels that maintained high activity for more than a week under continuous operation. The modular design also allowed parallelization and scale-up, demonstrating the potential of AEHs for semipreparative synthesis. Application to other enzyme classes such as phenolic acid decarboxylases further expanded the concept to defunctionalization reactions of renewable feedstocks. In this case, continuous production of p-hydroxystyrene from p-coumaric acid reached conversions above 98% and STY of 58 g L^–1 d^–1, while systematic variation of cross-linking density provided insight into how elasticity and mesh size correlate with flow performance.

Later developments transferred AEH formation into the cellular environment, creating intracellular enzyme hydrogels by coexpression of complementary fusion enzymes from polycistronic vectors in E. coli . The resulting nanoscale aggregates, 50–120 nm in diameter, could be isolated directly from crude lysates and used for continuous biocatalysis for several days without loss of activity. This simplified preparation process by combining enzyme expression and immobilization in a single step.

A microfluidic air-in-water droplet approach then enabled the fabrication of monodisperse enzyme foams based on the ADH/GDH system composed almost entirely of covalently linked enzymes. These foams could be dried and integrated directly into microreactors, providing high stability, open-pore architectures for efficient mass transport, and long-term operational performance (Figure B). Building on this concept, subsequent work replaced the covalent ST/SC system with noncovalent interaction modules such as PDZ, SH3, and GBD domains that mediate specific protein–protein binding. The resulting noncovalent AEHs (ncAEHs) formed either in living cells or from purified enzymes. While in-cell assemblies showed limited benefits under flow conditions, the purified ncAEH foams achieved stable conversions above 70% over 30 h of continuous operation. Drying the foams significantly improved mechanical robustness and activity (Figure C). Extending the method to additional biocatalysts, including phenolic acid decarboxylase, xylose reductase, and other unrelated enzyme classes, confirmed the generality and modularity of the approach.

Quantitative benchmarking using the NADP­(H)-dependent diketone model reaction allows comparison of AEHs with conventional immobilization formats. In these studies, covalently assembled AEH monoliths achieved the highest STY among tested systems, outperforming enzyme immobilization on beads by approximately 5-fold. Foam-based AEHs, whether covalently or noncovalently assembled, reached STYs around 180 g L^–1 d^–1 at a flow rate of 10 μL min^–1, roughly twice that of monolithic AEHs and well above the industrial threshold of 16 g L^–1 d^–1. ,, These foams operated stably for at least 30 h, exceeding the minimum runtime of 5–10 h typically required for continuous biocatalytic processes. While optimized industrial ketoreductases can reach STYs near 1000 g L^–1 d^–1, the AEH foams already operate at roughly one-fifth of this value without enzyme or process engineering, underscoring their potential as a robust and scalable platform for industrial biocatalysis.

From a practical perspective, considerations of catalyst reuse and cost are best interpreted in the context of continuous-flow operation and are discussed here using AEHs as a representative example of carrier-free immobilized enzymes. Carrier-free catalysts are typically employed under flow until the end of their operational lifetime rather than recovered and reused in discrete cycles, rendering classical recovery-based reuse experiments of limited relevance. Nevertheless, AEHs can be stored and applied in sequential catalytic campaigns, and both covalent and noncovalent enzyme foams have demonstrated high stability under storage and process conditions. , Reusability has also been demonstrated for AEH-packed reactor columns in sequential stereoselective reductions of different ketone substrates. ,

With respect to cost considerations, carrier-free immobilization eliminates the need for inert support materials and associated functionalization steps. While enzymes themselves constitute both the catalytic and structural components of AEHs, and not all enzyme molecules may be catalytically active in dense networks, this trade-off is partly offset by exceptionally high space–time yields and simplified reactor integration. In addition, AEH formation avoids the use of external cross-linking reagents such as glutaraldehyde and can reduce formulation complexity and processing steps, which is expected to translate into cost and sustainability benefits. Ultimately, systematic techno-economic analyses will be required to delineate application regimes in which carrier-free architectures provide clear economic advantages over conventional immobilization strategies.

Despite these advances, AEHs, as a representative class of carrier-free immobilized enzymes, also face several inherent limitations that need to be considered when translating such concepts to broader applications. As protein-based materials, their long-term operational stability is ultimately bounded by the intrinsic stability of the enzyme subunits and of the protein–protein interaction or conjugation domains forming the network. In addition, modification of enzymes with interaction modules can reduce intrinsic catalytic activity, although such effects can often be compensated by the high enzyme loading achievable in AEHs or mitigated through enzyme engineering. Dense AEH architectures may further impose diffusional constraints related to pore size and internal mass transport, while strong partitioning of certain substrates or products within the protein network could limit extraction efficiency under flow. Across these aspects, enzyme engineering and rational network design provide powerful strategies to alleviate AEH-specific limitations by improving stability, preserving activity, and tuning interactions in confined catalytic environments.

The chronological evolution of AEHs reflects a clear trajectory from covalent monoliths to intracellular nanogels and noncovalent foams, paralleled by substantial gains in catalytic density, stability, and process flexibility. These systems exemplify the convergence of molecular engineering, materials science, and reactor design in the creation of carrier-free biocatalytic materials. Beyond foams and porous monoliths, AEHs could be further developed into alternative structural formats tailored to specific process requirements. Possible designs include formulation as discrete beads for packed-bed or fluidized reactors, porosity tuning through the introduction of molecular or particulate spacers to control internal mass transport, and biohybrid architectures incorporating inorganic components such as magnetic particles or protective shells to enable retention, manipulation, or enhanced robustness under harsh flow conditions. In addition, fiber-based AEHs produced by electrospinning could offer high surface-to-volume ratios and directional mass transport in flow-through configurations.

Beyond the specific AEH implementations described above, the underlying design principle of carrier-free, enzyme-assembled catalytic materials has increasingly been adopted and independently implemented by other groups. These studies encompass diverse realizations, including SpyTag/SpyCatcher-mediated enzyme coassembly for intracellular and extracellular cofactor regeneration systems, enzyme-assembled gel monoliths formed directly from crude enzyme preparations, and alternative cross-linking strategies based on metal coordination or small-molecule bridging. Applications range from continuous-flow asymmetric reductions and the synthesis of chiral building blocks to in-cell assemblies and functional catalytic biomaterials. While differing in molecular details, these approaches share the central concept of transforming enzymes into both the catalytic and structural components of a hydrogel material, underscoring the generality and growing relevance of artificial enzyme-based architectures. −

Future developments are likely to exploit dynamic, multienzyme networks that mimic the compartmentalization and adaptability of natural metabolic assemblies, potentially integrating AEHs with additive manufacturing and microfluidic control for scalable, modular production platforms. Through such advances, AEHs are poised to bridge the gap between laboratory-scale demonstrations and truly industrial catalytic architectures for sustainable continuous-flow bioprocessing.

4. Summary and Outlook

The development of carrier-free enzyme immobilization strategies reveals a progression toward increasing structural definition, catalytic density, and process integration. Early cross-linked enzyme crystals and aggregates demonstrated that enzymes can be converted into self-supporting solids with high catalytic-site density and enhanced stability. Catalytically active inclusion bodies extended this concept into the cellular environment, showing that dense, functional protein aggregates can form directly in vivo with minimal downstream processing. Genetically encoded protein crystals and related supramolecular assemblies then introduced highly ordered, programmable scaffolds with exceptional mechanical robustness and precise lattice-defined architectures. All-enzyme hydrogels (AEHs) finally added a complementary, markedly more adaptable class of materials in which catalytic and cofactor-regenerating functions merge into continuous porous networks. Collectively, these developments illustrate how enzymes can be transformed from dispersed molecular catalysts into structured catalytic matter tailored for continuous operation.

Genetically encoded crystalline materials, including Cry-based scaffolds and CipA-driven aggregates, offer highly ordered, mechanically resilient matrices with strong resistance to denaturation or leaching. Their regular pore architectures promote long-term operation and efficient cofactor retention but can restrict mass transport and limit compatibility with bulky substrates or complex cascades. Continued refinement of pore dimensions, surface chemistry, and hybrid designs may help overcome such limitations, generating crystalline platforms that balance structural precision with improved substrate accessibility.

Within this evolving landscape, AEHs have emerged as the most flexible experimental framework. Their formation under mild aqueous conditions, compatibility with diverse enzyme classes, and tunable viscoelasticity enable integration into both batch and continuous-flow operation. Porous monolithic and foam-like variants facilitate efficient mass transport, fill reactor volumes without dead space, and can be readily incorporated into microfluidic or modular reactor architectures. These properties make AEHs particularly suitable for systematic structure–function studies and for maximizing volumetric productivity in carrier-free biocatalysis.

Taken together, these carrier-free materials exemplify the emergence of engineered catalytic matter in which mesoscale enzyme organization governs activity, stability, and diffusion. Progress in the field increasingly depends on combining material design with advanced process control. AEH-based reactors have already served as model systems for this integration: coupling AEHs with inline NMR spectroscopy and algorithmic feedback regulation enabled autonomous optimization of reaction parameters, outlining a blueprint for precision biocatalysis in continuous systems.

Future advances will likely arise from the convergence of enzyme engineering, additive manufacturing, and data-driven reactor optimization. Digital feedback loops can guide the design of hydrogel networks or crystalline assemblies with predictable mechanical and kinetic behavior, while automated microreactors will enable long-term, self-regulated operation. Through these developments, carrier-free enzyme materials are expected to mature into integral components of sustainable, programmable flow-biocatalytic platforms.

In parallel to these technological advances, the reproducibility and comparability of biocatalytic experiments remain critical challenges. Inaccuracies in analytics, incomplete reporting of reaction conditions, and inconsistent data management often complicate the interpretation and benchmarking of results. To improve transparency and credibility across the field, recent efforts have focused on implementing research data management frameworks that comply with the FAIR principles (Findable, Accessible, Interoperable, Reusable) and with initiatives such as the National Research Data Infrastructure (NFDI) consortium. These frameworks emphasize structured documentation of experimental data and metadata, as well as standardized formats for data exchange. While the systematic adoption of such practices remains the exception in biocatalysis, pioneering studies have already demonstrated their feasibility and benefit for process optimization and autonomous experimentation. ,

Carrier-free enzyme immobilization has thus evolved from a means of catalyst recovery into a design principle for constructing high-density catalytic materials that integrate molecular precision with process efficiency. The emerging architectures, ranging from cross-linked aggregates and inclusion bodies to genetically encoded protein crystals and all-enzyme hydrogels, illustrate how biological function can be translated into structured materials for continuous operation. Among these, AEHs exemplify the balance of catalytic density, mass transport, and adaptability required for next-generation continuous-flow biocatalysis. Their integration with digital reactor control marks the beginning of precision biocatalysis, where catalyst structure and process dynamics coevolve. Advancing these materials toward scalable, autonomous operation, supported by transparent and FAIR data practices, will transform enzyme catalysis from an empirical craft into a predictable engineering discipline for sustainable chemical manufacturing.

CRediT: Severin P. Stalter writing - original draft; Kersten S. Rabe writing - review & editing; Christof M. Niemeyer writing - original draft.

The authors declare the following competing financial interest(s): Several patents related to AEH materials have been filed, naming KSR and CMN as inventors.

Published as part of JACS Au special issue “Continuous Flow Chemistry”.