Synergistic integration of catalytically active inclusion bodies and cross-linked enzyme aggregates for high-performance immobilization of L-phenylserine aldolase.

From biopolymers to microcompartments: A structured review of protein-based scaffolds for enzyme immobilization.

This study presents an innovative methodology for fabricating polymeric beads via 3D printing to enhance cell immobilization strategies in bioprocess engineering. Using fused filament fabrication (FFF), beads composed of acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) were fabricated with precisely tailored geometries, enabling the systematic evaluation of how material type and internal design influence both manufacturing feasibility and structural stability. The methodology demonstrates high reproducibility, dimensional accuracy and flexibility, allowing researchers to fabricate immobilization beads adapted to specific bioprocess conditions. In contrast to conventional immobilization matrices, this FFF-based approach offers a scalable, cost-effective and customizable alternative, capable of producing complex porous architectures that promote microbial adhesion and mass transfer. The method stands out for extending additive manufacturing applications beyond enzyme immobilization toward whole-cell biocatalyst systems, providing a valuable framework for future biotechnological process development. • A reproducible FFF-based workflow for 3D-printed cell immobilization beads. • ABS and PLA beads fabricated with tunable geometry and internal porosity. • Method extends 3D printing applications from enzymes to whole-cell biocatalysts. • Cost-effective and scalable alternative to conventional immobilization matrices.

Magnetic MOF derivatives from Tunable MIL-88A for enhanced lipase immobilization and phosphatidyl EPA/DHA synthesis.

The excessive discharge of wastewater containing methylene blue (MB) has caused certain adverse effects on human health and daily life. This highlights the urgent need for the development of safe and environmentally friendly methods of degradation. In this study, two catalytic systems based on Zn-doped g-C3N4, namely, ZCN-1%/PMS and ZCN-1%/LC, were constructed for efficient activation of peroxymonosulfate (PMS) and increased laccase stability. Experimental results show that Zn-doped g-C3N4 shows much better photocatalytic activity, combined with good enzyme immobilization ability. At a catalyst dosage of 0.2 g/L, both systems were able to remove more than 97% of MB (0.2 g/L) in 30 and 60 min of irradiation, respectively. Quenching experiments and electron paramagnetic resonance (EPR) analysis further revealed singlet oxygen (1O2) and superoxide radicals (O2•-) as the main active species in the ZCN-1%/PMS and ZCN-1%/LC systems, respectively. Based on these results, the catalytic mechanisms of the two systems are systematically elucidated in this paper. This work not only proposes new methods for PMS activation and Photoenzymatic Synergistic Catalysis but also offers feasible technical strategies for treating MB-contaminated wastewater.

The Effect of Reduced Graphene Oxide in Chitosan-Based Nanoparticles on the Enzymatic Properties of the Immobilized Enzyme.

Possessing superior water retention capacity, exceptional biocompatibility, and versatile design flexibility, hydrogels have emerged as a prominent engineering platform for cell and enzyme immobilization. The core advantage of hydrogels lies in the structure-activity relationship between their physicochemical properties-governed by crosslinking mechanisms and fabrication techniques-and the biological performance of the immobilized biocatalysts. This relationship is central to constructing microenvironments that either promote cell proliferation or stabilize enzymatic reactions. This paper systematically reviews this structure-activity paradigm, emphasizing how raw material selection and manufacturing technologies determine hydrogel functionality. We further analyze the critical design criteria for immobilization-oriented hydrogels, which must balance physical performance, support biological activities, and exhibit environmental adaptability. Distinct design requirements for the immobilization of living cells versus enzymes are explicitly compared. Building upon this foundation, the review elaborates on the unique application value of this technology in medical diagnostics, therapeutics, biosensors, industrial biotransformation, and environmental remediation. Finally, we identify core challenges and propose a concrete development roadmap to provide more efficient and stable engineering solutions for related fields.

Amine-reactive hydrogels via nucleophilic thiol-yne reaction: A functional scaffold for laccase immobilization and dye degradation.

Enzyme immobilization represents a critical approach to address enzyme instability and limited recyclability in practical biocatalytic systems. However, simultaneously improving enzyme stability and catalytic activity remains a major bottleneck, restricting the broader implementation of immobilized enzymes. Herein, we report a mild in situ encapsulation strategy using layered double hydroxides (LDHs) as a structurally tailored host matrix for enzyme confinement. Benefiting from the hydrophilic and layered structure of MgAl-LDH, the HRP@MgAl-LDH composite preserved the native conformation of encapsulated HRP while delivering exceptional structural stability. The MgAl-LDH not only created a biocompatible microenvironment that sustained high enzymatic activity but also acted as a protective scaffold to enhance stability and reusability. Electrochemical characterization revealed that HRP@MgAl-LDH promoted rapid and efficient electron transfer between the enzyme active sites and the electrode surface, enabling faster reaction kinetics relative to free HRP and conventionally immobilized enzyme systems. Leveraging these favorable structural and electrochemical properties, we fabricated a portable electrochemical sensor for the sensitive detection of H2O2 in human urine. The sensor exhibited a wide linear detection range (50 pM-250 μM) and a low detection limit of 36.96 pM, along with rapid response, strong anti-interference performance, portability, and low cost. This LDH-based in situ encapsulation strategy effectively balanced the often-competing demands of high enzymatic activity and long-term stability, overcoming key limitations of traditional encapsulation methods. This work offered a promising high conductive material-based strategy for the development of practical point-of-care devices for H2O2 detection in complex biological samples.

A nano-MOF-based electrochemical microfluidic immunosensor for dengue E protein detection in human serum.

The development of immobilised biocatalysts for continuous‐flow processes is still largely based on laborious trial‐and‐error screening, and conditions optimised on conventional carriers are often not directly transferable to structured reactors. Here, we introduce a 3D‐printed methodology that integrates high‐throughput screening of enzyme immobilisation with implementation in a continuous‐flow reactor using the same photopolymeric formulation. The surface of the printed objects was modified through imidazolium‐based supported ionic liquid phases. Immobilisation conditions for an enzymatic solution with alcohol dehydrogenase (ADH‐200) were rapidly evaluated in a 96‐well format using a colorimetric assay that enables quantitative comparison of activity and loading across multiples of conditions in a parallel fashion. Methyl‐imidazolium‐modified supports showed markedly higher enzymatic activity and immobilisation efficiency than other imidazolium‐based ionic liquids, and the best‐performing formulation was directly applied to functionalise the 3D‐printed honeycomb‐structured reactor. The resulting heterogeneous biocatalyst catalysed the oxidation of 1‐phenylethanol to acetophenone under continuous flow with high conversion at moderate residence times and sustained performance over several hundred hours on stream. This integrated 3D‐printed platform facilitates the identification of effective immobilisation chemistries and their translation to robust flow reactors, providing a general strategy for the rapid development of immobilised biocatalysts.

Herein, a multifunctional nanocomposite was developed through a green synthesis route using Dolomiaea costus extract to produce ZnFe2O4 nanoparticles, which were subsequently integrated with multi-walled carbon nanotubes (MWCNTs) and cellulose acetate (CA) to form a stable ZnFe2O4-MWCNT-CA matrix for horseradish peroxidase (HRP) immobilization. The eco-friendly synthesis avoided toxic precursors by employing plant-derived biomolecules as natural reducing and stabilizing agents. Structural and morphological analyses revealed characteristic XRD reflections of cubic spinel ZnFe2O4 at 2θ ≈ 30.4°, 35.4°, 54.1°, 56.6°, and 62.2°, along with graphitic carbon peaks at 24.1° and 42.8°, confirming the successful integration of ZnFe2O4 and MWCNTs within the cellulose acetate matrix. FTIR spectra showed metal-oxygen vibrations at 565 cm⁻¹, cellulose acetate bands at 1735 and 1040-1240 cm⁻¹, and amide bands after immobilization, while SEM-EDX confirmed a porous interconnected morphology and the presence of C, O, Fe, and Zn as the main elements. The immobilized HRP exhibited an immobilization efficiency of 82%, a 0.5-unit alkaline shift in optimum pH (7.0 to 7.5), and an enhanced optimum temperature (50 to 60 °C) compared with the free enzyme. Kinetic analysis revealed an increased apparent Km (4.6 to 10.6 mM) and Vmax (20 to 30 μmol min⁻¹), indicating reduced substrate affinity but improved catalytic turnover after immobilization. The immobilized enzyme maintained 81% activity after ten reuse cycles and retained 71% activity after 57 days of storage, outperforming comparable magnetic or carbon-based supports. These results demonstrate that the green-synthesized ZnFe2O4-MWCNT-CA hybrid is a robust, recyclable, and eco-compatible platform for enzyme immobilization, offering significant promise for biocatalysis, pollutant degradation, and industrial wastewater treatment applications.

A flexible, lightweight and low-cost enzymatic bioanode was developed using a screen-printed silver conductive transparency sheet for enzymatic biofuel cell (EBFC) applications. In this work, indole was electrochemically polymerized directly onto the conductive substrate to form a polyindole (PIn) matrix capable of simultaneously entrapping glucose oxidase (GOx) and redox mediator vitamin K3 (VK3). The study integrates a disposable transparency-sheet platform with an electroactive PIn network that promotes efficient enzyme immobilization and enhanced electron transfer for glucose bioelectrocatalysis. The synergistic interaction between PIn, VK3 and GOx significantly improved charge-transfer kinetics and stabilized the bioelectrocatalytic interface, resulting in enhanced electrochemical performance. The fabricated PIn/VK3/GOx bioanode exhibited a current density of 1.18mA cm- 2 in 40 mM glucose solution, demonstrating efficient glucose-dependent electrocatalytic activity. The conductive PIn framework facilitated rapid electron transport between the buried active sites of GOx and the electrode surface, while VK3 acted as an efficient and biocompatible electron shuttle. The bioanode also displayed good electrical stability, semiconducting behavior, and favorable electrochemical characteristics, highlighting its suitability for flexible and wearable bioelectronic applications.

Genipin based green chemistry immobilization cascade towards enzyme based antibacterial biomaterial surfaces.

Preparation of antimicrobial biopolymer particles through co-precipitation of hen egg white lysozyme and poly(hydroxy butyrate).

Biomimetic immobilization of α-glucosidase in double-shelled ZIF-90 for inhibitor screening from traditional Chinese medicines.

Integrative simulation and spectroscopy illuminates L-asparaginase stability on nanocomposite surface for biosensing.

To meet the global pursuit of carbon neutrality, electroenzymatic reduction of CO2 has been a potential technology that can convert CO2 into useful chemicals or fuels. However, the low solubility of CO2 in water inhibits the reaction rate, and free enzyme suffers from poor stability and reusability and requires the expensive cofactor NADH. Herein, a spherical covalent organic framework (COF) TPB-DMTP was synthesized for both CO2 enrichment and enzyme immobilization. The electron mediator neutral red was covalently attached to a carbon paper electrode for electrochemical NADH regeneration, thereby avoiding damage to the enzyme. The constructed electrocatalytic NADH regeneration was coupled with enzyme-catalyzed CO2 reduction. After immobilizing formate dehydrogenase (FDH) in the COF, the CO2 was enriched near the enzyme molecules, and the stability and reusability of enzyme were enhanced. The optimized system produced up to 2.312 mM formate, which was 4.01 times that of the free enzyme system.

Fast and efficient protein digestion is important in proteomics to improve the performance of protein analysis. In this work, a series of magnetic macroporous silica carriers with pore sizes ranging from 32 nm to 184 nm was synthesized, among which the silica bead with the most suitable pore size and pore structure (SiO2@Fe3O4-650 °C/650 °C, 184 nm) exhibits high enzyme immobilization capacity (76.96 µg mg-1) and great digestion activity. Trypsin was adsorbed on the silica beads through the formation of a protein corona to prepare an IMER (SiO2@Fe3O4-nIMER). The non-covalent immobilization strategy could maximally preserve the enzymatic activity. SiO2@Fe3O-nIMER retained 97% of its enzymatic hydrolysis efficiency even after five cycles, demonstrating excellent reusability. SiO2@Fe3O4-nIMER was further immobilized in the capillary (75 µm i.d.) with the assistance of permanent magnets, constructing an open-tube immobilized enzyme microreactor (SiO2@Fe3O4-cIMER) for dynamic protein digestion. Under the optimal enzymatic digestion conditions (effective length of the enzyme reactor: 5.4 cm and flow rate of the protein sample: 10 µL min-1), the enzymatic digestion performance of SiO2@Fe3O4-cIMER was evaluated using bovine serum albumin (BSA) and cytochrome C (Cyt C) as model proteins, and the sequence coverages were 97% for BSA (168 peptides matched) and 92% for Cyt C (26 peptides matched). Using a dynamic enzymatic digestion mode, SiO2@Fe3O4-cIMER identified 509 proteins from human serum samples, which was significantly higher than that identified by SiO2@Fe3O4-nIMER (444 proteins) and free-solution enzymatic digestion (359 proteins). The novel enzyme reactor has the advantages of a simple preparation process, good reproducibility, stability and high digestion efficiency, making it suitable for large-scale proteomics research.

Development of a magnetic β-glucosidase biocatalyst: Structural and functional characterization with enhanced thermal stability.