A simple and high-performance cellulose nanocrystal based biocatalytic Pickering emulsion system for pharmaceutical molecule vitamin E nicotinate.

The use of Pickering emulsions for biocatalysis is gaining increased attention. However, the extensive application is greatly limited due to the enzyme inactivation. Herein, a biocatalytic Pickering emulsion with high-performance utilizing cellulose nanocrystals immobilized lipases (CNCs-Lps) particles as stabilizer is advanced and applied for the synthesis of Vitamin E nicotinate. CNCs-Lps display high activity and reusability due to the construction of biocatalytic microreactor in the O/W emulsion system. The yield of vitamin E nicotinate ester reached up to 83 %. More importantly, the CNCs-Lps can be reused due to the similar principles to microreactors in Pickering emulsions. Reusability test showed that the CNCs-Lps could be recovered from the emulsion system by centrifugation and the yield of vitamin E nicotinate retains 78 % of initial value after five cycles, demonstrating overwhelming advantage than the fair counterpart with free lipases.

Biocatalyst immobilization on magnetic nano-architectures for potential applications in condensation reactions.

In this review article, a perspective on the immobilization of various hydrolytic enzymes onto magnetic nanoparticles for synthetic organic chemistry applications is presented. After a first part giving short overview on nanomagnetism and highlighting advantages and disadvantages of immobilizing enzymes on magnetic nanoparticles (MNPs), the most important hydrolytic enzymes and their applications were summarized. A section reviewing the immobilization techniques with a particular focus on supporting enzymes on MNPs introduces the reader to the final chapter describing synthetic organic chemistry applications of small molecules (flavour esters) and polymers (polyesters and polyamides). Finally, the conclusion and perspective section gives the author's personal view on further research discussing the new idea of a synergistic rational design of the magnetic and biocatalytic component to produce novel magnetic nano-architectures.

Enhancing Chymotrypsin Activity and Stability of Capillary Immobilized Enzyme Microreactors Using Zeolitic Imidazolate Frameworks as Encapsulation Materials.

Open-tubular immobilized enzyme microreactors (OT-IMERs) are some of the most widely used enzyme reaction devices due to the advantages of simple preparation and fast sample processing. However, the traditional approaches for OT-IMERs preparation had some defects such as limited enzyme loading amount, susceptibility to complex sample interference, and less stability. Here, we report a strategy for the preparation of highly active and stable OT-IMERs, in which the single-stranded DNA-enzyme composites were immobilized in capillaries and then encapsulated in situ in the capillaries via zeolitic imidazolate frameworks (ZIF-L). The phosphate groups of the DNA adjusted the surface potential of the enzyme to negative values, which could attract cations, such as Zn2+, to promote the formation of ZIF-L for enzyme encapsulation. Using chymotrypsin (ChT) as a model enzyme, the prepared ChT@ZIF-L-IMER has higher activity and better affinity than the free enzyme and ChT-IMER. Moreover, the thermal stability, pH stability, and organic solvent stability of ChT@ZIF-L-IMER were much higher than those of free enzyme and ChT-IMER. Furthermore, the activity of ChT@ZIF-L-IMER was much higher than that of ChT-IMER after ten consecutive reactions. To demonstrate the versatility of this preparation method, we replaced ChT with glucose oxidase (GOx). The stability of GOx@ZIF-L-IMER was also experimentally demonstrated to be superior to that of GOx and GOx-IMER. Finally, ChT@ZIF-L-IMER was used for proteolytic digestion analysis. The results showed that ChT@ZIF-L-IMER had a short digestion time and high digestive efficiency compared with the free enzyme. The present study broadened the synthesis method of OT-IMERs, effectively integrating the advantages of metal-organic frameworks and IMER, and the prepared OT-IMERs significantly improved enzyme stability. All of the results indicated that the IMER prepared by this method had a broad application prospect in capillary electrophoresis-based high-performance enzyme analysis.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

A review of nanotechnology in enzyme cascade to address challenges in pre-treating biomass.

Nanotechnology has become a revolutionary technique for improving the preliminary treatment of lignocellulosic biomass in the production of biofuels. Traditional methods of pre-treatment have encountered difficulties in effectively degrading the intricate lignocellulosic composition, thereby impeding the conversion of biomass into fermentable sugars. Nanotechnology has enabled the development of enzyme cascade processes that present a potential solution for addressing the limitations. The focus of this review article is to delve into the utilization of nanotechnology in the pretreatment of lignocellulosic biomass through enzyme cascade processes. The review commences with an analysis of the composition and structure of lignocellulosic biomass, followed by a discussion on the drawbacks associated with conventional pre-treatment techniques. The subsequent analysis explores the importance of efficient pre-treatment methods in the context of biofuel production. We thoroughly investigate the utilization of nanotechnology in the pre-treatment of enzyme cascades across three distinct sections. Nanomaterials for enzyme immobilization, enhanced enzyme stability and activity through nanotechnology, and nanocarriers for controlled enzyme delivery. Moreover, the techniques used to analyse nanomaterials and the interactions between enzymes and nanomaterials are introduced. This review emphasizes the significance of comprehending the mechanisms underlying the synergy between nanotechnology and enzymes establishing sustainable and environmentally friendly nanotechnology applications.

Surface-Displayed Mannanolytic and Chitinolytic Enzymes Using Peptidoglycan Binding LysM Domains.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum. The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

Microgels with Immobilized Glycosyltransferases for Enzymatic Glycan Synthesis.

Glycans, composed of linked monosaccharides, play crucial roles in biology and find diverse applications. Enhancing their enzymatic synthesis can be achieved by immobilizing enzymes on materials such as microgels. Here, we present microgels with immobilized glycosyltransferases, synthesized through droplet microfluidics, immobilizing enzymes either via encapsulation or postattachment. SpyTag-SpyCatcher interaction was used for enzyme binding, among others. Fluorescamine and permeability assays confirmed enzyme immobilization and microgel porosity, while enzymatic activities were determined using HPLC. The potential application of microgels in cascade reactions involving multiple enzymes was demonstrated by combining ß4GalT and α3GalT in an enzymatic reaction with high yields. Moreover, a cascade of ß4GalT and ß3GlcNAcT was successfully implemented. These results pave the way toward a modular membrane bioreactor for automated glycan synthesis containing the presented biocatalytic microgels.

Controllable radical polymerization of TEMPO redox for stable and sensitive enzyme electrode interface.

Assembling functional molecules on the surface of an enzyme electrode is the most basic technique for constructing a biosensor. However, precise control of electron transfer interface or electron mediator on the electrode surface remains a challenge, which is a key step that affects the stability and sensitivity of enzyme-based biosensors. In this study, we propose the use of controllable free radical polymerization to grow stable 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) polymer as electron mediator on enzyme surface for the first time. Through scanning electron microscopy (SEM), Raman spectroscopy, electrode surface coverage measurement, static contact angle (SCA), and a series of electrochemical methods, it has been demonstrated that the TEMPO-based enzyme electrode exhibits a uniform hydrophilic morphology and stable electrochemical performance. Furthermore, the results show that the sensor demonstrates high sensitivity for detecting glucose biomolecules in artificial sweat and serum. Attributing to the quantitative and controllable radical polymerization of TEMPO redox assembled enzyme electrode surface, the as-proposed biosensor providing a use, storage, and inter-batch sensing stability, providing a vital platform for wearable/implantable biochemical sensors.

Amino Functionality Enables Aqueous Synthesis of Carboxylic Acid-Based MOFs at Room Temperature by Biomimetic Crystallization.

Enzyme immobilization within metal-organic frameworks (MOFs) is a promising solution to avoid denaturation and thereby utilize the desirable properties of enzymes outside of their native environments. The biomimetic mineralization strategy employs biomacromolecules as nucleation agents to promote the crystallization of MOFs in water at room temperature, thus overcoming pore size limitations presented by traditional postassembly encapsulation. Most biomimetic crystallization studies reported to date have employed zeolitic imidazole frameworks (ZIFs). Herein, we expand the library of MOFs suitable for biomimetic mineralization to include zinc(II) MOFs incorporating functionalized terephthalic acid linkers and study the catalytic performance of the enzyme@MOFs. Amine functionalization of terephthalic acids is shown to accelerate the formation of crystalline MOFs enabling new enzyme@MOFs to be synthesized. The structure and morphology of the enzyme@MOFs were characterized by PXRD, FTIR, and SEM-EDX, and the catalytic potential was evaluated. Increasing the linker length while retaining the amino moiety gave rise to a family of linkers; however, MOFs generated with the 2,2'-aminoterephthalic acid linker displayed the best catalytic performance. Our data also illustrate that the pH of the reaction mixture affects the crystal structure of the MOF and that this structural transformation impacts the catalytic performance of the enzyme@MOF.

Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose.

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.

Aspergillus oryzae ß-D-galactosidase immobilization on glutaraldehyde pre-activated amino-functionalized magnetic mesoporous silica: Performance, characteristics, and application in the preparation of sesaminol.

Aspergillus oryzae ß-D-galactosidase (ß-Gal) efficiently hydrolyzes sesaminol triglucoside into sesaminol, which has higher biological activity. However, ß-Gal is difficult to be separate from the reaction mixture and limited by stability. To resolve these problems, ß-Gal was immobilized on amino-functionalized magnetic nanoparticles mesoporous silica pre-activated with glutaraldehyde (Fe3O4@mSiO2-ß-Gal), which was used for the first time to prepare sesaminol. Under the optimal conditions, the immobilization yield and recovered activity of ß-Gal were 57.9 ± 0.3 % and 46.5 ± 0.9 %, and the enzymatic loading was 843 ± 21 Uenzyme/gsupport. The construction of Fe3O4@mSiO2-ß-Gal was confirmed by various characterization methods, and the results indicated it was suitable for heterogeneous enzyme-catalyzed reactions. Fe3O4@mSiO2-ß-Gal was readily separable under magnetic action and displayed improved activity in extreme pH and temperature conditions. After 45 days of storage at 4 °C, the activity of Fe3O4@mSiO2-ß-Gal remained at 92.3 ± 2.8 %, which was 1.29 times than that of free enzyme, and its activity remained above 85 % after 10 cycles. Fe3O4@mSiO2-ß-Gal displayed higher affinity and catalytic efficiency. The half-life was 1.41 longer than free enzymes at 55.0 °C. Fe3O4@mSiO2-ß-Gal was employed as a catalyst to prepare sesaminol, achieving a 96.7 % conversion yield of sesaminol. The excellent stability and catalytic efficiency provide broad benefits and potential for biocatalytic industry applications.

Time-Separated Pulse Release-Activation of an Enzyme from Alginate-Polyethylenimine Hydrogels Using Electrochemically Generated Local pH Changes.

ß-Glucosidase (EC 3.2.1.21) from sweet almond was encapsulated into pH-responsive alginate-polyethylenimine (alginate-PEI) hydrogel. Then, electrochemically controlled cyclic local pH changes resulting from ascorbate oxidation (acidification) and oxygen reduction (basification) were used for the pulsatile release of the enzyme from the composite hydrogel. Activation of the enzyme was controlled by the very same pH changes used for ß-glucosidase release, separating these two processes in time. Importantly, the activity of the enzyme, which had not been released yet, was inhibited due to the buffering effect of PEI present in the gel. Thus, only a portion of the released enzyme was activated. Both enzymatic activity and release were monitored by confocal fluorescence microscopy and regular fluorescent spectroscopy. Namely, commercially available very little or nonfluorescent substrate 4-methylumbelliferyl-ß-d-glucopyranoside was hydrolyzed by ß-glucosidase to produce a highly fluorescent product 4-methylumbelliferone during the activation phase. At the same time, labeling of the enzyme with rhodamine B isothiocyanate was used for release observation. The proposed work represents an interesting smart release-activation system with potential applications in biomedical field.

Enzyme encapsulation in metal-organic frameworks using spray drying for enhanced stability and controlled release: A case study of phytase.

Encapsulating enzymes in metal-organic frameworks is a common practice to improve enzyme stability against harsh conditions. However, the synthesis of enzyme@MOFs has been primarily limited to small-scale laboratory settings, hampering their industrial applications. Spray drying is a scalable and cost-effective technology, which has been frequently used in industry for large-scale productions. Despite these advantages, its potential for encapsulating enzymes in MOFs remains largely unexplored, due to challenges such as nozzle clogging from MOF particle formation, utilization of toxic organic solvents, controlled release of encapsulated enzymes, and high temperatures that could compromise enzyme activity. Herein, we present a novel approach for preparing phytase@MIL-88 A using solvent-free spray drying. This involves atomizing two MOF precursor solutions separately using a three-fluid nozzle, with enzyme release controlled by manipulating defects within the MOFs. The physicochemical properties of the spray dried particles are characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Leveraging the efficiency and scalability of spray drying in industrial production, this scalable encapsulation technique holds considerable promise for broad industrial applications.

Propitious catalytic response of immobilized α-amylase from G. thermoleovorans in modified APTES-Fe3O4 NPs for industrial bio-processing.

Challenges in enzyme and product recovery are currently intriguing in modern biotechnology. Coping enzyme stability, shelf life and efficiency, nanomaterials-based immobilization were epitomized of industrial practice. Herein, a α-amylase from Geobacillus thermoleovorans was purified and bound effectively on to a modified 3-Aminopropyltriethoxysilane (APTES)-Fe3O4 nanoparticle. It was revealed that the carrier-bound enzyme catalysis (pH 8 and 60 °C) was significant in contrast to the free enzyme (pH 7.5 and 55 °C). Furthermore, Zn2+ and Cu2+ were shown to cause inhibitory effects in both enzyme states. Unlike chloroform, toluene, benzene, and butanol, minimal effects were observed with ethanol, acetone, and hexane. The bound enzyme retained 27.4 % of its initial activity after being stored for 36 days. In addition, the reusability of the bound enzyme showed a gradual decline in activity after the first cycle; however, after 13 cycles, its residual activity at 53 % was observed. These data proved significant enough to use this enzyme for industrial starch and analogous substrate bio-processing.

Artificial antibody-antigen-directed immobilization of lipase for consecutive catalytic synthesis of ester: Benzyl acetate case study.

A strategy based on artificial antibody-antigen recognition was proposed for the specific directed immobilization of lipase. The artificial antibody was synthesized using catechol as a template, α-methacrylic acid as a functional monomer, and Fe3O4 as the matrix material. Lipase was modified with 3,4-dihydroxybenzaldehyde as an artificial antigen. The artificial antibody can specifically recognize catechol fragment in the enzyme structure to achieve the immobilization of lipase. The immobilization amount, yield, specific activity, and immobilized enzyme activity were 13.2 ± 0.2 mg/g, 78.9 ± 0.4 %, 7.9 ± 0.2 U/mgprotein, and 104.6 ± 1.7 U/gcarrier, respectively. Moreover, the immobilized lipase exhibited strong reusability and regeneration ability. Additionally, the immobilized lipase successfully catalyzed the synthesis of benzyl acetate and demonstrated robust continuous catalytic activity. These results fully demonstrate the feasibility of the proposed artificial antibody-antigen-directed immobilization of lipase.

Application of Hierarchically Porous Chitosan Monolith for Enzyme Immobilization.

Enzyme immobilization is a crucial technique for improving the stability of enzymes. Compared with free enzymes, immobilized enzymes offer several advantages in industrial applications. Efficient enzyme immobilization requires a technique that integrates the advantages of physical absorption and covalent binding while addressing the limitations of conventional support materials. This study offers a practical approach for immobilizing α-amylase on a hierarchically porous chitosan (CS) monolith. An optimized CS monolith was fabricated using chemically modified chitin by thermally induced phase separation. By combining physical adsorption and covalent bonding, this technique leverages the amino and hydroxy groups present in CS to facilitate effective enzyme binding and stability. α-Amylase immobilized on the CS monolith demonstrated excellent stability, reusability, and increased activity compared to its soluble counterpart across various pH levels and temperatures. In addition, the CS monolith exhibited a significant potential to immobilize other enzymes, namely, lipase and catalase. Immobilized lipase and catalase exhibited higher loading capacities and enhanced activities than their soluble forms. This versatility highlights the broad applicability of CS monoliths as support materials for various enzymatic processes. This study provides guidelines for fabricating hierarchical porous monolith structures that can provide efficient enzyme utilization in flow systems and potentially enhance the cost-effectiveness of enzymes in industrial applications.

Nanodiamonds and natural deep eutectic solvents as potential carriers for lipase.

This study investigates the use of nanodiamonds (ND) as a promising carrier for enzyme immobilization and compares the effectiveness of immobilized and native enzymes. Three different enzyme types were tested, of which Rhizopus niveus lipase (RNL) exhibited the highest relative activity, up to 350 %. Under optimized conditions (1 h, pH 7.0, 40 °C), the immobilized ND-RNL showed a maximum specific activity of 0.765 U mg-1, significantly higher than native RNL (0.505 U mg-1). This study highlights a notable enhancement in immobilized lipase; furthermore, the enzyme can be recycled in the presence of a natural deep eutectic solvent (NADES), retaining 76 % of its initial activity. This aids in preserving the native conformation of the protein throughout the reusability process. A test on brine shrimp revealed that even at low concentrations, ND-RNL had minimal toxicity, indicating its low cytotoxicity. The in silico molecular dynamics simulations performed in this study offer valuable insights into the mechanism of interactions between RNL and ND, demonstrating that RNL immobilization onto NDs enhances its efficiency and stability. All told, these findings highlight the immense potential of ND-immobilized RNL as an excellent candidate for biological applications and showcase the promise of further research in this field.

Utilizing immobilized recombinant serine alkaline protease from Bacillus safensis lab418 in wound healing: Gene cloning, heterologous expression, optimization, and characterization.

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.

Functionalized electrospun nanofibers to enhance ß-Galactosidase immobilization and catalytic activity for efficient galactooligosaccharide synthesis.

This study aimed to immobilize ß-galactosidase (ß-GAL) into enhanced polystyrene (PS) electrospun nanofiber membranes (ENMs) with functionalized graphene oxide (GO). Initially, GO sheets were functionalized by salinization with 3-aminopropyl triethoxysilane (APTES). Then the ENMs (PS, PS/GO, and PS/GO-APTES) were prepared and characterized. Then, the ß-GAL was immobilized in the different ENMs to produce the ß-GAL-bound nanocomposites (PS-GAL, PS/GO-GAL, and PS/GO-APTES-GAL). Immobilization of ß-GAL into PS/GO-APTES significantly improved enzyme adsorption by up to 87 %. Also, PS/GO-APTES-GAL improved the enzyme activity, where the highest enzyme activity was obtained at enzyme concentrations of 4 mg/L, 50 °C, and pH 4.5. Likewise, the storage stability and reusability of immobilized ß-GAL were improved. Furthermore, this process led to enhanced catalytic behavior and transgalactosylation efficiency, where GOS synthesis (72 %) and lactose conversion (81 %) increased significantly compared to the free enzyme. Overall, the immobilized ß-GAL produced in this study showed potential as an effective biocatalyst in the food industry.

Microfluidic isolation of extrachromosomal circular DNA through selective digestion of plasmids and linear DNA using immobilized nucleases.

Extrachromosomal circular DNA (eccDNA) refers to small circular DNA molecules that are distinct from chromosomal DNA and play diverse roles in various biological processes. They are also explored as potential biomarkers for disease diagnosis and precision medicine. However, isolating eccDNA from tissues and plasma is challenging due to low abundance and the presence of interfering linear DNA, requiring time-consuming processes and expert handling. Our study addresses this by utilizing a microfluidic chip tailored for eccDNA isolation, leveraging microfluidic principles for enzymatic removal of non-circular DNA. Our approach involves integrating restriction enzymes into the microfluidic chip, enabling selective digestion of mitochondrial and linear DNA fragments while preserving eccDNA integrity. This integration is facilitated by an in situ photo-polymerized emulsion inside microchannels, creating a porous monolithic structure suitable for immobilizing restriction and exonuclease enzymes (restriction enzyme MssI and exonuclease ExoV). Evaluation using control DNA mixtures and plasma samples with artificially introduced eccDNA demonstrated that our microfluidic chips reduce linear DNA by over 99%, performing comparable to conventional off-chip methods but with substantially faster digestion times, allowing for a remarkable 76-fold acceleration in overall sample preparation time. This technological advancement holds great promise for enhancing the isolation and analysis of eccDNA from tissue and plasma and the potential for increasing the speed of other molecular methods with multiple enzymatic steps.