Efficient Enzyme Immobilization Research Articles

In situ immobilization multi-enzyme biocatalytic system on covalent organic frameworks for efficient conversion of lignocellulose to glucose

Efficient enzyme immobilization is crucial for addressing the resource utilization challenges associated with lignocellulose. However, the widespread application of immobilized enzyme systems faces significant obstacles, including low enzyme activity and the limited pore structure of existing carriers. To overcome these challenges, a novel multi-enzyme biocatalytic system (multi-enzymes@COF) was developed for the in situ immobilization of cellulose and β-glucosidase on covalent organic frameworks (COFs). Results showed that multi-enzyme@COF exhibits good crystallinity and a mesoporous structure, leading to an increased enzyme loading rate of 0.6 g/g and enhanced cellulose conversion efficiency of up to 78.7 %. Additionally, multi-enzymes@COF demonstrated remarkable stability a broader pH range (4−7) and temperature range (50–70 ℃), with the actively above 70 %. Moreover, the enzymes maintained approximately 74.7 % of their activity even after seven cycles. This research presents an innovative strategy for the effective utilization of lignocellulose through enzymatic processes, promoting sustainable and efficient resource utilization.

Spacer arm of ionic liquids facilitated laccase immobilization on magnetic graphene enhancing its stability and catalytic performance

To fulfill the requirements of environmental protection, a magnetically recoverable immobilized laccase has been developed for water pollutant treatment. In order to accomplish this objective, we propose a polydopamine-coated magnetic graphene material that addresses the challenges associated with accumulation caused by electrostatic interactions between graphene and enzyme molecules, which can lead to protein denaturation and inactivation. To achieve this, we present a polydopamine-coated magnetic graphene material that binds to the enzyme molecule through flexible spacer arms formed by ionic liquids. The immobilized laccase exhibited a good protective effect on laccase and showed a high stability and recycling ability. Laccase-ILs-PDA-MGO has a wider pH and temperature range and retains about 80% of its initial activity even after incubation at 50 °C for 2 h, which is 2.2 times more active than free laccase. Furthermore, the laccase-ILs-PDA-MGO exhibited a remarkable removal efficiency of 97.0% and 83.9% toward 2,4-DCP and BPA within 12 h at room temperature. More importantly, laccase-ILs-PDA-MGO can be recovered from the effluent and used multiple times for organic pollutant removal, while maintaining a relative removal efficiency of 80.6% for 2,4-DCP and 81.4% for BPA after undergoing seven cycles. In this study, a strategy for laccase immobilization by utilizing ILs spacer arms to modify GO aims to provide valuable insights into the advancement of efficient enzyme immobilization techniques and the practical application of immobilized enzymes in wastewater treatment.

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.

An enzymatic continuous-flow reactor based on a pore-size matching nano- and isoporous block copolymer membrane

Continuous-flow biocatalysis utilizing immobilized enzymes emerged as a sustainable route for chemical synthesis. However, inadequate biocatalytic efficiency from current flow reactors, caused by non-productive enzyme immobilization or enzyme-carrier mismatches in size, hampers its widespread application. Here, we demonstrate a general-applicable and robust approach for the fabrication of a high-performance enzymatic continuous-flow reactor via integrating well-designed scalable isoporous block copolymer (BCP) membranes as carriers with an oriented and productive immobilization employing material binding peptides (MBP). Densely packed uniform enzyme-matched nanochannels of well-designed BCP membranes endow the desired nanoconfined environments towards a productive immobilized phytase. Tuning nanochannel properties can further regulate the complex reaction process and fortify the catalytic performance. The synergistic design of enzyme-matched carriers and efficient enzyme immobilization empowers an excellent catalytic performance with >1 month operational stability, superior productivity, and a high space-time yield (1.05 × 105 g L−1 d−1) via a single-pass continuous-flow process. The obtained performance makes the designed nano- and isoporous block copolymer membrane reactor highly attractive for industrial applications.

Alloyed Trimetallic Nanocomposite as an Efficient and Recyclable Solid Matrix for Ideonella sakaiensis Lipase Immobilization.

In this work, a trimetallic (Ni/Co/Zn) organic framework (tMOF), synthesized by a solvothermal method, was calcinated at 400 and 600 °C and the final products were used as a support for lipase immobilization. The material annealed at 400 °C (Ni-Co-Zn@400) had an improved surface area (66.01 m2/g) and pore volume (0.194 cm3/g), which showed the highest enzyme loading capacity (301 mg/g) with a specific activity of 0.196 U/mg, and could protect the enzyme against thermal denaturation at 65 °C. The optimal pH and temperature for the lipase were 8.0 and 45 °C but could tolerate pH levels 7.0-8.0 and temperatures 40-60 °C. Moreover, the immobilized enzyme (Ni-Co-Zn@Lipase, Ni-Co-Zn@400@Lipase, or Ni-Co-Zn@600@Lipase) could be recovered and reused for over seven cycles maintaining 80, 90, and 11% of its original activity and maintained a residual activity >90% after 40 storage days. The remarkable thermostability and storage stability of the immobilized lipase suggest that the rigid structure of the support acted as a protective shield against denaturation, while the improved pH tolerance toward the alkaline range indicates a shift in the ionization state attributed to unequal partitioning of hydroxyl and hydrogen ions within the microenvironment of the active site, suggesting that acidic residues may have been involved in forming an enzyme-support bond. The high enzyme loading capacity, specific activity, encouraging stability, and high recoverability of the tMOF@Lipase indicate that a multimetallic MOF could be a better platform for efficient enzyme immobilization.

Cation-assisted stabilization of carbonic anhydrase one-step in situ loaded in diatom-inspired silica nanospheres for potential applications in CO2 capture and utilization

Carbonic anhydrase (CA) is a powerful green catalyst for CO2 capture and utilization due to its ultrafast kinetics and biobased nature. For industrial utilization, immobilization of CA is needed to increase enzyme stability and recovery. Diatom-inspired silica nanoparticle provides a green platform for the efficient immobilization of enzymes in a fast and facile manner. Herein, we describe a simple and effective method of salt addition during diatom-inspired silicification for the development of an immobilized and highly stabilized catalyst with bovine CA (bCA). Silica synthesis was facilitated by the silica-forming R5 peptide fused to the bCA; bCA-R5 was immobilized in situ with an excellent immobilization yield. The thermal stability of bCA-R5 was improved via salt supplementation, which was controlled by the cation-assisted increase in silica synthesis with a high packing density. The salt effect was dependent on both the pH and the enzyme’s electrostatic nature, suggesting the interplay among all the reaction components. The thermal stability of immobilized bCA-R5 was improved 11-fold in a biological buffer and 18-fold in 4.2 M MDEA (an organic solvent used as a CO2 absorbent) via the cation-assisted method. The suggested strategy is useful for developing CO2-capturing nanomaterials and can be widely applicable to immobilizing and stabilizing various proteins, maximally exploiting the potential of diatom-inspired silicification.

Synergistic co-catalytic nanozyme system for highly efficient one-pot colorimetric sensing at neutral pH: Combining molybdenum trioxide and Fe(III)-Modified covalent triazine framework

This study investigates the co-catalytic capabilities of MoO3 nanosheets in enhancing the enzyme-like catalytic activity of a two-dimensional ultrathin Fe(III)-modified covalent triazine framework (Fe-CTF) under neutral pH conditions. The unique physicochemical surface properties and two-dimensional structures of Fe-CTF enable the direct immobilization of native enzymes (glucose oxidase (GOD) and xanthine oxidase (XOD)) through adsorption, eliminating the need for chemical processes. Efficient immobilization of the native enzymes within the Fe-CTF/GOD(XOD) hybrid is achieved through multipoint attachment involving various interactions. The Fe-CTF/MoO3 co-catalytic system exhibits enzyme-mimicking activity at neutral pH and, when combined with the high catalytic activity of the immobilized native enzymes, enables the development of a colorimetric method for glucose detection. This method demonstrates excellent facilitation, rapidity, sensitivity, and selectivity, with a linear detection range of 50–1000 μM and a limit of detection of 8.8 μM for glucose. Furthermore, a straightforward one-pot colorimetric method is established for screening XOD inhibitors. The inhibitory potential of a crude extract derived from Chinese water chestnut peel on XOD activity is evaluated using this method. The findings of this study pave the way for the utilization of nanozyme/native enzyme hybrids in pH-neutral conditions for one-pot colorimetric sensing. This work contributes to the advancement of enzyme-based sensing technologies and holds promise for various applications in biosensing and biomedical research.

Enhanced stability and catalytic performance of laccase immobilized on magnetic graphene oxide modified with ionic liquids

Graphite oxide (GO) is an excellent laccase immobilization material. However, the electrostatic interaction between graphene leads to the accumulation of GO, as well as the interaction with the surface of enzyme molecules causing protein denaturation and deactivation, which limits its further industrial application. In this study, the ionic liquids (ILs) modification strategy was proposed to improve the stability and catalytic performance of immobilized laccase. The laccase-ILs-MGO exhibited remarkable enzymatic properties, with significant enhancements in organic solvent tolerance, thermal and operational stability. The laccase-ILs-MGO system exhibited a remarkable removal efficiency of 95.5% towards 2,4-dichlorophenol (2,4-DCP) within 12 h and maintained over 70.0% removal efficiency after seven reaction cycles. In addition, the efficient elimination of other phenolic compounds and recalcitrant polycyclic aromatic hydrocarbons could also be accomplished. Molecular dynamics simulation and molecular docking studies demonstrated that immobilized laccase exhibited superior structural rigidity and stronger hydrogen bond interactions with substrates compared to free laccase, which was beneficial for the stability of both the laccase and substrate degradation efficiency. Therefore, this study proposed a simple and practical strategy for modifying GO with ILs, providing novel insights into developing efficient enzyme immobilization techniques.

Enzyme-immobilized hierarchically porous covalent organic framework biocomposite for catalytic degradation of broad-range emerging pollutants in water

Efficient enzyme immobilization is crucial for the successful commercialization of large-scale enzymatic water treatment. However, issues such as lack of high enzyme loading coupled with enzyme leaching present challenges for the widespread adoption of immobilized enzyme systems. The present study describes the development and bioremediation application of an enzyme biocomposite employing a cationic macrocycle-based covalent organic framework (COF) with hierarchical porosity for the immobilization of horseradish peroxidase (HRP). The intrinsic hierarchical porous features of the azacalix[4]arene-based COF (ACA-COF) allowed for a maximum HRP loading capacity of 0.76 mg/mg COF with low enzyme leaching (<5.0 %). The biocomposite, HRP@ACA-COF, exhibited exceptional thermal stability (∼200 % higher relative activity than the free enzyme), and maintained ∼60 % enzyme activity after five cycles. LCMSMS analyses confirmed that the HRP@ACA-COF system was able to achieve > 99 % degradation of seven diverse types of emerging pollutants (2-mercaptobenzothiazole, paracetamol, caffeic acid, methylparaben, furosemide, sulfamethoxazole, and salicylic acid)in under an hour. The described enzyme-COF system offers promise for efficient wastewater bioremediation applications.

Efficient immobilization of horseradish peroxidase enzyme on transition metal carbides

In this study, the molecular dynamics simulation is performed to investigate the adsorption of horseradish peroxidase (HRP, a model enzyme) on Ti3C2 and Ti3C2OH (as templates for enzyme immobilization). It has been found that van der Waals and electrostatic interactions regulate enzyme immobilization, whereas the hydrogen bonding between enzyme and MXene is involved in the adsorption process. The simulation outcomes indicate that various MXene can effectually adsorb HRP, and the adsorption strength is more for the Ti3C2@HRP complex, as revealed by energy analysis (interaction energy = −710.92 vs −449.02 kJ mol−1). Moreover, the root means square deviation, root means square fluctuation and secondary structure analysis indicate that Ti3C2 protects the conformation of enzymes more than Ti3C2OH. Hence, HRP can be effectually immobilized after the Ti3C2 in comparison to the Ti3C2OH. In the following, the evaluation of 2-thiouracil (2-Thi) ability to modulate HRP activity is investigated. The obtained results show that 2-Thi inhibitor has a great capacity to inhibit HRP enzyme. Eventually, the stability of immobilized HRP at higher temperatures (330 and 363 K) and acidic pH is examined. The present work suggests theoretical insights intorealizing the biocompatibility of MXenes and instructs for modeling efficacious synthetic inhibitors.

A Dynamic Defect Generation Strategy for Efficient Enzyme Immobilization in Robust Metal‐Organic Frameworks for Catalytic Hydrolysis and Chiral Resolution

AbstractEnzyme immobilization has been demonstrated to be a favorable protocol for promoting the industrialization of bioactive molecules, but still with formidable challenge. Addressing this challenge, we create a dynamic defect generation strategy for enzyme immobilization by using the dissociation equilibrium of metal‐organic frameworks (MOFs) mediated by enzymes. Enzymes can act as “macro ligands” to generate competitive coordination against original ligands, along with the release of metal clusters of MOFs to generate defects, hence promoting the gradual transport of enzymes from the surface to inside. Various enzymes can be efficiently immobilized in MOFs to afford composites with good enzymatic activities, protective performances and exceptional reusabilities. Moreover, multienzyme bioreactors capable of efficient cascade reactions can also be generated. This study provides new opportunities to construct highly efficient biocatalysts incorporating different types of enzymes.

A Dynamic Defect Generation Strategy for Efficient Enzyme Immobilization in Robust Metal-Organic Framework forCatalytic Hydrolysis and Chiral Resolution.

Enzyme immobilization has been demonstrated to be a favorable protocol for promoting the industrialization of bioactive molecules, but still with formidable challenge. Addressing this challenge, we create a dynamic defect generation strategy for enzyme immobilization by using the dissociation equilibrium of metal-organic frameworks (MOFs) mediated by enzymes. Enzymes can act as "macro ligands" to generate competitive coordination against original ligands, along with the release of metal clusters of MOFs to generate defects, hence promoting the gradual transport of enzymes from the surface to inside. Various enzymes can be efficiently immobilized in MOFs to afford composites with good enzymatic activities, protective performances and exceptional reusabilities. Moreover, multienzyme bioreactors capable of efficient cascade reactions can also be generated. This study provides new opportunities to construct highly efficient biocatalysts incorporating different types of enzymes.

Enhanced Activity of Enzyme Immobilized on Hydrophobic ZIF-8 Modified by Ni2+ Ions.

The development of efficient enzyme immobilization to promote their recyclability and activity is highly desirable. Zeolitic imidazolate framework-8 (ZIF-8) has been proved to be an effective platform for enzyme immobilization due to its easy preparation and biocompatibility. However, the intrinsic hydrophobic characteristic hinders its further development in this filed. Herein, a facile synthesis approach was developed to immobilize pepsin (PEP) on the ZIF-8 carrier by using Ni2+ ions as anchor (ZIF-8@PEP-Ni). By contrast, the direct coating of PEP on the surface of ZIF-8 (ZIF-8@PEP) generated significant conformational changes. Electrochemical oxygen evolution reaction (OER) was employed to study the catalytic activity of immobilized PEP. The ZIF-8@PEP-Ni composite attains remarkable OER performance with an ultralow overpotential of only 127 mV at 10 mA cm-2 , which is much lower than the 690 and 919 mV overpotential values of ZIF-8@PEP and PEP, respectively.

Enhanced Activity of Enzyme Immobilized on Hydrophobic ZIF‐8 Modified by Ni2+ Ions

AbstractThe development of efficient enzyme immobilization to promote their recyclability and activity is highly desirable. Zeolitic imidazolate framework‐8 (ZIF‐8) has been proved to be an effective platform for enzyme immobilization due to its easy preparation and biocompatibility. However, the intrinsic hydrophobic characteristic hinders its further development in this filed. Herein, a facile synthesis approach was developed to immobilize pepsin (PEP) on the ZIF‐8 carrier by using Ni2+ ions as anchor (ZIF‐8@PEP‐Ni). By contrast, the direct coating of PEP on the surface of ZIF‐8 (ZIF‐8@PEP) generated significant conformational changes. Electrochemical oxygen evolution reaction (OER) was employed to study the catalytic activity of immobilized PEP. The ZIF‐8@PEP‐Ni composite attains remarkable OER performance with an ultralow overpotential of only 127 mV at 10 mA cm−2, which is much lower than the 690 and 919 mV overpotential values of ZIF‐8@PEP and PEP, respectively.

Supported ionic liquid phase facilitated catalysis with lipase from Aspergillus oryzae for enhance enantiomeric resolution of racemic ibuprofen

Supported ionic liquid phase (SILP) was used as a carrier for lipase from Aspergillus oryzae (LAO) and used as a biocatalyst for enantiomeric resolution of racemic ibuprofen via esterification leading to (S) -(+)-ibuprofen ester. Using native form of lipase, outstanding results were achieved, obtaining (S) -(+)-ibuprofen propyl ester with enantiomeric excess ( ee ) of 99.9% and high conversion of racemic ibuprofen after 24 h ( α = 34 . 8 % ) and respectively ee = 99.9% with α = 45 . 2 % after 48 h. Several hybrid materials composited with silica and metal-based oxides including magnesium, calcium, and zirconia were evaluated as supports for LAO with various surface characteristics. The selected ionic liquid 1-methyl-3-(triethoxysilylpropyl)imidazolium bis(trifluoromethylsulfonyl)imide was immobilized via the covalent bound onto the surface of solid material and in the second step LAO was anchored. Optimized results in enantiomeric resolution of racemic ibuprofen (35.23% conversion of rac -ibuprofen after 7 days with 95% ee of ester) were obtained for SILP biocatalyst based on MgO ⋅ SiO 2 (1:1) (ionic liquid loading 6.79%, enzyme loading 3.96%). This is proposed as a generic approach to tailoring supported ionic liquids phase biocatalysts for industrially-relevant reactions, to generate both environmentally and economically sustainable processes. • Aspergillus oryzae lipase was used in enantiomeric resolution of racemic ibuprofen. • Free enzyme showed over 45% reaction yield and 99.9% enantiomeric excess after 48 h. • Supported ionic liquid phase with lipase was used to improve system feasibility. • Modification of surface with ionic liquid enabled efficient immobilization of enzyme. • High reaction rate and enantioselectivity were maintained in the reusability test.

Halohydrin dehalogenase immobilization in magnetic biochar for sustainable halocarbon biodegradation and biotransformation

Regarding the fact that halohydrin dehalogenase (HHDH) displays great potential for both biotransformation and biodegradation, developing immobilized HHDH that is suitable for those applications will be of great importance. Herein, the functionalized magnetic biochar was prepared for efficient enzyme immobilization, easy separation, and reuse. The immobilized halohydrin dehalogenase (HheC) was characterized using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM). The results showed that the relative enzyme activity retention was 85% upon immobilization in biochar (HheC-N-MBC600). After 70 days of storage at 4 °C, the HheC-N-MBC600 retained 50% of its initial activity, while only 8% of the activity was retained for the free enzyme. HheC-N-MBC600 displayed a strong organic solvent tolerance as it retained activity above 92% after incubation in 50% organic solvent for 12 h. Moreover, the immobilized catalyst retained more than 70% activity after a consecutive 30 cycles of reuse, indicating excellent recyclability of the immobilized biocatalyst. Moreover, the immobilized enzyme displayed the same enantioselective behavior as the free enzyme. Together with the immobilized epoxide hydrolyase (EchA-MBC600), HheC-N-MBC600 completely converted 1,3-dichloro-2-propanol (1,3-DCP) to a nontoxic compound. Our results showed that the immobilized HheC-N-MBC600 could be a green and industrially viable tool to degrade halogenated compounds and make useful chiral compounds.

Efficient immobilization of catalase on mesoporous MIL-101 (Cr) and its catalytic activity assay

Enzyme immobilization using metal-organic frameworks (MOFs) as carriers has aroused significant interest owing to the unique pore structure and versatile surface functional groups of MOFs. Catalase (CAT) is an important industrial enzyme that is widely used in the catalytic decomposition of hydrogen peroxide in the fields of food and biological products. In this study, mesoporous MIL-101 (Cr), synthesized through a facile hydrothermal process, was applied for CAT immobilization for the first time. The immobilization capacity of MIL-101 (Cr) for CAT was studied systematically by batch adsorption tests under different adsorption conditions, including the variation of the solution pH, operation temperature, adsorption time, and initial concentration of CAT. Based on these test findings, the optimum adsorption conditions and maximum adsorption capacity were determined. The adsorption kinetics were simulated to further explore the adsorption mechanism, and they suggest that chemical adsorption, rather than physical adsorption, is the main CAT adsorption mechanism. A comparison of Fourier transform infrared (FT-IR) spectra of MIL-101 (Cr) without and with adsorbed CAT reveals the formation of amide bonding between the -NH of CAT and the uncoordinated -CO of MIL-101(Cr). Finally, the stability and activity of the immobilized CAT were assessed, and an improved insensitivity against changes in pH and a prolonged storage time demonstrate the enhanced stability of immobilized CAT by MIL-101 (Cr) carriers. This study demonstrates the application of MOFs as functional supports for the efficient immobilization of versatile enzymes.

Modeling enzymes interacting with functionalized surfaces: lessons from multiscale modeling approaches

The efficient immobilization of enzymes on surfaces remains a complex but central issue in the biomaterials field. For example, the design of efficient biofuel cells requires us to grasp details of the interaction between the enzymes and the electrode surfaces on the molecular level. Such information can be obtained using molecular modeling approaches on different scales, either with classical all-atom Molecular Dynamics simulations, or with coarse-grain calculations based on Elastic Network Models.

Between Two Walls: Modeling the Adsorption Behavior of β-Glucosidase A on Bare and SAM-Functionalized Gold Surfaces.

The efficient immobilization of enzymes on surfaces remains a complex but central issue in the biomaterials field, which requires us to understand this process at the atomic level. Using a multiscale approach combining all-atom molecular dynamics and coarse-grain Brownian dynamics simulations, we investigated the adsorption behavior of β-glucosidase A (βGA) on bare and self-assembled monolayer (SAM)-functionalized gold surfaces. We monitored the enzyme position and orientation during the molecular dynamics (MD) trajectories and measured the contacts it forms with both surfaces. While the adsorption process has little impact on the protein conformation, it can nonetheless perturb its mechanical properties and catalytic activity. Our results show that compared to the SAM-functionalized surface, the adsorption of βGA on bare gold is more stable, but less specific, and more likely to disrupt the enzyme's function. This observation emphasizes the fact that the structural organization of proteins at the solid interface is a key point when designing devices based on enzyme immobilization, as one must find an acceptable stability-activity trade-off.

One-pot synthesis and biochemical characterization of a magnetic collagenase nanoflower and evaluation of its biotechnological applications

Recently, hierarchical magnetic enzyme nanoflowers have been found extensive attention for efficient enzyme immobilization due to high surface area, low mass transfer limitations, active site accessibility, promotion of the enzymatic performance, and facile reusing. Herein, we report the purification of the Bacillus collagenase and then synthesis of magnetic cross-linked collagenase-metal hybrid nanoflowers (mcCNFs). The catalytic efficiency (kcat/Km) value of the immobilized collagenase was 2.2 times more than that of the free collagenase. The collagenase activity of mcCNFs enhanced about 2.9 and 4.6 at 85 and 90 °C, respectively, compared to free collagenase. Thermal stability of mcCNFs increased about 31% and 24% after 3 h of incubation at 50 and 60 °C, respectively. After 10 cycles of reusing, the mCNFs collagenase showed 83% of its initial activity. Results showed that the mcCNFs revealed 1.4 times more activity than the free collagenase in 0.16% protein waste. Furthermore, the hydrolysis value of chicken pie protein wastes by the immobilized enzyme obtained 4 times more than the free collagenase after 240 min incubation at 40 °C. Finally, our results showed that the construction of mcCNFs is an efficient method to increase the enzymatic performance and has excessive potential for the hydrolysis of protein wastes in the food industry.