Carrier For Enzyme Immobilization Research Articles

High-Efficiency Enzyme Assay and Screening of Enzyme-Inhibiting Nanomaterials Using Capillary Electrophoresis with Hierarchically Porous Metal-Organic Framework-Based Immobilized Enzyme Microreactor.

Enzyme-inhibiting nanomaterials have significant potential for regulating enzyme activity. However, a universal and efficient method for systematically screening and evaluating the inhibitory effects of various nanomaterials on drug target enzymes has not been established. While the integrated technique of immobilized enzyme microreactor (IMER) with capillary electrophoresis (CE) serves as an effective tool for enzyme analysis, it still faces challenges such as low enzyme loadability, unsatisfactory stability, and limited applicability. Herein, hierarchical porous metal-organic frameworks (HP-MOFs) were explored as high-performance enzyme immobilization carriers and stationary phases to develop a novel HP-MOFs-based IMER-CE microanalysis system for efficient online enzyme assay and systematic screening of enzyme-inhibiting nanomaterials. As a proof-of-concept demonstration, the model enzyme xanthine oxidase (XOD) was immobilized on a HP-UiO-66-NH2 coated capillary, serving as an efficient and durable IMER for screening potential XOD-inhibiting nanomaterials. The hierarchically micro- and mesoporous structure and superior enzyme loadability of as-prepared HP-UiO-66-NH2-IMER was intensively characterized, followed by systematic evaluation of the separation performance of HP-UiO-66-NH2 coated column and the enzyme kinetics of the immobilized XOD. Compared to the microporous UiO-66-NH2-IMER, the HP-UiO-66-NH2-IMER-CE system showed significant improvements in enzyme loading, maximum reaction rate, repeatability, and long-term stability. Furthermore, the established method was effectively employed to screen the XOD inhibitory activity of various nanomaterials, revealing that graphene oxide, single wall carbon nanotube and three other nanomaterials exhibited inhibitory potentials. The HP-MOFs-based microanalysis system can be easily expanded by modifying the types of immobilized enzymes and holds the potential to accelerate the identification and rational design of effective enzyme-inhibiting nanomaterials.

Chitosan-Based Charge-Controllable Supramolecular Carrier for Universal Immobilization of Enzymes with Different Isoelectric Points.

Electrostatic adsorption is an enzyme immobilization method that effectively maintains enzyme activity and exhibits considerable binding efficiency. However, enzymes carry different charges at their respective reaction pH levels, which prevents the use of the same carrier to immobilize enzymes with different charges. In this study, we employed a template-mediated polysaccharide-enzyme coupling self-assembly strategy to develop a charge-controllable supramolecular immobilization carrier by regulating the charge properties of carboxymethyl chitosan, enabling the universal immobilization of enzymes with different charge levels across a range of reaction pH values. By using silica nanoparticles of certain sizes as templates, the size of the carrier can be precisely controlled and the hollow network structure formed after removing the template can effectively reduce mass transfer resistance. Trypsin and papain are used as model enzymes, and the experimental results show that the supramolecular self-assembly immobilization strategy does not disrupt the secondary structure of the enzyme molecules. After 2 h of reaction, the enzyme activities of immobilized papain and immobilized trypsin are 13.2% and 7.7% higher than those of the free enzymes, respectively. After 10 consecutive reactions, the enzyme activities of immobilized papain and immobilized trypsin retained 56.3% and 64.3% of their initial values, respectively.

Purification and immobilization of β-glucosidase using surface modified mesoporous silica Santa Barbara Amorphous 15 for eco-friendly preparation of sagittatoside A

Functionalized mesoporous materials have become a promising carrier for enzyme immobilization. In this study, Santa Barbara Amorphous 15 (SBA-15) was modified by N-aminoethyl-γ-aminopropyl trimethoxy (R). R-SBA-15 was employed to purify and immobilize recombinant β-glucosidase from Terrabacter ginsenosidimutans (BgpA) in one step for the first time. Optimum pH of the constructed R-SBA-15@BgpA were 7.0, and it has 20 ℃ higher optimal temperature than free enzyme. Relative activity of R-SBA-15@BgpA still retained > 70% at 42 ℃ after 8-h incubation. The investigation on organic reagent resistance revealed that the immobilized enzyme can maintain strong stability in 15% DMSO. In leaching test and evaluation of storage stability, only trace amount of protein was detected in buffer of the immobilized enzyme after storage at 4 ℃ for 33 days, and the immobilized BgpA still maintained > 50% relative activity. It also demonstrated good reusability, with 76.1% relative activity remaining after fourteen successive enzymatic hydrolyses of epimedin A to sagittatoside A. The newly proposed strategy is an effective approach for the purification and immobilization of BgpA concurrently. In addition, R-SBA-15@BgpA was demonstrated to have high efficiency and stability in this application, suggesting its great feasibility and potential to produce bioactive compounds such as secondary glycosides or aglycones from natural products.Graphical

Enhanced Interfacial Electron Transfer in Photocatalyst-Natural Enzyme Coupled Artificial Photosynthesis System: Tuning Strategies and Molecular Simulations.

Laccase is capable of catalyzing a vast array of reactions, but its low redox potential limits its potential applications. The use of photocatalytic materials offers a solution to this problem by converting absorbed visible light into electrons to facilitate enzyme catalysis. Herein, MIL-53(Fe) and NH2-MIL-53(Fe) serve as both light absorbers and enzyme immobilization carriers, and laccase is employed for solar-driven chemical conversion. Electron spin resonancespectroscopy results confirm that visible light irradiation causes rapid transfer of photogenerated electrons from MOF excitation to T1 Cu(II) of laccase, significantly increasing the degradation rate constant of tetracycline (TC) from 0.0062 to 0.0127 min-1. Conversely, there is only minimal or no electron transfer between MOF and laccase in the physical mixture state. Theoretical calculations demonstrate that the immobilization of laccase's active site and its covalent binding to themetal-organic frameworksurface augment the coupled system's activity, reducing the active site accessible from 27.8 to 18.1 Å. The constructed photo-enzyme coupled system successfully combines enzyme catalysis' selectivity with photocatalysis's high reactivity, providing a promising solution for solar energy use.

Eggshell waste transformation to calcium chloride anhydride as food-grade additive and eggshell membranes as enzyme immobilization carrier

Abstract In continuation of our efforts to fully utilize eggshell waste (ESW), here we report the possibility of ESW transformation to calcium chloride (CaCl2) anhydride of food-grade additive purity and eggshell membranes (ESMs) as potential enzyme immobilization carriers. ESW chemical transformation by 5% (w/v) hydrochloric acid to CaCl2 solution and ESM completely devoid of the remnants of ESW calcium carbonate was performed in the constructed 15 L batch reactor during 4 h at room temperature, followed by separation of ESM from CaCl2 solution by filtration. ESW-derived CaCl2 solution containing the excess hydrochloric acid was neutralized by adding calcium hydroxide, concentrated to approximately 1/8th of volume, and spray dried. Separated ESM was washed with water and acetone, dried, and ground to a size of less than 0.5 mm. The ESW transformation process produced 102.42 ± 3.31 g of CaCl2 anhydrous and 2.48 ± 0.28 g of ESM per 100 g of ESW dry matter. ESW-derived CaCl2 fulfilled all criteria for food-grade additive, while obtained ESM showed their suitability for Burkholderia cepacia lipase immobilization by adsorption.

Hydroxyapatite: An Eco-Friendly Material for Enzyme Immobilization.

Biocatalysis has emerged in the last decade as a valuable and eco-friendly tool in chemical synthesis, allowing in several instances to reduce or eliminate the use of hazardous reagents, environmentally dangerous solvents and harsh reaction conditions. Enzymes are indeed able to catalyse chemical transformations on non-natural substrates under mild reaction conditions, still maintaining their high chemo-, regio-, and stereoselectivity. Enzyme immobilization, i. e. the grafting of enzymes on solid supports, can be viewed as an enabling technology, as it allows a better control of the reaction and the recycling of the biocatalyst, thus rendering economically viable the use of expensive enzymes also on a large scale. To pursue a sustainable approach, the supports for enzyme immobilization should be eco-friendly and possibly renewable. This review highlights the use of hydroxyapatite (HAP), an inorganic biomaterial able to confer strength and stiffness to the bone tissue in animals, as carrier for enzyme immobilization. HAP is a cheap, non-toxic and biocompatible material, with high surface area and protein affinity. Different enzyme classes, immobilization strategies, and the use of diverse HAP-based supports will be discussed, underlining the immobilization conditions and the properties of the obtained biocatalysts.

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.

Multivariate mesoporous MOFs with regulatable hydrophilic/hydrophobic surfaces as a versatile platform for enzyme immobilization

Zeolitic imidazole frameworks materials-8 (ZIF-8), a member of the metal-organic framework (MOFs) series, have been extensively used as a host matrix for enzyme immobilization. However, the microporous structure and hydrophobicity, as well as the protonation of the precursor 2-methylimidazole (2-MeIm) of ZIF-8, remain considerable challenges in maintaining the activity of immobilized enzymes. Here, novel multivariate mesoporous MOFs (mMOFs) with regulatable hydrophilic/hydrophobic surfaces were designed by the multivariate competitive strategy and pore modification engineering. 3-Methyl-1H-1,2,4-triazole (3-MTZ) and 5-methyltetrazole (5-MTA) were employed to partially replace 2-MeIm. These were then combined with zinc sulfate heptahydrate (soft templates) to yield mMOFs in a methanol solution. As a proof-of-concept application, we used mMOFs as carriers for enzyme immobilization and investigated the properties of the immobilized enzymes. Benefiting from their mesoporous structure, hydrophilic surface, and improved microenvironment, multivariate mMOFs exhibit a strong ability to stabilize enzyme conformation and increase enzyme activity compared with traditional ZIF-8. Our study offers an avenue for the controllable preparation of well-designed MOF structures, which will further broaden the application opportunities of MOF materials for enzyme immobilization.

Tannic acid mediated hollow biocatalytic zeolitic imidazolate frameworks as a “three-in-one” label for enhancing the sensitivity of fluorescence immunosensor

Benefiting from the special structure feature, zeolitic imidazolate frameworks (ZIFs) have emerged as a carrier for enzyme immobilization. However, the sensitivity of the immunosensor with ZIFs needs further enhancement. Herein, a “three-in-one” label mediated by tannic acid (TA) was designed, demonstrating high enzyme activity, cascade signal amplification capability, and high coupling effectivity with antibodies. The acid property of TA was used as an etchant to etch ZIFs in situ for the generation of hollow ZIFs, which retained the conformational freedom of glucose oxidase for preserving their excellent catalytic activity. The TA on the surface of ZIFs coordinated Fe3+ ions through its phenol groups, forming metal polyphenol network films, which possessed peroxidase-like activity for cascade signal amplification. Meanwhile, the large amount of catechol groups of TA improved the coupling efficiency of antibodies. Based on the proposed “three-in-one” label, a novel fluorescence immunosensor was developed for the sensitive detection of Escherichia coli O157:H7. The limit of detection of the fluorescence immunosensor was 1.35 × 103 CFU mL−1 and 12.51-fold lower than that of traditional immunoassay. This proposed strategy provides a convenient method for preparing hollow ZIFs, facilitating the applications of ZIFs in the fields of biosensors.

Immobilization of L-asparaginase on Oxidized Bacterial Cellulose to Improve the Thermal Stability of the Enzyme

Bacterial cellulose (BC) membranes can be modified for covalent immobilization of macromolecules. One type of modification is oxidation, after which the oxidized BC membrane (OBC) could be used as a matrix for covalent immobilization of enzymes. In this work, the BC membrane was chemically oxidized with sodium periodate (NaIO4) to increase the stability of immobilized mesophilic L-asparaginase (L-ASNase) from Erwinia carotovora (EwA). IR spectroscopy confirmed the immobilization of L-ASNase EwA on OBC membranes. Immobilization of the enzyme increased its temperature optimum for its activity by 15°C and raised the inactivation temperature to 60°C. The OBC membrane could be used as a potential carrier for covalent immobilization of enzymes to improve their pharmacological properties by increasing their thermostability.

Enzyme-triggered approach to reduce water bodies' contamination using peroxidase-immobilized ZnO/SnO2/alginate nanocomposite

Enzyme immobilization on solid support offers advantages over free enzymes by overcoming characteristic limitations. To synthesize new stable and hyperactive nano-biocatalysts (co-precipitation method), ginger peroxidase (GP) was surface immobilized (adsorption) on ZnO/SnO2 and ZnO/SnO2/SA nanocomposite with immobilization efficacy of 94 % and 99 %, respectively. Thereafter, catalytic and biochemical characteristics of free and immobilized GP were investigated by deploying various techniques, i.e., FTIR, PXRD, SEM, and PL. Diffraction peaks emerged at 2θ values of 26°, 33°, 37°, 51°, 31°, 34°, 36°, 56°, indicating the formation of SnO2 and ZnO. The OH stretching of the H2O molecules was attributed to broad peaks between 3200 and 3500 cm−1, whereas ZnO/SnO2 spikes occurred in the 1626–1637 cm−1 range. SnO stretching mode and ZnO terminal vibrational patterns have been verified at corresponding wavelengths of 625 cm−1 and 560 cm−1. Enzyme entrapment onto substrate was verified via interactions between GP and ZnO/SnO2/SA as corroborated by signals beneath 1100 cm−1. GP-immobilized fractions were optimally active at pH 5, 50 °C, and retained maximum activity after storage of 4 weeks at −4 °C. Kinetic parameters were determined by using a Lineweaver-Burk plot and Vmax for free GP, ZnO/SnO2/GP and ZnO/SnO2/SA/GP with guaiacol as a substrate, were found to be 322.58, 49.01 and 11.45 (μM/min) respectively. A decrease in values of Vmax and KM indicates strong adsorption of peroxidase on support and maximum affinity between nano support and enzyme, respectively. For environmental remediation, free ginger peroxidase (GP), ZnO/SnO2/GP and ZnO/SnO2/SA/GP fractions effectively eradicated highly intricate dye. Multiple scavengers had a significant impact on the depletion of the dye. In conclusion, ZnO/SnO2 and ZnO/SnO2/SA nanostructures comprise an ecologically acceptable and intriguing carrier for enzyme immobilization.

Ultrathin 2D-MOFs for dual-enzyme cascade biocatalysis with sensitive glucose detection performances.

In recent years, two-dimensional nanosheet metal-organic frameworks (2D MOFs) have been widely considered as promising carriers for enzyme immobilization owing to their large surface area, designable and tunable structures, and other properties that enhance enzyme loading and modulate interactions with enzymes. In this study, a series of ultrathin 2D M-TCPP (M = Co, Ni, Zn, Cu) nanosheets were synthesized employing a surfactant-assisted bottom-up approach, and the effect of solvent ratio on the morphology and properties of 2D MOFs was explored. After systematic characterization, Cu-based 2D MOFs with large specific surface areas and excellent water stability was selected as the carrier for the co-immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP). The effects of adsorption and covalent immobilization strategies on bis-enzyme loading and enzyme activity, as well as their applications in rapid glucose detection, were systematically investigated. The results showed that A-CTGH and C-CTGH owned enzyme loadings of 187.9 and 249.1mg/g, respectively, and exhibited superior enzymatic activity when exposed to harsh environments compared to free enzymes. In addition, the covalently immobilized biocatalyst based on GOx demonstrated a more sensitive glucose detection performance, including a wide linear range from 5.0 to 16μM with a detection limit of 0.6μM.

Recent trends in metal‐organic frameworks mediated lipase immobilization: A state‐of‐the‐art review

AbstractImmobilized lipase is a powerful biocatalytic system with numerous applications in industries, particularly in the energy, pharmaceutical, cosmetic, and food industries. Reusability, simple recovery, and high chemical and thermal stability make it an attractive alternative to traditional chemical catalysts in industrial applications. Novel methods and support materials for immobilizing lipases have recently attracted much attention. Metal‐organic frameworks (MOFs) are a promising class of materials for enzyme immobilization carriers due to their appealing features, including a high specific surface area, high specific porosity, a stable framework structure, and a wide variety of functional sites. Due to the protection provided to enzymes by MOFs, several reported MOFs‐lipase composites display exceptional catalytic characteristics relative to free lipases. This includes increased enzyme efficiency, stability, selectivity, and recyclability. Herein, we summarize an updated review of the most recent advances in MOFs immobilizing lipases. This review sheds light on the numerous aspects of lipase‐MOF immobilization, with special emphasis on different techniques of designing lipase‐MOF platforms and the advantages of lipase‐MOF composites. Subsequently, molecular simulation approaches in lipase‐MOF immobilization are briefly introduced. Moreover, practical applications of MOFs‐lipase composites have been outlined. Finally, potential limitations and future directions for MOFs‐lipase immobilization research are highlighted.

Preparation of polyhydroxyalkanoate-based magnetic microspheres for carbonyl reductase purification and immobilization

A polyhydroxyalkanoate (PHA) magnetic microsphere was designed for one-step purification and immobilization of a novel carbonyl reductase (RLSR5) from recombinant Escherichia coli lysate. The hydrophobic core of this microsphere was composed of a highly biocompatible polymer, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), in which magnetic Fe3O4 particles were embedded during solvent evaporation. The hydrophilic shell of the fusion protein formed by PHA particle-binding protein (PhaP) and RLSR5 (PR) was expressed in recombinant E. coli. The magnetic core of Fe3O4@PHBHHx directly purified the hydrophilic shell from the E. coli lysate, and the two self-assembled to form Fe3O4@PHBHHx-PR through hydrophobic and hydrophilic interactions, eliminating the separation of the fusion protein. The microstructure, magnetic properties, morphology, size, and dispersion of Fe3O4@PHBHHx-PR were investigated by XRD, VSM, SEM, TEM, elemental mapping and DLS. It was found that Fe3O4@PHBHHx-PR correctly assembled, with a well dispersed spherical structure at the nanoscale and superparamagnetism properties. The amount of RLSR5 immobilized on PHA microspheres reached 121.9 mg/g. The Fe3O4@PHBHHx-PR was employed to synthesize (R)-tolvaptan with 99 % enantiomeric excess and 97 % bioconversion efficiency, and the catalyst maintained 78.6 % activity after 10 recovery cycles. These PHA magnetic microspheres are versatile carriers for enzyme immobilization and demonstrate improved stability and reusability of the free enzyme.

Multistep Extraction Transformation of Spent Coffee Grounds to the Cellulose-Based Enzyme Immobilization Carrier

The present study investigated the possibility of spent coffee ground (SCG) transformation to a cellulose-based enzyme immobilization carrier using a multistep extraction procedure. In the first step, SCGs were extracted with n-hexane by Soxhlet extraction in order to obtain SCG oil, while the remaining solid residue was subjected to continuous solvent flow sequential subcritical extraction with 96% and 50% ethanol and water. Afterwards, the obtained solid residue was subjected to alkaline liquefaction with 8% NaOH in order to remove lignin and produce cellulose-enriched SCGs as a potential enzyme immobilization carrier. Multistep extraction transformation of SCGs was monitored by chemical analysis of extracts and obtained solid residues. Soxhlet extraction of 100 g of SCGs yielded 10.58 g of SCG oil rich in linoleic and palmitic acid, while continuous solvent flow sequential subcritical extraction of 100 g of defatted SCGs yielded a total of 1.63 g of proteins, 5.58 g of sugars, 204 mg of caffeine, 76 mg of chlorogenic acid, and 11.97 mg of 5-(hydroxymethyl)furfural. Alkaline liquefaction of 100 g of sequentially extracted defatted SCGs by 8% NaOH yielded 7.45 g of proteins, 8.63 g of total polyphenols, 50.73 g of sugars, and 20.83 g of cellulose-enriched SCGs. Based on the characteristics of cellulose-enriched SCGs including a volume-weighted mean particle size of 277 μm, relative narrow particle size distribution with a span value of 1.484, water holding capacity of 7.55 mL/g, and a lack of carrier leakage, it could be safely concluded that produced cellulose-enriched SCGs fulfills criteria to be used as potential enzyme immobilization carrier. Overall, it seems that the proposed multistep extraction transformation of SCGs has great potential to be used for the production of several high-value added products.

Preparation and Catalytic Properties of Lipase Immobilized on Epoxy‐functionalized Starch Nanoparticles

AbstractAs a natural macromolecular material, starch is an ideal carrier for enzyme immobilization because of its widely available source, easy regeneration and excellent biodegradability. However, the natural starch cannot be directly used for immobilization due to the large particle size and excessive hydrophilicity. In this paper, the epoxy groups were grafted onto esterified starch by bifunctional reagents. The grafting efficiency of epoxy groups was increased by starvation drop addition. The changes of contact angle and particle size are conducive to the adsorption of immobilized enzyme at the water oil interface in the subsequent enzyme catalytic reaction, hence improve of the lipase activity. The maximum amount of lipase mobilized on such modified starch was ∼143.7 mg/g, which was near 40 mg/g higher than that of those from direct crosslinking. The immobilized enzymes showed excellent resistance to organic solvents and good reusability. Immobilization of lipases on epoxy‐functionalized starch nanoparticles can potentially improves the possibility of enzymes industrial application.

A novel bienzymatic bioreactor based on magnetic hierarchical porous MOF for improved catalytic activity and stability: Kinetic analysis, thermodynamics properties and biocatalyst applications

Metal-organic frameworks (MOFs) have been regarded as ideal carriers for enzyme immobilization owing to their unique structural characteristics. Whereas, enzyme activity and catalytic efficiency are seriously influenced owing to the conformation change of enzyme molecules during immobilization and the increase of substrate mass transfer resistance during catalysis. Herein, for the first time, we designed a novel magnetic hierarchical micro- and mesoporous metal-organic framework (mH-UiO-66(Zr)) as a platform for co-immobilizing horseradish peroxidase (HRP) and glucose oxidase (GOx). The obtained mH-UiO-66(Zr) with magnetic performance and hierarchical structure can effectively inhibit enzyme leakage, and reduce the material loss caused by centrifugation and the mass transfer resistance of substrate. Notably, the bienzyme bioreactor (GOx&HRP@mH-UiO-66(Zr)) was accurately constructed online in situ by microcalorimetry, which exhibited outstanding catalytic efficiency and superior stability against organic solvents, pH and temperature and favourable reusability, maintaining 95.1% of the original activity after 1 h incubation at 70 °C and over 90% initial activity after 15 cycles. Kinetics and thermodynamics investigation revealed the enhanced affinity of GOx&HRP@mH-UiO-66(Zr) for substrates. Furthermore, GOx&HRP@mH-UiO-66(Zr) has been utilized for the rapid detection and efficient degradation of 2,4-dichlorophenol (2,4-DCP), ensuring bio-catalytic material a bright potential application in environmental remediation.

Nano-sized mesoporous hydrogen-bonded organic frameworks for in situ enzyme immobilization

Hydrogen-bonded organic frameworks (HOFs) are promising carriers for enzyme immobilization. HOFs suitable for enzyme reactions containing nicotinamide cofactors and/or larger molecule substrates are to be explored. Herein, we report the first example of nano-sized mesoporous HOFs (nmHOFs) for in situ enzyme immobilization. Taking tetrakis(4-amidiniumphenyl) methane (TAM) and 1,3,6,8-tetrakis(p-benzoic acid) pyrene (H4TBAPy) as building blocks, enzymes induce the assembly of TAM and H4TBAPy into enzyme-nmHOF (named enzyme@TaTb) in aqueous solution. The larger π-conjugated H4TBAPy building block and the electrostatic/hydrogen-bonding interactions between TAM in nmHOF and –COOH/–NH2 residues in enzymes trigger the formation of TaTb with pore aperture of 2.4 nm and particle size from 19.9 ± 6.4 to 56.4 ± 20.1 nm. As a demonstration, lactate dehydrogenase (LDH) that can convert pyruvate into lactate in the presence of NADH is immobilized in TaTb nmHOF. Compared with LDH@ZIF-8 and LDH@Bio-HOF-1, LDH@TaTb affords faster diffusion of NADH and pyruvate, thus exhibiting ultrahigh activity close to the free LDH. Meanwhile, the exoskeleton of TaTb nmHOF is capable of stabilizing enzymes through spatial confinement, rendering superior stability against external negative stimuli. The TaTb nmHOF is also used for immobilizing α-amylase and horseradish peroxidase. Our study may facilitate the development of HOFs into platform carriers for enzyme immobilization.

Vacuum-assisted adsorption of Candida rugosa lipase with nanotubes for monooleoglycerol synthesis

A surplus of crude glycerol has been produced as a by-product due to the escalating production of biofuels. Utilization of crude glycerol for monoolein production which offers a different approach using the abandoned crude glycerol from the biodiesel sector is the main aim of this study. Immobilized lipase was used in this investigation to catalyse the direct esterification of oleic acid and glycerol, which resulted in monoolein. The immobilized Candida rugosa lipase onto the halloysite nanotubes (HNTs) has shown remarkably promising attainment of lipase activity and loading capacity. The immobilised lipase was optimised for crucial parameters (HNT to lipase ratio, pH, temperature, and pressure) via response surface methodology (RSM). The recommended optimum conditions obtained are pH 6, 38.6 °C, and 0.33 atm (81.92% lipase activity). To analyse influences of reaction period, temperature, mixing rate, and crude glycerol to oleic acid molar ratio on the esterification reaction was carried out using the optimized conditions of immobilization process. The optimum conditions for physically immobilised lipase were to yield 88.97% monoolein produced at 42 °C in a substrate ratio of 1:4 (oleic acid: glycerol) stirred at 200 rotations per minute (rpm) with 16 h’ incubation time. Based on the research's findings, it was confirmed that HNT has good application potential for enzyme immobilization carriers.

Fabrication of a Magnetic Porous Organic Polymer for α-Glucosidase Immobilization and Its Application in Inhibitor Screening

The technology based on immobilized enzymes was employed to screen the constituents inhibiting disease-related enzyme activity from traditional Chinese medicine, which is expected to become an important approach of innovative drug development. Herein, the Fe3O4@POP composite with a core-shell structure was constructed for the first time with Fe3O4 magnetic nanoparticles as the core, 1,3,5-tris (4-aminophenyl) benzene (TAPB) and 2,5-divinylterephthalaldehyde (DVA) as organic monomers, and used as the support for immobilizing α-glucosidase. Fe3O4@POP was characterized by transmission electron microscopy, energy-dispersive spectrometry, Fourier transform infrared, powder X-ray diffraction, X-ray photoelectron spectroscopy, and vibrating sample magnetometry. Fe3O4@POP exhibited a distinct core-shell structure and excellent magnetic response (45.2 emu g-1). α-Glucosidase was covalently immobilized on core-shell Fe3O4@POP magnetic nanoparticles using glutaraldehyde as the cross-linking agent. The immobilized α-glucosidase possessed improved pH stability and thermal stability as well as good storage stability and reusability. More importantly, the immobilized enzyme exhibited a lower Km value and enhanced affinity for the substrate than the free one. The immobilized α-glucosidase was subsequently used for inhibitor screening from 18 traditional Chinese medicines in combination with capillary electrophoresis analysis among which Rhodiola rosea exhibited the highest enzyme inhibitory activity. These positive results demonstrated that such magnetic POP-based core-shell nanoparticles were a promising carrier for enzyme immobilization and the screening strategy based on immobilized enzyme provided an effective way to rapidly explore the targeted active compounds from medicinal plants.