Immobilized Enzyme Reactor Research Articles

Development of a Dual-Stage CIM® CDI Reactor with Immobilized Glucuronan Lyases and Laccases for Sustainable Synthesis of Antioxidant Phenolized Oligoglucuronan

Immobilized enzyme reactors (IMERs) are critical tools for developing novel oligosaccharides based on the enzymatic catalysis of polysaccharides. In this paper, a novel glucuronan lyase from Peteryoungia rosettiformans was produced, purified, and then immobilized on a CIM® CDI disk for cleaving glucuronan. The results showed that around 63.6% of glycuronan lyases (800.9 μg) were immobilized on the disk. The Vmax values of immobilized glucuronan lyases did not significantly change (56.9 ± 4.7 μM∙min−1), while the Km values (0.310 ± 0.075 g∙L−1) increased by 2.5 times. It is worth noting that immobilized glucuronan lyases overcame the catalytic inhibition of free enzymes observed under high glucuronan concentrations (0.5–2 g∙L−1). circumscribed central composite design (CCCD) and response surface methodology (RSM) showed that glucuronan concentration, flow rate, and reaction time significantly affected the yield of oligoglucuronans. The degree of polymerization (DP) of degraded glucuronan ranged from DP 2–8 according to the results obtained by high performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD). The IMER retained 50.9% activity after running 2373 column volumes of glucuronan. Finally, this glucuronan lyase reactor was tentatively connected to an immobilized laccase reactor to depolymerize, and gallic acid (GA) was added to glucuronan. Approximately 8.5 mg of GA was added onto 1 g of initial glucuronan, and the GA–oligoglucuronan conjugates showed notable antioxidant activity.

Sustainable Enzymatic Production of Omega-3 Oil from Squid Viscera

Fish oils are widely consumed around the world to increase omega-3 fatty acid intake. Due to negative impacts on marine resources and ecosystems from an increasing demand for fish, alternative sustainable sources are under investigation. Squid viscera contains up to 10% oil by mass and is available as a byproduct from squid processing. Squid viscera oil is a source of EPA and DHA and contains the xanthophyll carotenoid astaxanthin, known for its significant anticancer, antioxidant, antidiabetic, and cardiovascular properties. In the raw form, squid viscera oil has a high free fatty acid (FFA) content, so conventional alkaline refining results in low yield and loss of astaxanthin. As a higher-yielding alternative, the current study optimized lipase-catalyzed glycerolysis of squid viscera oil to convert FFA into acylglycerol using a custom-built one-liter immobilized enzyme reactor. To monitor the reaction progress and assess its impact on the oil, we analyzed lipid classes, fatty acid composition and astaxanthin levels. Under optimized conditions, FFA was reduced from 40% to 2.7% in 10 h and 1.7% in 24 h, with no significant effect on EPA and DHA levels, and astaxanthin being retained. Squid viscera presents a safe and sustainable additional source of marine-derived EPA and DHA oil.

Molar-scale formate production via enzymatic hydration of industrial off-gases

Decarbonizing the steel industry, a major CO2 emitter, is crucial for achieving carbon neutrality. Escaping the grip of CO combustion methods, a key contributor to CO2 discharge, is a seemingly simple yet formidable challenge on the path to industry-wide net-zero carbon emissions. Here we suggest enzymatic CO hydration (enCOH) inspired by the biological Wood‒Ljungdahl pathway, enabling efficient CO2 fixation. By employing the highly efficient, inhibitor-robust CO dehydrogenase (ChCODH2) and formate dehydrogenase (MeFDH1), we achieved spontaneous enCOH to convert industrial off-gases into formate with 100% selectivity. This process operates seamlessly under mild conditions (room temperature, neutral pH), regardless of the CO/CO2 ratio. Notably, the direct utilization of flue gas without pretreatment yielded various formate salts, including ammonium formate, at concentrations nearing two molar. Operating a 10-liter-scale immobilized enzyme reactor feeding live off-gas at a steel mill resulted in the production of high-purity formate powder after facile purification, thus demonstrating the potential for decarbonizing the steel industry.

Insights into nucleoside hydrolase from Leishmania donovani inhibition: A new bioaffinity chromatography-based screening assay and docking studies

Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(β-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(β-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (Ki = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors.

Ultrafast protein digestion using an immobilized enzyme reactor following high-resolution mass spectrometry analysis for rapid identification of abrin toxin.

Abrin toxin, highly dangerous with an estimated human lethal dose of 0.1-1 μg per kg body weight, has attracted much attention regarding criminal and terroristic misuse over the past decade. Therefore, developing a rapid detection method for abrin toxin is of great significance in the field of biosecurity. In this study, based on the specific dissociation method of an immobilized enzyme reactor, the trypsin immobilized reactor Fe3O4@CTS-GA-Try was prepared to replace free trypsin, and the immobilized enzyme digestion process was systematically investigated and optimized by using bovine serum albumin as the simulant of abrin. After 5 min one-step denaturation and reduction, a satisfactory peptide number and coverage were yielded with only 15 s assisted by an ultrasound probe to identify model proteins. Subsequently, abrin was rapidly digested using the established method, resulting in a stable and highly reproducible characteristic peptide number of 39, which can be analyzed by nanoelectrospray ionization coupled with high-resolution mass spectrometry. With the acquisition mode of full MS scan coupled with PRM, not only MS spectroscopy of total abrin peptides but also the corresponding MS/MS spectroscopy of specific abrin peptides can achieve the characteristic detection of abrin toxin and its different isoforms in less than 10 minutes, with high repeatability. This assay provides a universal platform and has great potential for the development of on-site detection and rapid mass spectrometric analysis techniques for macromolecular protein toxins and can further be applied to the integrated detection of chemical and biological agents.

A multi-channel microfluidic platform based on human flavin-containing monooxygenase 3 for personalised medicine.

Human flavin-containing monooxygenase 3 (FMO3) is a drug-metabolizing enzyme (DME) which is known to be highly polymorphic. Some of its polymorphic variants are associated with inter-individual differences that contribute to drug response. In order to measure these differences, the implementation of a quick and efficient in vitro assay is highly desirable. To this end, in this work a microfluidic immobilized enzyme reactor (μ-IMER) was developed with four separate serpentines where FMO3 and its two common polymorphic variants (V257M and E158K) were covalently immobilized via glutaraldehyde cross-linking in the presence of a polylysine coating. Computational fluid dynamics simulations were performed to calculate the selected substrate retention time in serpentines with different surface areas at various flow rates. The oxidation of tamoxifen, an anti-breast cancer drug, was used as a model reaction to characterize the new device in terms of available surface area for immobilization, channel coating, and applied flow rate. The highest amount of product was obtained when applying a 10 μL min-1 flow rate on polylysine-coated serpentines with a surface area of 90 mm2 each. Moreover, these conditions were used to test the device as a multi-enzymatic platform by simultaneously assessing the conversion of tamoxifen by FMO3 and its two polymorphic variants immobilized on different serpentines of the same chip. The results obtained demonstrate that the differences observed in the conversion of tamoxifen within the chip are similar to those already published (E158K > WT > V257M). Therefore, this microfluidic platform provides a feasible option for fabricating devices for personalised medicine.

Precise Control of Trypsin Immobilization by a Programmable DNA Tetrahedron Designed for Ultrafast Proteome Digestion and Accurate Protein Quantification.

In proteomics research, with advantages including short digestion times and reusable applications, immobilized enzyme reactors (IMERs) have been paid increasing attention. However, traditional IMERs ignore the reasonable spatial arrangement of trypsin on the supporting matrixes, resulting in the partial overlapping of the active domain on trypsin and reducing digesting efficiency. In this work, a DNA tetrahedron (DNA TET)-based IMER Fe3O4-GO-AuNPs-DNA TET-Trypsin was designed and prepared. The distance between vertices of DNA TETs effectively controls the distribution of trypsin on the nanomaterials; thus, highly efficient protein digestion and accurate quantitative results can be achieved. Compared to the in-solution digestion (12-16 h), the sequence coverage of bovine serum albumin was up to 91% after a 2-min digestion by the new IMER. In addition, 3328 proteins and 18,488 peptides can be identified from HeLa cell protein extract after a 20-min digestion. For the first time, human growth hormone reference material was rapidly and accurately quantified after a 4-h digestion by IMER. Therefore, this new IMER has great application potential in proteomics research and SI traceable quantification.

A-202 Establishing of Lipid-Apolipoprotein Connectivity Map in Hyperglycemia

Abstract Background Hyperglycemia, a condition characterized by a significantly increased glucose level in the blood, can lead to insulin deficiency and pre-diabetes. Lack of insulin production also interferes with lipid metabolism and increases the risk for cardiovascular disease. Thus, diagnosis and treatment of hyperglycemic patients can benefit from advanced proteomics and lipidomics testing approaches. Methods Disease-focused serum samples from 50 specimens were collected and evaluated, including 29 specimens with a glucose level>180 mg/dL and 21 normal lipidemic specimens. The sample proteomics analysis was conducted with a Perfinity IDP workstation (Shimadzu Scientific, Columbia, MD, USA) using on-line protein digestion with an immobilized enzyme reactor (IMER) directly coupled to a HALO-C18 column (Advanced Materials Technology, Wilmington, DE, USA). Lipidomics data were collected with the Lipidyzer platform (AB SCIEX, Framingham, MA, USA). Statistical analysis was conducted using JMP software (SAS Institute, USA) and custom scripts. Results Using targeted lipidomics and proteomics platforms we identified 20 proteins and 575 lipid species across 10 lipid subclasses. We identified specific lipids and lipoproteins dysregulated in hyperglycemic specimens as compared to the normolipidemic samples and linked them to major metabolic pathways. Lipoprotein particles related-proteins apos A1/B/C2/C3/E and lecithin–cholesterol acyltransferase (LCAT) were significantly dysregulated and associated with changes in HDL-mediated lipid transport, very-low-density lipoprotein particle remodeling, acylglycerol homeostasis, and triglyceride homeostasis. Conclusion The conducted integrated analysis of lipid and protein concentrations in hyperglycemia specimens to provide new insights into molecular changes associated with hyperglycemia that may inform new diagnostic and therapeutic strategies. The network analysis can allow improved categorization of patients that can lead to more effective and individualized therapeutic intervention approaches. This study provides new experimental and informatics approaches to better assess underlying metabolic conditions and related diseases, including cardiovascular disease, diabetes, and metabolic syndrome. Disclaimer The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the CDC.

Nucleoside hydrolase immobilized on magnetic particles as a tool for onflow screening and characterization of inhibitors

Nucleoside Hydrolases (NH) are considered a target for the development of new antiprotozoal agents. The development of new and automated screening assays for the identification of NH inhibitors can accelerate the first stages of the drug discovery process. In this work, NH from Leishmania donovani (LdNH) was covalently immobilized onto magnetic particles (LdNH-MPs) and trapped by magnets into a TFE tube to yield an immobilized enzyme reactor (IMER). For an automated assay, the LdNH-MP-IMER was connected in-line to an analytical column in an HPLC-DAD system to monitor the enzyme activity through quantification of the product hypoxanthine. Kinetic studies provided a KM value of 2079 ± 87 µmol.L−1 for the inosine substrate. Validation of the LdNH-MP-IMER for onflow screening purposes was performed with a library containing 12 quinolone ribonucleosides. Among them, three were identified as new competitive LdNH inhibitors, with Ki values between 83.5 and 169.4 µmol.L−1. This novel in-line screening assay has proven to be reliable, fast, low cost, and applicable to large libraries of compounds.

Modeling glucose isomerization in a packed- bed reactor: A complete theoretical and numerical approach

Immobilized enzymes are used in many applications in the food industry presently. Some researchers employ apparent kinetic parameters in immobilized enzyme reactor systems, which only apply to these specific case studies. To increase productivity in a packed bed reactor, it is vital to establish a thorough understanding of all the factors affecting the process, including axial dispersion, convective mass transport, intra-particle diffusive mass transfer, and kinetics. This paper discusses the packed-bed reactor's steady-state model of immobilized glucose isomerase. This model is based on a nonlinear reaction-diffusion equation containing nonlinear terms associated with the enzymatic reaction of Michaelis–Menten kinetics. Akbari-Ganji's method (AGM) and Taylors series with Pade's approximation method are used to calculate the species concentration and normalized current for all possible kinetic parameters and Thiele modulus. The saturated and unsaturated kinetics also have been investigated analytically. A significant agreement between the simulation results and the analytical expression of concentration is observed.

Immobilization of the Amidohydrolase MxcM and Its Application for Biocatalytic Flow Synthesis of Pseudochelin A

The chemical synthesis of heterocycles typically requires elevated temperature and acid or base addition to form the desired product. Moreover, these reactions often involve hazardous reagents, which is why biocatalytic routes for heterocycle formation have gained increasing attention. In recent years, several enzymes belonging to the amidohydrolase superfamily have been identified to generate heterocycles via cyclocondensation reactions. Of particular interest is the amidohydrolase MxcM, which catalyzes the formation of an imidazoline moiety in the biosynthesis of the anti-inflammatory natural product pseudochelin A. In this study, we present a concept for the immobilization of this enzyme using a fused hexahistidine tag for fixation onto a solid, porous carrier. Notably, the immobilization improves the enzyme’s tolerance to organic solvents. The immobilized MxcM exhibits a residual activity of 169% in the polar solvent acetonitrile compared to the free enzyme, and the storage stability in the presence of 20 vol% acetonitrile was ameliorated. In addition, an immobilized enzyme reactor (IMER) was designed that can be operated under flow conditions. The MxcM-IMER retains its biocatalytic activity and mechanic stability over the tested operation time. These results provide important insights for the integration of heterocycle-forming amidohydrolases in chemical processes.

Study on Construction and Performance of Immobilized Enzyme Reactors by Carboxyl-functionalized Fe3O4

Study on Construction and Performance of Immobilized Enzyme Reactors by Carboxyl-functionalized Fe₃O₄

Kinetics of Enzymatic Reactions at the Solid/Liquid Interface in Nanofluidic Channels.

Nanostructures can realize highly efficient reactions due to their structural advantages. However, the mechanism of accelerating enzyme reactions in a nanospace is still unknown from a kinetic perspective because it is difficult to control a well-defined nanospace, enzyme density, and reaction time. Here, we investigated kinetic parameters of an immobilized enzyme in micro- and nanochannels using nanofabrication, partial enzyme patterning, fluidic control, and a high sensitivity detection system. Devices with channel depths of 300 nm, 4.4 μm, and 13.6 μm were fabricated. Kinetic parameters were determined by the Michaelis-Menten model. Compared to the bulk reaction, all kcats for immobilized enzyme reactors were decreased, although the kcats were approximately the same for the immobilized enzyme reactors of different depths. An ultrafast enzyme reaction could overcome the drawback due to immobilization by an increase of the apparent [E]0 due to the decreased channel depth.

Ten Years Milestones in Xanthine Oxidase Inhibitors Discovery: Febuxostat-Based Inhibitors Trends, Bifunctional Derivatives, and Automatized Screening Assays

Xanthine oxidase (XO) is an enzyme involved in the oxidative process of hypoxanthine and xanthine to uric acid (UA). This process also produces reactive oxygen species (ROS) as byproducts. Both UA and ROS are dangerous for human health, and some health conditions trigger upregulation of XO activity, which results in many diseases (cancer, atherosclerosis, hepatitis, gout, and others) given the worsened scenario of ROS and UA overproduction. So, XO became an attractive target to produce and discover novel selective drugs based on febuxostat, the most recent XO inhibitor out of only two approved by FDA. Under this context, high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have been successfully applied to rapidly and easily screen for bioactive compounds, isolated or in complex natural matrixes, that act as enzyme inhibitors through the use of an immobilized enzyme reactor (IMER). This article’s goal is to present advances comprising febuxostat-based XO inhibitors as a new trend, bifunctional moieties capable of inhibiting XO and modulating ROS activity, and in-flow techniques employing an IMER in HPLC and CE to screen for synthetic and natural compounds that act as XO inhibitors.

On-flow enzymatic inhibitor screening: The emerging success of liquid chromatography-based assays

Enzymes are targets commonly explored in screening assays aiming to discover new leads in the drug development process. Among the diverse assay models to identify new enzymatic inhibitors, on-flow assays based on liquid chromatography (LC) can be highlighted. In these approaches, the ligand-enzyme interaction can be examined by monitoring the catalytic activity or the affinity/retention. Most applications use the biological target immobilized in solid supports resulting in the acquisition of an immobilized enzymatic reactor (IMER). Coupling IMERs to LC or mass spectrometry (MS) systems allows monitoring enzyme activity online and studying binding events between target and ligands. On-flow screening assays present many advantages for the hit-to-lead process, such as the possibility of system automation, reusability, and high stability. This review covers articles from the last decade that combine the use of varied immobilization methods on different solid supports and several equipment setups in on-flow systems, emphasizing the performance and capacity of recognizing and identifying biologically active compounds in various matrices.

Transient response of immobilized enzyme reactors - the effects of reactor type and shape of core-shell bio-catalytic pellets

Transient response of immobilized enzyme reactors - the effects of reactor type and shape of core-shell bio-catalytic pellets

3D-Printed High-Pressure-Resistant Immobilized Enzyme Microreactor (μIMER) for Protein Analysis.

Additive manufacturing (3D printing) has greatly revolutionized the way researchers approach certain technical challenges. Despite its outstanding print quality and resolution, stereolithography (SLA) printing is cost-effective and relatively accessible. However, applications involving mass spectrometry (MS) are few due to residual oligomers and additives leaching from SLA-printed devices that interfere with MS analyses. We identified the crosslinking agent urethane dimethacrylate as the main contaminant derived from SLA prints. A stringent washing and post-curing protocol mitigated sample contamination and rendered SLA prints suitable for MS hyphenation. Thereafter, SLA printing was used to produce 360 μm I.D. microcolumn chips with excellent structural properties. By packing the column with polystyrene microspheres and covalently immobilizing pepsin, an exceptionally effective microscale immobilized enzyme reactor (μIMER) was created. Implemented in an online liquid chromatography-MS/MS setup, the protease microcolumn enabled reproducible protein digestion and peptide mapping with 100% sequence coverage obtained for three different recombinant proteins. Additionally, when assessing the μIMER digestion efficiency for complex proteome samples, it delivered a 144-fold faster and significantly more efficient protein digestion compared to 24 h for bulk digestion. The 3D-printed μIMER withstands remarkably high pressures above 130 bar and retains its activity for several weeks. This versatile platform will enable researchers to produce tailored polymer-based enzyme reactors for various applications in analytical chemistry and beyond.

Flowing on-line preparation of deglycosylation, labeling and purification for N-glycan analysis

The current in-solution analysis of N-glycans suffers from several disadvantages including tedious de-glycosylation time and multi-step pre-treatment procedures. Here, an ultra-simple flowing on-line analysis of labeled N-glycans for high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for eliminating the deficiencies. This on-line analysis consisted of an immobilized enzyme reactor (IMER) of PNGase F for efficient release of N-glycans, labeling of released N-glycans and following purification of derivatives on microfluidic chip. Notably, efficient preparations for all type of N-glycans were completed within ∼30 min. To our best knowledge, this is the first time to integrated the whole preparation of N-glycan deglycosylation, labeling and purification only by a simple fluidic flow with our developed device. Good reproducibility and stability were achieved with the relative standard deviation (RSD) less than 10%. Furthermore, the glycome studies with human serum revealed a good adaptability for biological samples. Our work provides an efficient N-glycomic strategy that can be applied to further multilayered clinical analysis.

Síntesis benigna y sencilla de dímeros de tipo lignano usando peroxidasa cruda de rábano rojo (Raphanus sativus var sativus)

Nowadays, immobilized enzymes are utilized in several food industry applications. Some researches use apparent kinetic parameters in immobilized enzyme reactor systems, which are limited to such case studies. To enhance productivity in a packed bed reactor (PBR), a clear description of all the mechanisms (kinetic, intra-particle diffusive mass transport, fluid-particle convective mass transport, and axial dispersion) affecting the process should be established. The objective of this study was to model the isomerization of glucose in a PBR with calcium alginate beads (CAB), using an approach where the kinetic and diffusional mechanisms are described independently. The convective mass transfer (kL) and axial dispersion (Dz) coefficients were calculated from correlations. Validation was performed comparing predictions against experimental data (R2 = 0.907) of glucose conversion at the reactor’s outlet once steady state was reached. Under the study conditions, in contrast to the effect exerted by diffusive mass transport on fructose specific productivity, the effect of axial dispersion and convective mass transport is negligible. Analyzing different operation parameters via simulation, the particle size had the highest impact on the glucose bioconversion. By reducing the CAB size, the surface area is increased and thus the conversion. It is recommended to test new immobilizing agents or decreasing the CAB size, monitoring that immobilizing support preserves its stability and functionality.

The Power of Trypsin Immobilized Enzyme Reactors (IMERs) Deployed in Online MDLC–MS Applications

Immobilized enzyme reactors (IMERs) are a powerful and essential part of multidimensional liquid chromatography–tandem mass spectrometry (MDLC–MS/MS) approaches that enable online identification, characterization, and quantification of post-translational modifications of therapeutic antibodies. This review gives an overview of commercially available and selected trypsin IMERs in regard to their application in LC-based and automated sample preparation. Additionally, we address the challenges of IMER application in online systems and the advantages of self-made IMERs.