Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Towards Greener and More Cost-efficient Biosynthesis of Pharmaceuticals and Fragrance Molecules.

Enzymes are natural catalysts which are gaining momentum in chemical synthesis due to their exquisiteselectivity and their biodegradability. However, the cost-efficiency and the sustainability of the overall biocatalytic process must be enhanced to unlock completely the potential of enzymes for industrial applications. To reach this goal, enzyme immobilization and the integration into continuous flow reactors have been the cornerstone of our research. We showed key examples of the advantages of those tools for the biosynthesis of antivirals, anticancer drugs, and valuable fragrance molecules. By combining new strategies to immobilize biocatalysts, innovative bioengineering approaches, and process development, the performance of the reactions could be boosted up to 100-fold.

Integrated Process for Schizochytrium Oil Extraction, Enzymatic Modification of Lipids and Concentration of DHA Fatty Acid Esters Using Alternative Methodologies.

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

Green manufacturing of a hypoxanthine enzyme sensor for fish freshness based on modified nitrocellulose surface with chito-oligosaccharide.

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.

Reagentless Glucose Biosensor Based on Combination of Platinum Nanostructures and Polypyrrole Layer.

Two types of low-cost reagentless electrochemical glucose biosensors based on graphite rod (GR) electrodes were developed. The electrodes modified with electrochemically synthesized platinum nanostructures (PtNS), 1,10-phenanthroline-5,6-dione (PD), glucose oxidase (GOx) without and with a polypyrrole (Ppy) layer-(i) GR/PtNS/PD/GOx and (ii) GR/PtNS/PD/GOx/Ppy, respectively, were prepared and tested. Glucose biosensors based on GR/PtNS/PD/GOx and GR/PtNS/PD/GOx/Ppy electrodes were characterized by the sensitivity of 10.1 and 5.31 µA/(mM cm2), linear range (LR) up to 16.5 and 39.0 mM, limit of detection (LOD) of 0.198 and 0.561 mM, good reproducibility, and storage stability. The developed glucose biosensors based on GR/PtNS/PD/GOx/Ppy electrodes showed exceptional resistance to interfering compounds and proved to be highly efficient for the determination of glucose levels in blood serum.

Enzymes in "Green" Synthetic Chemistry: Laccase and Lipase.

Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes and alternatives to many common catalysts. Their essential advantages are high catalytic efficiency, substrate specificity, minimal formation of byproducts, and low energy demand. All of these benefits make enzymes highly desirable targets of academic research and industrial development. This review has the modest aim of briefly overviewing the classification, mechanism of action, basic kinetics and reaction condition effects that are common across all six enzyme classes. Special attention is devoted to immobilization strategies as the main tools to improve the resistance to environmental stress factors (temperature, pH and solvents) and prolong the catalytic lifecycle of these biocatalysts. The advantages and drawbacks of methods such as macromolecular crosslinking, solid scaffold carriers, entrapment, and surface modification (covalent and physical) are discussed and illustrated using numerous examples. Among the hundreds and possibly thousands of known and recently discovered enzymes, hydrolases and oxidoreductases are distinguished by their relative availability, stability, and wide use in synthetic applications, which include pharmaceutics, food and beverage treatments, environmental clean-up, and polymerizations. Two representatives of those groups-laccase (an oxidoreductase) and lipase (a hydrolase)-are discussed at length, including their structure, catalytic mechanism, and diverse usage. Objective representation of the current status and emerging trends are provided in the main conclusions.

Optimizing the enzymatic production of biolubricants by the Taguchi method: Esterification of the free fatty acids from castor oil with 2-ethyl-1-hexanol catalyzed by Eversa Transform 2.0.

The solvent-free esterification of the free fatty acids (FFAs) obtained by the hydrolysis of castor oil (a non-edible vegetable oil) with 2-ethyl-1-hexanol (a branched fatty alcohol) was catalyzed by different free lipases. Eversa Transform 2.0 (ETL) features surpassed most commercial lipases. Some process parameters were optimized by the Taguchi method (L16'). As a result, a conversion over 95% of the FFAs of castor oil into esters with lubricants properties was achieved under optimized reaction conditions (15 wt% of biocatalyst content, 1:4 molar ratio (FFAs/alcohol), 30 °C, 180 rpm, 96 h). The substrates molar ratio had the highest influence on the dependent variable (conversion at 24 h). FFAs/2-ethyl-1-hexanol esters were characterized regarding the physicochemical and tribological properties. Interestingly, the modification of the FFAs with 2-ethyl-1-hexanol by ETL increased the oxidative stability of the FFAs feedstock from 0.18 h to 16.83 h. The biolubricants presented a lower friction coefficient than the reference commercial mineral lubricant (0.052 ± 0.07 against 0.078 ± 0.04). Under these conditions, ETL catalyzed the oligomerization of ricinoleic acid (a hydroxyl fatty acid) into estolides, reaching a conversion of 25.15% of the initial FFAs (for the first time).

Combined cross-linking of Rhizomucor miehei lipase and Candida antarctica lipase B for the effective enrichment of omega-3 fatty acids in fish oil.

The incorporation of a non-specific lipase and a sn-1,3 specific one in a single immobilized system can be a promising approach for the exploitation of both lipases. A one-step immobilization platform mediated by an isocyanide-based multi-component reaction was applied to create co-cross-linked enzymes (co-CLEs) of lipases from Rhizomucor miehei (sn-1,3 specific) and Candida antarctica (non-specific). Glutaraldehyde was found to be effective cross-linker by producing specific activity of 16.9 U/mg and immobilization yield of 99 %. High activity recovery of up to 404 % was obtained for immobilized derivatives. Leaking experiment showed covalent nature of the cross-linking processes. BSA had considerable effect on the immobilization process, providing 87-100 % immobilization yields and up to 10 times improvement in the specific activity of the immobilized derivatives. Scanning electron microscopy images showed flower-like and rod-like structures for the CLEs prepared by glutaraldehyde and undecanedicarboxylic acid, respectively. The prepared co-CLEs were examined in non-selective enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil, showing capability of releasing up to 100 % of both omega-3 fatty acids within 8 h of the reaction. The reusability of co-CLEs in five successive cycles presented retaining 63-72 % of their initial activities after the fifth reuse cycle in the hydrolysis reaction.

Regiospecificity of Immobilized Candida antarctica Lipase B (CAL-B) towards 2,3-Diacyl-1-O-alkyl Glyceryl Ether in Ethanol.

Highly pure 2,3-dioleoyl-1-O-alkyl glyceryl ether (DOGE), whose 1-position is a lipase-tolerant ether bond, was chemically synthesized and its detailed regioselectivity and acyl transfer were confirmed. During ethanolysis using immobilized Candida antarctica lipase B (CAL-B) with DOGE as the substrate, monooleoyl-1-O-alkyl glyceryl ethers (MOGEs) and a few 1-alkyl glyceryl ethers were formed upon consumption of the substrate. The structure of MOGE was confirmed using nuclear magnetic resonance spectroscopy and only the isomer of 2-MOGE was formed, indicating that CAL-B has complete α- regiospecificity. During ethanolysis, 3-MOGE was formed via acyl migration. These results indicate that the formation of 1-alkyl glyceryl ethers is not due to the imperfect regiospecificity of CAL-B, but rather due to ethanolysis of the formed 3-MOGE. The ethanolysis rate at the 3-α-position of DOGE was faster and the rate of acyl transfer was slightly slower for chain lengths greater than 14. These results show for the first time that both deacylation at the 3-position and acyl migration from the 2- to 3-position are affected by the structure of 1-position.

Amorphous-crystalline phase transition and intrinsic magnetic property of nickel organic framework for easy immobilization and recycling of ß-Galactosidase.

This work describes the synthesis of fibrous nickel-based metal organic framework (Ni-ZIF) via simple solvothermal method. The material formed was calcinated at 400, 600, 800 °C to improve its surface area, porosity and enzyme binding capacity. Changes in X-ray diffraction pattern after calcination revealed the Ni-ZIF transitioned from amorphous to crystalline structure. The surface area, pore volume and pore size for Ni-ZIF@600 were found to be 312.15 m2/g, 0.88 cm3/g and 10.28 nm, with an enzyme loading capacity of 593.85 mg/g after 30 h The free (ß-Gal-LEH) and immobilized ß-Galactosidase were stable at pH 7.5, temperature 50 °C, and yielded 70.70 and 63.95 mM glucose after milk lactose hydrolysis, respectively. The Ni-ZIF@600@ß-Gal-LEH exhibited high enzyme retention capacity, maintaining 59.44 % of its original activity after 6-cycles. The enhanced magnetic property, enzyme binding capacity and easy recoverability of the calcinated Ni-ZIF could guarantee its industrial significance as immobilization module for enzyme-mediated catalysis.

Comparative oil extraction from mutt (Myliobatis goodei) liver by enzymatic hydrolysis: free versus immobilized biocatalyst.

BACKGROUND: The development and fine-tuning of biotechnological processes for fish oil extraction constitute a very important focus to contribute to the development of a food industry based on fish consumption. This work lies in a comparative analysis of the oil extraction yield of Myliobatis goodei livers using free and immobilized enzymes. RESULTS: An immobilized biocatalyst was designed from the cell-free extract of a Bacillus sp. Mcn4. A complete factorial design was used to study the components of the bacterial culture medium and select the condition with the highest titers of extracellular enzymatic activities. Wheat bran had a significant effect on the culture medium composition for enzymatic production. The immobilized biocatalyst was designed by covalent binding of the proteins present in the cocktail retaining a percentage of different types of enzymatic activities (Mult.Enz@MgFe2 O4 ). Among the biocatalyst used, Alcalase® 2.4 L and Purazyme® AS 60 L (free commercial proteases) showed extraction yields of 87.39% and 84.25%, respectively, while Mult.Enz@MgFe2 O4 achieved a better one of 89.97%. The oils obtained did not show significant differences in their physical-chemical properties while regarding the fatty acid content, the oil extracted with Purazyme® AS 60 L showed a comparatively lower proportion of polyunsaturated fatty acids. CONCLUSIONS: Our results suggest that the use of by-products of M. goodei is a valid alternative and encourages the use of immobilized multienzyme biocatalysts for the treatment of complex substrates in the fishing industry. © 2023 Society of Chemical Industry.

Effect of structural stability of lipase in acetonitrile on its catalytic activity in EGCG esterification reaction: FTIR and MD simulation.

In this study, (-)-Epigallocatechin-3-O-gallate (EGCG) esterification reaction was catalyzed by Novozym 435, Lipozyme RM, Lipozyme TLIM, and lipase Amano 30SD in acetonitrile. Fourier transform infrared spectroscopy (FTIR) and molecular dynamic (MD) simulations were used to analyze the structural stability of different lipases in acetonitrile and their effect on EGCG esterification reaction. The results showed that conversion rate of EGCG catalyzed by Lipozyme RM was the highest, followed by Lipozyme TLIM. FTIR indicated that the secondary structure of Lipozyme RM was the most stable. MD simulations suggested that whole structural stability of Lipozyme RM in acetonitrile was superior to Novozym 435 and lipase Amano 30SD and similar to Lipozyme TLIM due to their similar conformation, while the active site of Lipozyme RM is more flexible than that of Lipozyme TLIM, which indicated that lipase with stable whole structure and flexible active site may be more conducive to the esterification of EGCG in acetonitrile. This study provided a direction for rapidly screening lipase to synthetize EGCG or other polyphenols esterified derivatives.

One-step, reagent-free construction of nano-enzyme as visual and reusable biosensor for oxidase substrates.

The substrates of oxidase are biologically essential substances that are closely associated with human physiological health. However, current biosensing methods suffer from tough recyclability and undesired denaturation of enzyme due to impurity interference. Herein, we have developed a visual and reusable biosensor for detecting substrate using glucose oxidase (GOx) as a model oxidase. GOx was immobilized onto gold nanoparticles (AuNPs) at -20 °C in one step without additional reagents. The resulting nano-enzyme generated coloimetric signals by coupling with horseradish peroxidase (HRP) using TMB as the substrate. Our results demonstrated that the immobilized GOx exhibited satisfactory sensitivity (0.68 µM) for glucose detection and higher inherent stability than free GOx under harsh conditions, enabling reliable detection of glucose in complex fluids (colored beverages and saliva). Furthermore, the nano-enzyme retained 80 % activity even after four cycles of catalytic oxidation. This strategy constructs a universal biosensor for substrates with nano-enzyme which rely only on intrinsic cysteine within the oxidase while avoiding functional handle modification.

Direct purification and immobilization of his-tagged enzymes using unmodified nickel ferrite NiFe2O4 magnetic nanoparticles.

Purification of valuable engineered proteins and enzymes can be laborious, costly, and generating large amount of chemical waste. Whilst enzyme immobilization can enhance recycling and reuse of enzymes, conventional methods for immobilizing engineered enzymes from purified samples are also inefficient with multiple-step protocols, regarding both the carrier preparation and enzyme binding. Nickel ferrite magnetic nanoparticles (NiFe2O4 MNPs) offer distinct advantages in both purification and immobilization of enzymes. In this work, we demonstrate the preparation of NiFe2O4 MNPs via a one-step solvothermal synthesis and their use in direct enzyme binding from cell lysates. These NiFe2O4 MNPs have showed an average diameter of 8.9 ± 1.7 nm from TEM analysis and a magnetization at saturation (Ms) value of 53.0 emu g-1 from SQUID measurement. The nickel binding sites of the MNP surface allow direct binding of three his-tagged enzymes, D-phenylglycine aminotransferase (D-PhgAT), Halomonas elongata ω-transaminase (HeωT), and glucose dehydrogenase from Bacillus subtilis (BsGDH). It was found that the enzymatic activities of all immobilized samples directly prepared from cell lysates were comparable to those prepared from the conventional immobilization method using purified enzymes. Remarkably, D-PhgAT supported on NiFe2O4 MNPs also showed similar activity to the purified free enzyme. By comparing on both carrier preparation and enzyme immobilization protocols, use of NiFe2O4 MNPs for direct enzyme immobilization from cell lysate can significantly reduce the number of steps, time, and use of chemicals. Therefore, NiFe2O4 MNPs can offer considerable advantages for use in both enzyme immobilization and protein purification in pharmaceutical and other chemical industries.

Tyrosinase enzyme purification and immobilization from Pseudomonas sp. EG22 using cellulose coated magnetic nanoparticles: characterization and application in melanin production.

Melanin is a brown-black pigment with significant roles in various biological processes. The tyrosinase enzyme catalyzes the conversion of tyrosine to melanin and has promising uses in the pharmaceutical and biotechnology sectors. This research aims to purify and immobilize the tyrosinase enzyme from Pseudomonas sp. EG22 using cellulose-coated magnetic nanoparticles. Various techniques were utilized to examine the synthesized nanoparticles, which exhibited a spherical shape with an average diameter of 12 nm and a negative surface potential of - 55.7 mV with a polydispersity index (PDI) of 0.260. Comparing the immobilized magnetic tyrosinase enzyme with the free enzyme, the study's findings showed that the immobilized tyrosinase enzyme had optimal activity at a pH of 6 and a temperature of 35 °C, and its activity increased as the concentration of tyrosine increased. The study investigated the antibacterial and anticancer bioactivity of the enzyme's melanin product and found that it exhibited potential antibacterial activity against a multi-drug resistant strain including S. aureus and E. coli. The produced melanin also demonstrated the potential to decrease cell survival and induce apoptosis in initiation cells.

Nature-Inspired Biomolecular Corona Based on Poly(caffeic acid) as a Low Potential and Time-Stable Glucose Biosensor.

Herein, we present a novel biosensor based on nature-inspired poly(caffeic acid) (PCA) grafted to magnetite (Fe3O4) nanoparticles with glucose oxidase (GOx) from Aspergillus niger via adsorption technique. The biomolecular corona was applied to the fabrication of a biosensor system with a screen-printed electrode (SPE). The obtained results indicated the operation of the system at a low potential (0.1 V). Then, amperometric measurements were performed to optimize conditions like various pH and temperatures. The SPE/Fe3O4@PCA-GOx biosensor presented a linear range from 0.05 mM to 25.0 mM, with a sensitivity of 1198.0 µA mM-1 cm-2 and a limit of detection of 5.23 µM, which was compared to other biosensors presented in the literature. The proposed system was selective towards various interferents (maltose, saccharose, fructose, L-cysteine, uric acid, dopamine and ascorbic acid) and shows high recovery in relation to tests on real samples, up to 10 months of work stability. Moreover, the Fe3O4@PCA-GOx biomolecular corona has been characterized using various techniques such as Fourier transform infrared spectroscopy (FTIR), high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Bradford assay.

Enzyme immobilized on magnetic fluorescent bifunctional nanoparticles for α-glucosidase inhibitors virtual screening from Agrimonia pilosa Ledeb extracts accompanied with molecular modeling.

Agrimonia pilosa Ledeb (APL) is a significant source of inhibitors for α-glucosidase, which is an essential target enzyme for the treatment of type 2 diabetes, cancer and acquired immune deficiency syndrome. Ligand fishing is a suitable approach for the highly selective screening of bioactive substances in complex mixtures. Yet it is unable to conduct biomedical imaging screening, which is crucial for real-time identification. In this case, a bioanalytical platform combining magnetic fluorescent ligand fishing and in-situ imaging technique was established for the screening and identification of α-glucosidase inhibitors (AGIs) from APL crude extract, utilizing α-glucosidase coated CuInS2/ZnS-Fe3O4@SiO2 (AG-CIZSFS) nanocomposites as extracting material and fluorescent tracer. The AG-CIZSFS nanocomposites prepared through solvothermal and crosslinking methods displayed fast magnetic separation, excellent fluorescence performance and high enzyme activity. The tolerance of immobilized enzyme to temperature and pH was stronger than that of free enzyme. Prior to proof-of-concept with APL crude extract, a number essential parameters (glutaraldehyde concentration, immobilized time, enzyme amount, reaction solution pH, incubation temperature, incubation time, percentage of methanol in eluen, elution times and eluent volume) were optimized using an artificial test mixture. The fished ligands were identified by UPLC-MS/MS and their biological activities were preliminarily evaluated by real-time cellular morphological imaging of human colon carcinoma (HCT-116) cells based on confocal laser scanning microscope (CLSM). Their α-glucosidase inhibitory activities were further verified and studied by classical pNPG method and molecular docking. The isolated compounds exhibited significant α-glucosidase inhibitory activities with a IC50 value of 11.57 µg·mL-1. Six potential AGIs including tribuloside, ivorengenin A, tormentic acid, 1ß, 2ß, 3ß, 19α-Tetra hydroxyurs-12-en-28-oic acid, corosolic acid and pomolic acid were ultimately screened out and identified from APL crude extracts. The proposed approach, which combined highly specific screening with in-situ visual imaging, provided a powerful platform for discovering bioactive components from multi-component and multi-target traditional Chinese medicine (TCM).

Preparation of a Mesoporous Biosensor for Human Lactate Dehydrogenase for Potential Anticancer Inhibitor Screening.

Cancer is the second leading cause of death worldwide, with a dramatic impact due to the acquired resistance of cancers to used chemotherapeutic drugs and treatments. The enzyme lactate dehydrogenase (LDH-A) is responsible for cancer cell proliferation. Recently the development of selective LDH-A inhibitors as drugs for cancer treatment has been reported to be an efficient strategy aiming to decrease cancer cell proliferation and increase the sensitivity to traditional chemotherapeutics. This study aims to obtain a stable and active biocatalyst that can be utilized for such drug screening purposes. It is conceived by adopting human LDH-A enzyme (hLDH-A) and investigating different immobilization techniques on porous supports to achieve a stable and reproducible biosensor for anticancer drugs. The hLDH-A enzyme is covalently immobilized on mesoporous silica (MCM-41) functionalized with amino and aldehyde groups following two different methods. The mesoporous support is characterized by complementary techniques to evaluate the surface chemistry and the porous structure. Fluorescence microscopy analysis confirms the presence of the enzyme on the support surface. The tested immobilizations achieve yields of ≥80%, and the best retained activity of the enzyme is as high as 24.2%. The optimal pH and temperature of the best immobilized hLDH-A are pH 5 and 45 °C for the reduction of pyruvate into lactate, while those for the free enzyme are pH 8 and 45 °C. The stability test carried out at 45 °C on the immobilized enzyme shows a residual activity close to 40% for an extended time. The inhibition caused by NHI-2 is similar for free and immobilized hLDH-A, 48% and 47%, respectively. These findings are significant for those interested in immobilizing enzymes through covalent attachment on inorganic porous supports and pave the way to develop stable and active biocatalyst-based sensors for drug screenings that are useful to propose drug-based cancer treatments.

Immobilized enzyme microreactors for analysis of tryptic peptides in ß-casein and ß-lactoglobulin.

In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: ß-casein (ßCN) and ß-lactoglobulin (ßLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (µ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The µ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the µ-IMER method demonstrated superior sequence coverage for oxidized molecules in ßCN (33 ± 1.5%) and ßLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for ßCN; 49 ± 2% for ßLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for ßCN; 60 ± 4% for ßLG) and µ-IMER (41 ± 3% for ßCN; 80 ± 5% for ßLG). Additionally, phosphorylations were identified. For ßCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With µ-IMER and µ-IMER-ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For ßLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and µ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and µ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of µ-IMER systems for enhanced sequence coverage and post-translational modification detection.

Orientation-Controllable Enzyme Cascade on Electrode for Bioelectrocatalytic Chain Reaction.

The accomplishment of concurrent interenzyme chain reaction and direct electric communication in a multienzyme-electrode is challenging since the required condition of multienzymatic binding conformation is quite complex. In this study, an enzyme cascade-induced bioelectrocatalytic system has been constructed using solid binding peptide (SBP) as a molecular binder that coimmobilizes the invertase (INV) and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) cascade system on a single electrode surface. The SBP-fused enzyme cascade was strategically designed to induce diverse relative orientations of coupling enzymes while enabling efficient direct electron transfer (DET) at the FAD cofactor of GDHγα and the electrode interface. The interenzyme relative orientation was found to determine the intermediate delivery route and affect overall chain reaction efficiency. Moreover, interfacial DET between the fusion GDHγα and the electrode was altered by the binding conformation of the coimmobilized enzyme and fusion INVs. Collectively, this work emphasizes the importance of interenzyme orientation when incorporating enzymatic cascade in an electrocatalytic system and demonstrates the efficacy of SBP fusion technology as a generic tool for developing cascade-induced direct bioelectrocatalytic systems. The proposed approach is applicable to enzyme cascade-based bioelectronics such as biofuel cells, biosensors, and bioeletrosynthetic systems utilizing or producing complex biomolecules.