Abstract
Enzymes are important catalysts in biochemical reactions with superior regio, stereo, and substrate selectivity. However, enzymatic reaction systems have drawbacks including product ...inhibition, difficulty recycling, and poor stability. Importantly, the rate of an enzyme catalyzed reaction diminishes rapidly due to product inhibition and substrate depletion, making it difficult for many enzymes to catalyze a reaction to completion. The outcome is a mixture of unreacted substrates being present in the final reaction, necessitating additional separation steps that increase costs. This study presents a microfluidic reactor for accelerating enzyme catalyzed reactions using a countercurrent design that continuously removes products and adds fresh substrate into the reaction, allowing enzymes to operate under better reaction conditions. It demonstrates that countercurrent flow accelerates enzymatic reactions in our system up to 36 % for horseradish peroxidase and 21 % for β‐glucosidase compared to cocurrent flow, and the resulting reaction solution contains highly pure product with minimal substrate contamination.
Protein antigenicity can be reduced by enzymatic hydrolysis, which can be performed either by free or immobilized enzyme. The immobilized enzyme is removed from the reaction medium and reused, while ...the free enzyme must be inactivated to stop the reaction, generally by heating. Here we have shown that hydrolysates produced with free or immobilized Alcalase on glyoxyl-agarose bead presented different physicochemical properties (hydrophilicity profile, molecular mass distribution, surface hydrophobicity) and different levels of residual milk allergens (α-lactalbumin and β-lactoglobulin). Although, under the studied conditions, the hydrolysis with immobilized enzyme did not reduce the residual allergen levels as efficiently as the free enzyme, the results suggest potential applications of immobilized Alcalase for production of hypoallergenic hydrolysates.
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•At same conditions free or immobilized Alcalase produced different hydrolysates.•Hydrolysates with Immobilized Alcalase (ImA) showed lower degree of hydrolysis (DH).•Hydrolysates with Free Alcalase (FA) showed lower surface hydrophobicity (S0).•Only at pH8.0/ 65°C ImA and FA produced similar hydrolysates.•Hydrolysates with higher DH and lower S0 presented lower residual α-La and β-Lg levels.
Orientationally immobilized enzyme microreactors (OIMERs), embedded in microfluidic paper-based analytical devices (μPADs) were developed for improved detection of pesticide residues in food. ...Acetylcholinesterase (AChE) was orientationally immobilized on the reusable Part I of the μPADs, using the specific affinity binding of concanavalin A (Con A) to a glycosyl group on AChE. Using the disposable Part II, facile colorimetric quantification was performed with a smartphone and software, or qualitative detection by a naked-eye visual test. The AChE immobilized in OIMERs not only had improved activity and stability, but also high sensitivity, with a limit of detection as low as (0.007 ± 0.003) μg/mL. The method was used to detect pesticides residues in real vegetable samples; the recovery (88.6-102.7%) showed high reliability for pesticide residues detection in foods. A molecular docking study and an enzyme kinetic analysis were conducted to characterize the mechanism of action of the OIMERs.
The use of enzymes immobilized on magnetic nanoparticles to detect contaminants in aqueous samples has gained interest, since it allows the magnetic control, concentration and reuse of the enzymes. ...In this work, the detection of trace amounts of organophosphate pesticides (chlorpyrifos) and antibiotics (penicillin G) in water was attained by developing a nanoassembly formed by either inorganic or biomimetic magnetic nanoparticles used as substrates to immobilize acetylcholinesterase (AChE) and β-lactamase (BL). Other than the substrate, the optimization of the nanoassembly was done by testing enzyme immobilization both through electrostatic interaction (also reinforced with glutaraldehyde) and covalent bonds (by carbodiimide chemistry). Temperature (25 °C), ionic strength (150 mM NaCl) and pH (7) were set to ensure enzymatic stability and to allow both the nanoparticles and the enzymes to present ionic charges that would allow electrostatic interaction. Under these conditions, the enzyme load on the nanoparticles was ⁓0.1 mg enzyme per mg nanoparticles, and the preserved activity after immobilization was 50–60% of the specific activity of the free enzyme, being covalent bonding the one which yielded better results. Covalent nanoassemblies could detect trace concentrations of pollutants down to 1.43 nM chlorpyrifos and 0.28 nM penicillin G. They even permitted the quantification of 14.3 μM chlorpyrifos and 2.8 μM penicillin G. Also, immobilization conferred higher stability to AChE (⁓94% activity after 20 days storage at 4 °C) and allowed to reuse the BL up to 12 cycles.
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•Acetylcholinesterase (AChE) and β-lactamase (BL) can be immobilized on biomimetic magnetic nanoparticles (BMNPs).•Such an immobilization increases enzyme stability and enables their magnetic concentration and reutilization.•AChE-BMNPs and BL-BMNP nanoassemblies can detect 1.43 nM chlorpyrifos and 0.28 nM penicillin G, respectively.
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•Novozym® 435 modification with 2,4,6-trinitrobenzensulfonic acid favors oil reaction.•Support swelling by solvent accelerates alcoholysis catalyzed by Novozym® 435 adducts.•TNBS ...derivative is also more stable than Novozym® 435 in methanolysis with solvent.•Breaking of support particles is reduced by the 7 different modifications studied.•Product deposits do not impose any significant mass transfer restrictions.
Alcoholysis of oils mediated by immobilized lipases are limited by mass transfer effects on substrates. In this work, Novozym® 435 lipase was subjected to seven different chemical derivatizations. The effects of changes in the enzyme surface and changes of the support particles size, on substrates mass transfer restrictions were studied on the alcoholysis of Camelina oil in the presence or not of t-butanol as co-solvent.
Significant changes of the support particle size were detected after their chemical modification. The particle size of Lewatit VP OC 1600 support of Novozym® 435 diminished in solvent-free systems. Alcoholysis rates in t-butanol media were enhanced caused by two favorable effects of this solvent: substrates dissolution and support swelling. This latter effect was not sufficient to promote protein desorption during processing. The hydrophobic environment created by 2,4,6-trinitrobenzensulfonic acid (TNBS) derivatization favoured the oil conversion. The TNBS derivative was also more stable than Novozym® 435 in methanolysis with solvent.
Scanning electron microscopy revealed that after 14 reaction cycles of 24h, a large proportion of biocatalyst particles were broken; however, matrix rupture did not cause biocatalysts inactivation. All modifications studied seemed to protect the support particles from breaking. Accumulated product particles on all biocatalysts surfaces did not impose significant mass transfer restrictions to substrates, but prevented protein desorption in urea solution.
Multi-enzyme biocatalysis is an important technology to produce many valuable chemicals in the industry. Different strategies for the construction of multi-enzyme systems have been reported. In ...particular, immobilization of multi-enzymes on the support materials has been proved to be one of the most efficient approaches, which can increase the enzymatic activity via substrate channeling and improve the stability and reusability of enzymes. A general overview of the characteristics of support materials and their corresponding attachment techniques used for multi-enzyme immobilization will be provided here. This review will focus on the materials-based techniques for multi-enzyme immobilization, which aims to present the recent advances and future prospects in the area of multi-enzyme biocatalysis based on support immobilization.
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•Coal fly ash-derived Ni-zeolite was developed as an enzyme support.•The enzyme immobilization yield was 77.5%.•Immobilized CphC-I catalyzes the initial step of 4-CP ...biodegradation.•The activity of immobilized CphC-I was 7.98 U/g Ni-zeolite.•vmax = 0.20 mM/min, KM = 0.44 mM, and kcat/KM = 0.02 mM−1.min−1.
A new type of biocatalyst was developed to facilitate the biochemical decomposition of 4-chlorophenol (4-CP) in this study. Oxydoreductases that catalyze the initial steps of 4-CP biodegradation were immobilized on a synthetic inorganic enzyme support. Type-X zeolite, a high-surface area support, was synthesized from coal fly ash, on which nickel ions were plated by impregnation (Ni-zeolite), followed by the effective immobilization (77.5% immobilization yield) of recombinant monooxygenase (CphC-I), dioxygenase (CphA-I), and flavin reductase (Fre) isolated from Pseudarthrobacter chlorophenolicus A6 and Escherichia coli K-12, respectively. The retained catalytic activity of the enzymes immobilized on Ni-zeolite was as high as 64% of the value for the corresponding free enzymes. The Michaelis–Menten kinetic parameters vmax and KM of the immobilized enzymes were determined to be 0.20 mM·min−1 and 0.44 mM, respectively. These results are expected to provide useful information with respect to the development of novel enzymatic treatments for phenolic hydrocarbon contaminants.
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•Synthesis of covalently bound glutathione (GSH) on magnetic nanoparticles (SPIONs).•The linkage of GSH on amino-grafted SPIONs was activated with maleic anhydride.•GST-tagged enzyme ...(sfULP1) was biospecifically immobilized on GSH-bound nanoparticles.•Immobilized sfULP1 retained 87 % of activity for the cleavage of SUMO-fused peptide.
Magnetic nanoparticles bound with glutathione (GSH) are useful for diagnostics, enzyme immobilization, and affinity precipitation by using the strong and specific interaction of GSH with glutathione S-transferase (GST)-fused proteins. Our studies revealed that GSH-bound magnetic nanoparticles could be obtained using the covalent bond linkage of GSH and nanoparticles to promote the stability of bound GSH. To yield this conjugate, superparamagnetic iron oxide nanoparticles (SPIONs) were prepared and modified using tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES), which introduced amino groups that were then activated with maleic anhydride (MA) for covalent binding of GSH. After MA was used to activate the amino-grafted SPION for 24 h, the yield of GSH conjugation increased over 4 days from 37 % to 74 % of the original amine density on the surface as the incubation of GSH with MA-activated SPION. These GSH-bound magnetic nanoparticles, designated as SPION@silica-GSH with approximately 103 nmol GSH/mg particles, were ready for coupling with GST-fused protein through the GSH–GST affinity interaction. A GST-tagged small fragment of ubiquitin-like-specific protease 1 (sfULP1) was used as the model protein for immobilization on SPION@silica-GSH. ULP1 is a small ubiquitin-like modifier (SUMO) protease. Results indicated that this immobilized GST-sfULP1 could retain 87 % ± 5 % enzyme activity of free protease before immobilization and could catalyze the cleavage of the SUMO-fused peptide (SUMO-GLP-1) to obtain glucagon-like peptide-1, a peptide hormone for type 2 diabetes therapy.
Abstract
Nanoparticles (NPs) have attracted interest recently as a promising support for the immobilization of enzymes.Xylanase (Xyl) has been utilized in the food industry for several purposes. To ...improve its stability and reusability,starch NPs (SNPs)are synthesized and assessed for Xyl immobilization for the first time. . The characterization results reveal that SNPs are successfully synthesized and Xyl@SNPs are effective as the immobilization carrier. The Xyl is immobilized on SNPs under optimized conditions leading to 91% immobilization efficiency. A shift in optimum pH (from 6.0 to 8.0) and temperature (from 70 to 60 °C) of Xyl is observed after immobilization. The Xyl@SNPs possess improve pH stability and thermal stability as well as operational stability. The Xyl@SNPs are easily reutilized and the residual activity is still higher than 62% after seven times. More importantly, the Xyl@SNPs have a lower
K
m
value and enhance affinity for the substrate compared to the free Xyl. Additionally, the clarity of orange juice is increased by 76.0% by using Xyl@SNPs after 2 h incubation at 60 °C, pH 8.0. These positive results show promising support for the Xyl immobilization of SNPs, confirming that this immobilized enzyme‐based strategy is an effective technique for rapid clarification of targeted juices.
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