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1.
Food Chem ; 366: 130616, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34311240

RESUMO

The lipase from Bacillus licheniformis NCU CS-5 was immobilized onto ß-cyclodextrin (CD) grafted and aminopropyl-functionalized chitosan-coated Fe3O4 magnetic nanocomposites (Fe3O4-CTS-APTES-GA-ß-CD). Fourier transform infrared spectroscopy, thermogravimetry analysis, X-ray diffraction, scanning electron microscopy and transmission electron microscopy showed that not only the functionalized magnetic nanoparticles were synthesized but also the immobilized lipase was successfully produced. The immobilized lipase exhibited higher optimal pH value (10.5) and temperature (60℃) than the free lipase. The pH and thermal stabilities of the immobilized lipase were improved significantly compared to the free lipase. The immobilized lipase remained more than 80% of the relative activity at temperature of 60 ℃ and pH 12.0. The immobilized lipase also remained over 80% of its relative activity after 28 days of storage and 15 cycles of application. The application of the immobilized lipase in esterification of isoamyl acetate and pentyl valerate showed that maximum esterification efficiency was achieved in n-hexane having 68.0% and 89.2% respectively. Therefore, these results indicated that the Fe3O4-CTS-APTES-GA-ß-CD nanoparticles are novel carriers for immobilizing enzyme, and the immobilized lipase can be used as an innovative green approach to the synthesis of fruity flavor esters in food industry.


Assuntos
Quitosana , Ciclodextrinas , Nanopartículas de Magnetita , Nanocompostos , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Esterificação , Ésteres , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Fenômenos Magnéticos
2.
Food Chem ; 366: 130699, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34348221

RESUMO

In this work, magnetic chitosan (MCH) beads were synthesized by phase-inversion method, and grafted with polydopamine (PDA) and then used for direct immobilization of Candida rugosa lipase by Schiff base reaction. The amount of immobilized enzyme and the retained activity were found to be 47.3 mg/g and 72.8%, respectively, at pH 7.0, and at 25 °C. The apparent Km (9.7 mmol/L), and Vmax (384 U/mg) values of the immobilized lipase were significantly changed compared to the free lipase. The MCH@PDA-lipase was better thermal and storage stability at different temperatures than those of the free lipase. In hexane medium, the esterification reaction results showed that the maximum conversions of isoamylalcohol and isopentyl alcohol to isoamyl acetate and isopentyl acetate using the MCH@PDA-lipase were found to be 98.4 ± 1.3% and 73.7 ± 0.7%, respectively. These results showed that the MCH@PDA-lipase can be used as an operative immobilized enzyme system for many biotechnological applications.


Assuntos
Quitosana , Lipase , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Ésteres , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Fenômenos Magnéticos , Saccharomycetales
3.
Food Chem ; 372: 131256, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34627092

RESUMO

In this study, a continuous-flow bioreactor packed with well-organized lipase microarrays was developed for the sustainable synthesis of functional lipid-phytosterol esters (PEs). Hollow mesoporous silicon spheres with a suitable pore size were prepared for lipase immobilization, and the hydrophobic modification endowed the lipase with excellent catalytic activity and stability. The results showed that the condensely packed lipase microarrays offered large specific surface areas and guaranteed the thorough interaction between the lipase and substrates in the continuous-flow bioreactor. Meanwhile, the substrate could pass through the reactor at 1 mL/min with a high conversion of 93.6% due to the hollow structure of the packing spheres. Moreover, the reactors were able to produce 1564 g PEs/g catalyst in a continuous 30-day processing period, which set the highest records for PEs synthesis. This sustainable and highly-converting flow system provided a feasible path for scale-up production of PEs in the food processing area.


Assuntos
Lipase , Fitosteróis , Reatores Biológicos , Enzimas Imobilizadas/metabolismo , Esterificação , Ésteres , Lipase/metabolismo , Fitosteróis/metabolismo
4.
J Colloid Interface Sci ; 606(Pt 2): 1229-1238, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34492461

RESUMO

Lipase is the most widely used enzyme in industry. Due to its unique "lid" structure, lipase can only show high activity at the oil-water interface, which means that water is needed in the catalytic esterification process. However, the traditional lipase catalytic system cannot effectively control "micro-water" in the esterification environment, resulting in the high content of free water, which hinders the esterification reaction and reduces the yield. In this paper, a promising strategy of esterification catalyzed by polyacrylamide hydrogel immobilized lipase is reported. The porous polyacrylamide hydrogel microspheres (PHM) prepared by inverse emulsion polymerization are used as carrier to adsorb lipase by hydrogen bonding interaction. These hydrogel microspheres provide a "micro-water environment" for lipase in the anhydrous reaction system, and further provide an oil-water interface for "interface activation" of lipase. The obtained lipase-porous polyacrylamide hydrogel microspheres (L-PHMs) exhibit higher temperature and pH stability compared with free lipase, and the optimum enzymatic activity reach 1350 U/g (pH 6, 40 °C). L-PHMs can still remain about 49% of their original activity after 20 reuses. Furthermore, L-PHMs have been successfully applied to catalyze the synthesis of conjugated linoleic acid ethyl ester. The results suggest that this immobilization method opens up a new way for the application of lipase in ester synthesis.


Assuntos
Hidrogéis , Lipase , Enzimas Imobilizadas/metabolismo , Esterificação , Hidrogênio , Lipase/metabolismo , Microesferas
5.
Enzyme Microb Technol ; 152: 109935, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34749020

RESUMO

Enzymes are particularly attractive as biocatalysts for the green synthesis of chemicals and pharmaceuticals. However, the traditional enzyme purification and separation process is complex and inefficient, which limits the wide application of enzyme catalysis. In this paper, an efficient strategy for enzyme purification and immobilization in one step is proposed. A novel poly (ionic liquid)-styrene microsphere is prepared by molecular design and synthesis for adsorbing and purifying high activity lipase from fermentation broth directly. By optimizing the surface morphologies and charge of the microspheres, the enzyme loading is significantly improved. In order to further stabilize the catalytic environment of lipase, the resulting lipase/poly (ionic liquid)-styrene microspheres are immobilized in physical crosslinking hydrogel to obtain a complex lipase catalytic system, which can be prepared into various shapes according to the requirements of catalytic environment. In the actual catalytic reaction process, this complex lipase catalytic system exhibits excellent catalytic activity (6314.69 ± 21.27 U mg-1) and good harsh environment tolerance compared with the lipase fermentation broth (1672.87 ± 36.68 U mg-1). Under the condition of cyclic catalysis, the complex lipase catalytic system shows the outstanding reusability (After 8 cycles the enzymatic activity is still higher than that of the lipase fermentation broth) and is easily separated from the products.


Assuntos
Líquidos Iônicos , Lipase , Enzimas Imobilizadas/metabolismo , Esterificação , Hidrogéis , Lipase/metabolismo , Microesferas , Estireno
6.
J Biotechnol ; 342: 13-27, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34634391

RESUMO

The present work evaluates the immobilization of LipC12 on different supports in tandem with bioimprinting technique, in order to improve its activity and stability in organic medium. Oleic acid was selected as the bioimprinting molecule. The immobilized LipC12 was applied in the synthesis of pentyl oleate by esterification reaction and in the production of fatty acids, mono, and diglycerides via hydrolysis of triacylglycerols, in n-heptane reaction media. For all immobilized lipase preparations, an increase in the conversion of oleic acid to pentyl oleate was observed when immobilization in tandem with bioimprinting treatment was carried out versus immobilization without bioimprinting. The highest conversions were achieved using LipC12 immobilized on hydrophobic supports. The reuse potential of the immobilized preparations was evaluated. The preparations were used in eight successive cycles of esterification reactions and the best results were obtained for LipC12 immobilized on Immobead 150 and chitosan. The activity for the hydrolysis of soybean oil was improved by bioimprinting treatment only for LipC12 immobilized on commercial polypropylene and Accurel MP-1000. LipC12 immobilized on hydrophilic supports or on Immobead150 could be used to hydrolyze tricaprylin to obtain diglycerides with a high proportion of 1,2-diglycerides in reaction times as short as 30 min.


Assuntos
Enzimas Imobilizadas , Lipase , Enzimas Imobilizadas/metabolismo , Esterificação , Lipase/metabolismo , Metagenômica , Ácido Oleico
7.
Colloids Surf B Biointerfaces ; 208: 112147, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34634655

RESUMO

Laccase from Aspergillus sp. (LC) was immobilized within Fe-BTC and ZIF-zni metal organic frameworks through a one-pot synthesis carried out under mild conditions (room temperature and aqueous solution). The Fe-BTC, ZIF-zni MOFs, and the LC@Fe-BTC, LC@ZIF-zni immobilized LC samples were characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The kinetic parameters (KM and Vmax) and the specific activity of the free and immobilized laccase were determined. Immobilized LCs resulted in a lower specific activity compared with that of the free LC (7.7 µmol min-1 mg-1). However, LC@ZIF-zni was almost 10 times more active than LC@Fe-BTC (1.32 µmol min-1 mg-1 vs 0.17 µmol min-1 mg-1) and only 5.8 times less active than free LC. The effect of enzyme loading showed that LC@Fe-BTC had an optimal loading of 45.2 mg g-1, at higher enzyme loadings the specific activity decreased. In contrast, the specific activity of LC@ZIF-zni increased linearly over the loading range investigated. The storage stability of LC@Fe-BTC was low with a significant decrease in activity after 5 days, while LC@ZIF retained up to 50% of its original activity after 30 days storage. The difference in activity and stability between LC@Fe-BTC and LC@ZIF-zni is likely due to release of Fe3+ and the low stability of Fe-BTC MOF. Together, these results indicate that ZIF-zni is a superior support for the immobilization of laccase.


Assuntos
Estruturas Metalorgânicas , Aspergillus/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Cinética , Lacase/metabolismo
8.
Molecules ; 26(19)2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34641510

RESUMO

Bile acids (BAs) are a family of steroids synthesized from cholesterol in the liver. Among bile acids, ursodeoxycholic acid (UDCA) is the drug of choice for treating primary biliary cirrhosis and dissolving cholesterol gallstones. The clinical effectiveness of UDCA includes its choleretic activity, the capability to inhibit hydrophobic bile acid absorption by the intestine under cholestatic conditions, reducing cholangiocyte injury, stimulation of impaired biliary output, and inhibition of hepatocyte apoptosis. Despite its clinical effectiveness, UDCA is poorly soluble in the gastro-duodeno-jejunal contents, and pharmacological doses of UDCA are not readily soluble in the stomach and intestine, resulting in incomplete absorption. Indeed, the solubility of 20 mg/L greatly limits the bioavailability of UDCA. Since the bioavailability of drug products plays a critical role in the design of oral administration dosages, we investigated the enzymatic esterification of UDCA as a strategy of hydrophilization. Therefore, we decided to enzymatically synthesize a glyceric ester of UDCA bile acid to produce a more water-soluble molecule. The esterification reactions between UDCA and glycerol were performed with an immobilized lipase B from Candida antarctica (Novozym 435) in solvent-free and solvent-assisted systems. The characterization of the UDCA-monoglyceride, enzymatically synthesized, has been performed by 1H-NMR, 13C-NMR, COSY, HSQC, HMBC, IR, and MS spectroscopy.


Assuntos
Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Monoglicerídeos/química , Pró-Fármacos/síntese química , Ácido Ursodesoxicólico/química , Basidiomycota/enzimologia , Catálise , Cromatografia Líquida de Alta Pressão , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Esterificação , Proteínas Fúngicas/metabolismo , Glicerol/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Solubilidade , Solventes/química , Temperatura
9.
Molecules ; 26(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34641385

RESUMO

α-Glucosidase was immobilized on magnetic nanoparticles (MNPs) for selective solid-phase extraction of the enzyme's ligands present in Aloe vera, which is a medicinal plant used for the treatment of various diseases and possesses anti-diabetic activity. One new compound, aloeacone (2), together with two known compounds, aloenin aglycone (1) and aloin A (3), were fished out as the enzyme's ligands. The structure of 2 was determined by HR-MS and comprehensive NMR techniques. Compound 3 exhibited a weak inhibitory effect on α-glucosidase, while compounds 1 and 2 were found to possess activation effects on the enzyme for the first time. It is interesting that both an inhibitor and agonists of α-glucosidase were fished out in one experiment.


Assuntos
Enzimas Imobilizadas/metabolismo , Glucosídeos/metabolismo , Nanopartículas de Magnetita/química , Extratos Vegetais/metabolismo , alfa-Glucosidases/metabolismo , Aloe , Catárticos/metabolismo , Emodina/análogos & derivados , Emodina/metabolismo , Enzimas Imobilizadas/química , Glucosídeos/isolamento & purificação , Ligantes , alfa-Glucosidases/química
10.
Molecules ; 26(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34641446

RESUMO

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1-2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.


Assuntos
Reatores Biológicos/estatística & dados numéricos , Eletroforese Capilar/métodos , Enzimas Imobilizadas/química , Proteólise , Dióxido de Silício/química , Tripsina/química , Enzimas Imobilizadas/metabolismo , Humanos , Tripsina/metabolismo
11.
Nanoscale ; 13(42): 17871-17880, 2021 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-34673862

RESUMO

Surface immobilization of enzymes on magnetic-recoverable carriers is of great interest and importance for the biocatalysis of relatively large molecules. In this work, the nanosized amino-rich filamentous M13 virus, a versatile biological scaffold, was applied as the unique soft backbone for lipase immobilization. Based on the structure and capsid proteins of M13 phages, the magnetic-recoverable mimic-free-lipases (MFLs) composed of the M13 hydrogels and magnetic particles were developed in two designs. In the first design, nanosized wild M13 phages were crosslinked into a phage hydrogel through the N-terminals of pVIII peptides while NH2-Fe3O4 magnetic nanoparticles (MNPs) were attached to the M13 virus through glutaraldehyde, forming the M13-(NH2-Fe3O4) magnetic phage hydrogel. In the second design, special M13 with Fe3O4 affinity pIII-peptide (FAP-M13) was biopanned for strongly binding towards bare Fe3O4 with the "hook"-like pIII-peptide (N-LPLSTQH-C). TEM observation confirmed the direct grasp of FAP-M13 on bare Fe3O4, forming the magnetic (FAP-M13)-Fe3O4 virus hydrogel. Lipases were uniformly anchored on the phage surface of nanoscale by crosslinking with the N-terminals of pVIII peptides, and then lipase@M13-(NH2-Fe3O4) and lipase@(FAP-M13)-Fe3O4 MFLs were constructed. For both MFLs, high activity recovery yield (>95%) and efficient magnetic separation were characterized. Significantly reduced MNP-usage-amount and enhanced lipase-loading-amount both by about 40 folds were obtained, compared with the conventional NH2-Fe3O4 carriers. The quantified Km and Vmax/Km values were almost equal to those of the free lipases, verifying free-enzyme-mimicking features of the MFLs. High pH-tolerance, wide temperature adaptability, enhanced thermal stability and stable magnetic separation capability of both MFLs were also observed. In particular, the (FAP-M13)-Fe3O4 magnetic virus hydrogel simply using bare Fe3O4 MNPs would be more convenient and economical in the scaled-up biocatalysis.


Assuntos
Lipase , Nanopartículas de Magnetita , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrogéis , Lipase/metabolismo , Fenômenos Magnéticos
12.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2936-2946, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472310

RESUMO

A stable Zr-based metal-organic framework (MOF, UiO-66-NH2) synthesized via micro-water solvothermal method was used to immobilize amidase by using the glutaraldehyde crosslinking method. The effect of immoblization conditions on enzyme immoblization efficiency was studied. An activity recovery rate of 86.4% and an enzyme loading of 115.3 mg/g were achieved under the optimal conditions: glutaraldehyde concentration of 1.0%, cross-linking time of 180 min, and the weight ratio of MOF to enzyme of 8:1. The optimal temperature and optimal pH of the immobilized amidase were determined to be 40 °C and 9.0, respectively, and the Km, Vmax and kcat of the immoblized amidase were 58.32 mmol/L, 16.23 µmol/(min·mg), and 1 670 s⁻¹, respectively. The immobilized enzyme was used for (S)-4-fluorophenylglycine synthesis and the optimal reaction conditions were 300 mmol/L of N-phenylacetyl-4-fluorophenylglycine, 10 g/L of immobilized enzyme loading, and reacting for 180 min at pH 9.0 and 40 °C. A conversion rate of 49.9% was achieved under the optimal conditions, and the conversion rate can be increased to 99.9% under the conditions of enantiomeric excess. The immobilized enzyme can be repeatedly used, 95.8% of its original activity can be retained after 20 cycles.


Assuntos
Estruturas Metalorgânicas , Amidoidrolases , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Glicina/análogos & derivados , Concentração de Íons de Hidrogênio , Temperatura
13.
Int J Mol Sci ; 22(18)2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34576071

RESUMO

The presented research is focused on an investigation of the effect of the addition of polyvinyl alcohol (PVA) to a gelatin-based hydrogel on the functional properties of the resulting material. The main purpose was to experimentally determine and compare the properties of hydrogels differing from the content of PVA in the blend. Subsequently, the utility of these matrices for the production of an immobilized invertase preparation with improved operational stability was examined. We also propose a useful computational tool to predict the properties of the final material depending on the proportions of both components in order to design the feature range of the hydrogel blend desired for a strictly specified immobilization system (of enzyme/carrier type). Based on experimental research, it was found that an increase in the PVA content in gelatin hydrogels contributes to obtaining materials with a visibly higher packaging density, degree of swelling, and water absorption capacity. In the case of hydrolytic degradation and compressive strength, the opposite tendency was observed. The functionality studies of gelatin and gelatin/PVA hydrogels for enzyme immobilization indicate the very promising potential of invertase entrapped in a gelatin/PVA hydrogel matrix as a stable biocatalyst for industrial use. The molecular modeling analysis performed in this work provides qualitative information about the tendencies of the macroscopic parameters observed with the increase in the PVA and insight into the chemical nature of these dependencies.


Assuntos
Simulação por Computador , Gelatina/química , Álcool de Polivinil/química , Animais , Força Compressiva , Módulo de Elasticidade , Enzimas Imobilizadas/metabolismo , Ligação de Hidrogênio , Teste de Materiais , Modelos Moleculares , Porosidade , Suínos , Água/química
14.
Enzyme Microb Technol ; 150: 109865, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489024

RESUMO

In this study, we cross-linked aminated Thermothelomyces thermophilus laccase onto Immobead 150P epoxy carrier, and achieved an immobilization yield of 99.84 %. The optimum temperature and pH values for the oxidation of ABTS by laccase were determined to be 70 °C and pH 3.0. After 6 h at 50 °C, laccase activity was diminished by about 13 % in the free form and 28 %, in the immobilized form. Km values for both free and cross-linked laccase were 0.051 and 0.567 mM, whereas Vmax values were 2.027 and 0.854 µmol. min-1, respectively. The immobilized laccase was able to preserve its full activity for 6 weeks, retaining approximately 95 % and 78 % of its initial activity after 8 and 20 weeks, respectively. The contact angles were two-fold higher when the laccase enzyme was occupied in the biografting reaction, revealing that the hydrophobic compound bonded stably onto beechwood samples.


Assuntos
Enzimas Imobilizadas , Lacase , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lacase/metabolismo , Sordariales , Temperatura
15.
PLoS One ; 16(9): e0257615, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34547058

RESUMO

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 µg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.


Assuntos
Biomarcadores/urina , Endopeptidase K/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Tuberculose/diagnóstico , Endopeptidase K/química , Ensaio de Imunoadsorção Enzimática/instrumentação , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Lipopolissacarídeos/urina , Papel , Temperatura
16.
Anal Chem ; 93(35): 11973-11981, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34428014

RESUMO

Biocatalysis has rapidly become an essential tool in the scientific and industrial communities for the development of efficient, safe, and sustainable chemical syntheses. Immobilization of the biocatalyst, typically an engineered enzyme, offers significant advantages, including increased enzyme stability and control, resistance to environmental change, and enhanced reusability. Determination and optimization of the spatial and chemical distribution of immobilized enzymes are critical for proper functionality; however, analytical methods currently employed for doing so are frequently inadequate. Machine learning, in the form of multivariate curve resolution, with Raman hyperspectral imaging is presented herein as a potential method for investigating the spatial and chemical distribution of evolved pantothenate kinase immobilized onto two diverse, microporous resins. An exhaustive analysis indicates that this method can successfully resolve, both spatially and spectrally, all chemical species involved in enzyme immobilization, including the enzyme, both resins, and other key components. Quantitation of the spatial coverage of immobilized enzymes, a key parameter used for process development, was accomplished. Optimal analytical parameters were determined by the evaluation of different excitation wavelengths. Exploratory chemometric approaches, including principal component analysis, were utilized to investigate the chemical species embedded within the data sets and their relationships. The totality of this information can be utilized for an enhanced understanding of enzyme immobilization processes and can allow for the further implementation of biocatalysis within the scientific and pharmaceutical communities.


Assuntos
Enzimas Imobilizadas , Aprendizado de Máquina , Biocatálise , Fenômenos Químicos , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo
17.
Dokl Biochem Biophys ; 499(1): 220-224, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34426915

RESUMO

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND-oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND-oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND-oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.


Assuntos
Basidiomycota/enzimologia , Técnicas Biossensoriais/métodos , Espaço Extracelular/enzimologia , Nanodiamantes/química , Oxirredutases/metabolismo , Fenol/análise , Água/química , Basidiomycota/citologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Oxirredutases/química
18.
Molecules ; 26(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34361678

RESUMO

The accurate determination of analyte concentrations with selective, fast, and robust methods is the key for process control, product analysis, environmental compliance, and medical applications. Enzyme-based biosensors meet these requirements to a high degree and can be operated with simple, cost efficient, and easy to use devices. This review focuses on enzymes capable of direct electron transfer (DET) to electrodes and also the electrode materials which can enable or enhance the DET type bioelectrocatalysis. It presents amperometric biosensors for the quantification of important medical, technical, and environmental analytes and it carves out the requirements for enzymes and electrode materials in DET-based third generation biosensors. This review critically surveys enzymes and biosensors for which DET has been reported. Single- or multi-cofactor enzymes featuring copper centers, hemes, FAD, FMN, or PQQ as prosthetic groups as well as fusion enzymes are presented. Nanomaterials, nanostructured electrodes, chemical surface modifications, and protein immobilization strategies are reviewed for their ability to support direct electrochemistry of enzymes. The combination of both biosensor elements-enzymes and electrodes-is evaluated by comparison of substrate specificity, current density, sensitivity, and the range of detection.


Assuntos
Técnicas Biossensoriais/métodos , Eletrodos , Elétrons , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Biocatálise , Monitoramento Biológico/métodos , Biomarcadores Tumorais/análise , Glicemia/análise , Automonitorização da Glicemia/métodos , Coenzimas/metabolismo , Eletroquímica/métodos , Transporte de Elétrons , Estrutura Molecular , Nanoestruturas/química
19.
Bioresour Technol ; 339: 125599, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34303095

RESUMO

L-asparaginase shows great potential as a food enzyme to reduce acrylamide formation in fried and baked products. But for food applications, enzymes must be stable at high temperatures and have higher catalytic efficiency. These desirable characteristics are conferred by the immobilization of enzymes on a suitable matrix. The present study aimed to immobilize the L-asparaginase enzyme on magnetic nanoparticles to reduce acrylamide content in the food system. Immobilized preparations were characterized using SEM, TEM, FTIR, UV-spectrometry, and XRD diffraction analyses. These nanoparticles enhanced the thermal stability of the enzyme up to four-fold at 70 °C compared to the free enzyme. Kinetic parameters exhibited an increase in Vmax, Km, and catalytic efficiency by ~ 38% than the free counterpart. The immobilized preparations were reusable for up to five cycles. Moreover, their application in the pre-treatment coupled with blanching of potato chips led to a significant reduction (greater than 95%) of acrylamide formation.


Assuntos
Asparaginase , Nanopartículas de Magnetita , Acrilamida , Asparaginase/metabolismo , Catálise , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Cinética
20.
Chem Biol Interact ; 346: 109577, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34274336

RESUMO

The introduction of enzyme nanoreactors in medicine is relatively new. However, this technology has already been experimentally successful in cancer treatments, struggle against toxicity of reactive oxygen species in inflammatory processes, detoxification of drugs and xenobiotics, and correction of metabolic and genetic defects by using encapsulated enzymes, acting in single or cascade reactions. Biomolecules, e.g. enzymes, antibodies, reactive proteins capable of inactivating toxicants in the body are called bioscavengers. In this review, we focus on enzyme-containing nanoreactors for in vivo detoxification of organophosphorous compounds (OP) to be used for prophylaxis and post-exposure treatment of OP poisoning. A particular attention is devoted to bioscavenger-containing injectable nanoreactors operating in the bloodstream. The nanoreactor concept implements single or multiple enzymes and cofactors co-encapsulated in polymeric semi-permeable nanocontainers. Thus, the detoxification processes take place in a confined space containing highly concentrated bioscavengers. The article deals with historical and theoretical backgrounds about enzymatic detoxification of OPs in nanoreactors, nanoreactor polymeric enveloppes, realizations and advantages over other approaches using bioscavengers.


Assuntos
Nanoestruturas/química , Compostos Organofosforados/metabolismo , Xenobióticos/metabolismo , Biocatálise , Coenzimas/química , Coenzimas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Inativação Metabólica , Nanotecnologia , Compostos Organofosforados/química , Polímeros/química
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