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1.
J Chromatogr A ; 1722: 464896, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38631224

RESUMO

In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted.


Assuntos
Simulação de Acoplamento Molecular , Inibidores da Monoaminoxidase , Monoaminoxidase , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Inibidores da Monoaminoxidase/isolamento & purificação , Monoaminoxidase/metabolismo , Monoaminoxidase/química , Ligantes , Indóis/química , Monoterpenos/química , Monoterpenos/isolamento & purificação , Cinética , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/antagonistas & inibidores , Humanos , Extratos Vegetais/química
2.
Int J Biol Macromol ; 266(Pt 2): 131329, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38574906

RESUMO

The bacterial nanocellulose (BnC) membranes were produced extracellularly by a novel aerobic acetic acid bacterium Komagataeibacter melomenusus. The BnC was modified in situ by adding carboxymethyl cellulose (CMC) into the culture media, obtaining a BnC-CMC product with denser fibril arrangement, improved rehydration ratio and elasticity in comparison to BnC. The proteolytic enzyme bromelain (Br) and antimicrobial peptide nisin (N) were immobilized to BnC matrix by ex situ covalent binding and/or adsorption. The optimal Br immobilization conditions towards the maximized specific proteolytic activity were investigated by response surface methodology as factor variables. At optimal conditions, i.e., 8.8 mg/mL CMC and 10 mg/mL Br, hyperactivation of the enzyme was achieved, leading to the specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1 %. The antimicrobial activity was observed against Gram-positive bacteria (S. epidermidis, S. aureus and E. faecalis) for membranes with immobilized N and was superior when in situ modified BnC membranes were used. N immobilized on the BnC or BnC-CMC membranes was cytocompatible and did not cause changes in normal human dermal fibroblast cell morphology. BnC membranes perform as an efficient carrier for Br or N immobilization, holding promise in wound debridement and providing antimicrobial action against Gram-positive bacteria, respectively.


Assuntos
Acetobacteraceae , Bromelaínas , Celulose , Nisina , Nisina/farmacologia , Nisina/química , Bromelaínas/química , Bromelaínas/farmacologia , Celulose/química , Celulose/farmacologia , Acetobacteraceae/química , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Cicatrização/efeitos dos fármacos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Nanoestruturas/química , Testes de Sensibilidade Microbiana
3.
Int J Biol Macromol ; 264(Pt 2): 130730, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38462111

RESUMO

Magnetic nanoparticles were functionalized with polyethylenimine (PEI) and activated with epoxy. This support was used to immobilize Lipase (Eversa® Transform 2.0) (EVS), optimization using the Taguchi method. XRF, SEM, TEM, XRD, FTIR, TGA, and VSM performed the characterizations. The optimal conditions were immobilization yield (I.Y.) of 95.04 ± 0.79 %, time of 15 h, ionic load of 95 mM, protein load of 5 mg/g, and temperature of 25 °C. The maximum loading capacity was 25 mg/g, and its stability in 60 days of storage showed a negligible loss of only 9.53 % of its activity. The biocatalyst demonstrated better stability at varying temperatures than free EVS, maintaining 28 % of its activity at 70 °C. It was feasible to esterify free fatty acids (FFA) from babassu oil with the best reaction of 97.91 % and ten cycles having an efficiency above 50 %. The esterification of produced biolubricant was confirmed by NMR, and it displayed kinematic viscosity and density of 6.052 mm2/s and 0.832 g/cm3, respectively, at 40 °C. The in-silico study showed a binding affinity of -5.8 kcal/mol between EVS and oleic acid, suggesting a stable substrate-lipase combination suitable for esterification.


Assuntos
Lipase , Nanopartículas de Magnetita , Lipase/química , Enzimas Imobilizadas/química , Óleos de Plantas/química , Esterificação , Estabilidade Enzimática
4.
J Pharm Biomed Anal ; 243: 116110, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38513498

RESUMO

In this study, thrombin was immobilized with magnetic particles modified by glutaraldehyde. The changes in secondary structures of immobilized enzyme revealed an increment in conformational rigidity and stability, which can be reflected in temperature and pH stability as well as the tolerance of organic reagents. The optimal reutilization times of magnetic particle immobilized thrombin were 7 times, and the half-life of enzyme activity preserved at room temperature was 5 days, which was 2.5 times higher than that of free enzyme. Ligusticum chuanxiong and Anemarrhenae Rhizoma with high enzyme inhibitory activity were selected for primary screening, and six potential inhibitors of thrombin were identified by HPLC/MS. The results showed that three compounds in Anemarrhenae Rhizoma had better predictive thrombin inhibitory activity. Through the in vitro thrombin activity inhibition experiment, it was also verified that mangiferin and neo-mangiferin had an ideal thrombin activity inhibition effect, which was consistent with the results of molecular docking.


Assuntos
Produtos Biológicos , Medicamentos de Ervas Chinesas , Nanopartículas de Magnetita , Medicamentos de Ervas Chinesas/química , Trombina , Produtos Biológicos/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Enzimas Imobilizadas/química , Anticoagulantes
5.
Enzyme Microb Technol ; 175: 110409, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38335559

RESUMO

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).


Assuntos
Óleo de Rícino , Ácidos Graxos não Esterificados , Hexanóis , Esterificação , Ésteres/química , Ácidos Graxos/química , Lipase/metabolismo , Etanol , Catálise , Enzimas Imobilizadas/química
6.
Molecules ; 29(3)2024 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-38338454

RESUMO

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts.


Assuntos
Quitosana , Nanotubos de Carbono , Selênio , Enzimas Imobilizadas/química , Peroxidase do Rábano Silvestre/química , Quitosana/química , Estabilidade Enzimática , Íons , Concentração de Íons de Hidrogênio
7.
Bioprocess Biosyst Eng ; 47(2): 249-261, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38197955

RESUMO

ß-galactosidase has been immobilized onto novel alginate/tea waste gel beads (Alg/TW) via covalent binding. Alg/TW beads were subjected to chemical modification through amination with polyethyleneimine (PEI) followed by activation with glutaraldehyde (GA). Chemical modification parameters including PEI concentration, PEI pH, and GA concentration were statistically optimized using Response Surface methodology (RSM) based on Box-Behnken Design (BBD). Analysis of variance (ANOVA) results confirmed the great significance of the model that had F value of 37.26 and P value < 0.05. Furthermore, the R2 value (0.9882), Adjusted R2 value (0.9617), and predicted R2 value (0.8130) referred to the high correlation between predicted and experimental values, demonstrating the fitness of the model. In addition, the coefficient of variation (CV) value was 2.90 that pointed to the accuracy of the experiments. The highest immobilization yield (IY) of ß-galactosidase (75.1%) was given under optimized conditions of PEI concentration (4%), PEI pH (9.5), and GA concentration (2.5%). Alg/TW beads were characterized by FT-IR, TGA, and SEM techniques at each step of immobilization process. Moreover, the immobilized ß-galactosidase revealed a very good reusability as it could be reused for 15 and 20 consecutive cycles keeping 99.7 and 72.1% of its initial activity, respectively. In conclusion, the environmental waste (tea waste) can be used in modern technological industries such as the food and pharmaceutical industry.


Assuntos
Alginatos , Enzimas Imobilizadas , Enzimas Imobilizadas/química , Microesferas , Concentração de Íons de Hidrogênio , Alginatos/química , Chá , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , beta-Galactosidase/química
8.
J Biotechnol ; 379: 78-86, 2024 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-38072327

RESUMO

This study presents a solvent-free enzymatic approach for the synthesis of fatty acid methyl esters (FAMEs), such as methyl oleate, for their application as adjuvant in plant protection products (PPP) formulations. The direct esterification between free fatty acid and methanol was optimized to achieve 98% acid conversion. The kinetics of this conversion was accurately described by a simple second order mechanism and non-linear regression was applied to calculate the rate constants of the forward and backward reactions based on full progress curves data. The rate constant of the forward reaction (synthesis) was one order of magnitude higher than the backward reaction (hydrolysis) and favored formation of the target methyl ester product, rendering the removal of water unnecessary. Enzymatically synthesized methyl oleate was benchmarked against the chemically synthesized compound, showing matching results in terms of stability, spreadability and emulsifying capacity in plant care formulations. The enzymatic synthesis of FAMEs under solvent free conditions allows to achieve a safer and more sustainable character for carrier solvents in PPP formulations.


Assuntos
Ésteres , Lipase , Lipase/química , Esterificação , Hidrólise , Ácidos Graxos , Solventes/química , Cinética , Enzimas Imobilizadas/química
9.
Int J Biol Macromol ; 257(Pt 1): 128804, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38101664

RESUMO

In this paper, using a coprecipitation method to prepare Fe3O4 magnetic nanoparticles (Fe3O4 MNPS), magnetic dialdehyde starch nanoparticles with immobilized phospholipase A1 (MDSNIPLA) were successfully prepared by using green dialdehyde starch (DAS) instead of glutaraldehyde as the crosslinking agent. The Fe3O4 MNPS was characterized by infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), the Brunauer-Emmett-Teller (BET) surface area analysis method, thermogravimetric analysis (TGA), and transmission electron microscopy (TEM) et al. The results showed that the alkaline resistance and acid resistance of the enzyme were improved after the crosslinking of DAS. After repeated use (seven times), the relative activity of MDSNIPLA reached 56 %, and the magnetic dialdehyde starch nanoparticles (MDASN) had good carrier performance. MDSNIPLA was applied to enzymatic hydrolysis of phospholipids in the soybean oil degumming process. The results showed that the acyl transfer rate of sn-2-HPA was 14.01 %, and the content of free fatty acids was 1.144 g/100 g after 2 h reaction at 50 °C and pH 5.0 with appropriate boric acid. The immobilized enzyme has good thermal stability and storage stability, and its application of soybean oil improves the efficiency of the oil.


Assuntos
Enzimas Imobilizadas , Óleo de Soja , Amido/análogos & derivados , Espectroscopia de Infravermelho com Transformada de Fourier , Enzimas Imobilizadas/química , Fosfolipases , Fenômenos Magnéticos
10.
Anal Methods ; 15(45): 6220-6228, 2023 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-37942997

RESUMO

Cellulose filter paper (CFP) is expected to be an ideal carrier for enzyme immobilization due to its sustainability and biocompatibility. However, the interaction between the carrier and enzyme might change the spatial conformation of the enzyme and its microenvironment, and thus the flexibility of the enzyme molecule or the transport of the substrate to the active site would be hampered. In this work, a two-component system of catechol and tetraethylene pentamine was used to replace dopamine, and a polydopamine-like composite layer was deposited on the surface of CFP to introduce amino groups, which was similar to the self-polymerization-adhesion behavior of dopamine. Using polyethylene glycol diglycidyl ether with flexible spacer arms as the cross-linking agent, α-glucosidase was covalently bonded to amino-modified CFP through an epoxy ring-opening reaction. The immobilized α-glucosidase exhibited greater tolerance to pH and high temperature. After 10 repeated uses, the immobilized α-glucosidase maintained relatively high enzyme activity. Its kinetic behavior was investigated to illustrate the reliability for enzyme inhibitor screening. Finally, a screening method combining an immobilized enzyme and capillary electrophoresis analysis was proposed and applied to screening inhibitors from 11 kinds of traditional Chinese medicines, among which Chebulae Fructus, Phyllanthi Fructus and Terminaliae Relliricae Fructus exhibited strong enzyme inhibitory activities.


Assuntos
Medicina Tradicional Chinesa , alfa-Glucosidases , alfa-Glucosidases/química , Reprodutibilidade dos Testes , Dopamina/farmacologia , Celulose/química , Enzimas Imobilizadas/química
11.
J Chromatogr A ; 1711: 464433, 2023 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-37847969

RESUMO

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).


Assuntos
Agrimonia , Diabetes Mellitus Tipo 2 , Nanopartículas , Humanos , Inibidores de Glicosídeo Hidrolases/química , Simulação de Acoplamento Molecular , alfa-Glucosidases/química , Cromatografia Líquida , Ligantes , Dióxido de Silício , Espectrometria de Massas em Tandem , Enzimas Imobilizadas/química , Fenômenos Magnéticos , Extratos Vegetais/farmacologia , Extratos Vegetais/química
12.
Bioprocess Biosyst Eng ; 46(8): 1195-1208, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37329348

RESUMO

Acidified oil is obtained from by-product of crops oil refining industry, which is considered as a low-cost material for fatty acid production. Hydrolysis of acidified oil by lipase catalysis for producing fatty acid is a sustainable and efficient bioprocess that is an alternative of continuous countercurrent hydrolysis. In this study, lipase from Candida rugosa (CRL) was immobilized on magnetic Fe3O4@SiO2 via covalent binding strategy for highly efficient hydrolysis of acidified soybean oil. FTIR, XRD, SEM and VSM were used to characterize the immobilized lipase (Fe3O4@SiO2-CRL). The enzyme properties of the Fe3O4@SiO2-CRL were determined. Fe3O4@SiO2-CRL was used to catalyze the hydrolysis of acidified soybean oil to produce fatty acids. Catalytic reaction conditions were studied, including amount of catalyst, reaction time, and water/oil ratio. The results of optimization indicated that the hydrolysis rate reached 98% under 10 wt.% (oil) of catalyst, 3:1 (v/v) of water/oil ratio, and 313 K after 12 h. After 5 cycles, the hydrolysis activity of Fe3O4@SiO2-CRL remained 55%. Preparation of fatty acids from high-acid-value by-products through biosystem shows great industrial potential.


Assuntos
Ácidos Graxos , Lipase , Lipase/química , Hidrólise , Óleo de Soja , Dióxido de Silício , Enzimas Imobilizadas/química , Água , Estabilidade Enzimática
13.
J Sep Sci ; 46(14): e2300195, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37232227

RESUMO

The inhibition of tyrosinase is considered to be a common therapeutic strategy for some hyperpigmentation disorders. Screening of tyrosinase inhibitors is of great significance to the treatment of pigmentation diseases. In this study, tyrosinase was covalently immobilized on magnetic multi-walled carbon nanotubes for the first time, and the immobilized tyrosinase was applied for ligand fishing of tyrosinase inhibitors from complex medicinal plants. The immobilized tyrosinase was characterized by transmission electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, vibrating sample magnetometry, and thermo-gravimetric analyzer, which indicated that tyrosinase was immobilized onto magnetic multi-walled carbon nanotubes. The immobilized tyrosinase showed better thermal stability and reusability than the free one. The ligand was fished out from Radix Paeoniae Alba and identified as 1,2,3,4,6-pentagalloylglucose by ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry. 1,2,3,4,6-pentagalloylglucose was found to be a tyrosinase inhibitor with similar half maximal inhibitory concentration values of 57.13 ± 0.91 µM compared to kojic acid (41.96 ± 0.78 µM). This work not only established a new method for screening tyrosinase inhibitors but also holds considerable potential for exploring the new medicinal value of medicinal plants.


Assuntos
Monofenol Mono-Oxigenase , Nanotubos de Carbono , Monofenol Mono-Oxigenase/química , Nanotubos de Carbono/química , Ligantes , Fenômenos Magnéticos , Enzimas Imobilizadas/química
14.
Int J Biol Macromol ; 242(Pt 2): 124807, 2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-37178887

RESUMO

The hydrolysis of natural oils (vegetable oils and fats) by lipase has significant applications in food and medicine. However, free lipases are usually sensitive to temperature, pH and chemical reagents in aqueous solutions, which hinders their widespread industrial application. Excitingly, immobilized lipases have been widely reported to overcome these problems. Herein, inspired by lipase interface activation, a hydrophobic Zr-MOF (UiO-66-NH2-OA) with oleic acid was synthesized for the first time in an emulsion consisting of oleic acid and water, and the Aspergillus oryzae lipase (AOL) was immobilized onto the UiO-66-NH2-OA through hydrophobic interaction and electrostatic interaction to obtain immobilized lipase (AOL/UiO-66-NH2-OA). 1H NMR and FT-IR data indicated that oleic acid was conjugated with the 2-amino-1,4-benzene dicarboxylate (BDC-NH2) by amidation reaction. As a result, the Vmax and Kcat values of AOL/UiO-66-NH2-OA were 179.61 µM﹒min-1 and 8.27 s-1, which were 8.56 and 12.92 times higher than those of the free enzyme, respectively, due to the interfacial activation. After treated at 70 °C for 120 min, the immobilized lipase maintained 52 % of its original activity, but free AOL only retained 15 %. Significantly, the yield of fatty acids by the immobilized lipase reached 98.3 % and still exceeded 82 % after seven times of recycling.


Assuntos
Lipase , Ácido Oleico , Lipase/química , Hidrólise , Espectroscopia de Infravermelho com Transformada de Fourier , Enzimas Imobilizadas/química , Óleos de Plantas/química , Ácidos Graxos Insaturados , Água , Interações Hidrofóbicas e Hidrofílicas
15.
Langmuir ; 39(15): 5239-5249, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-37014629

RESUMO

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


Assuntos
Enzimas Imobilizadas , Nanopartículas de Magnetita , Enzimas Imobilizadas/química , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo , Polímeros , Porosidade , Cinética , Fenômenos Magnéticos , Nanopartículas de Magnetita/química , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , Temperatura
16.
Anal Bioanal Chem ; 415(4): 615-625, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36445454

RESUMO

Enzyme-based electrochemical biosensors have been widely deployed for the detection of a range of contaminants in different food products due to their significant advantages over other (bio)sensing techniques. Nevertheless, their performance is greatly affected by the sample matrix itself or by the matrix they are presented with in pretreated samples, both of which can impact the accuracy as well as the sensitivity of the measurements. Therefore, and in order to acquire reliable and accurate measurements, matrix effects and their influence on sensor performance should be taken into consideration. Herein, acetylcholinesterase (AChE)-modified electrochemical sensors were employed for the detection of pesticides in vegetable oils. Sensor interrogation with pretreated oil samples, spiked with carbofuran, revealed the inhibitory potential of the extracted matrix varies between different types of vegetable oil and their fatty acid content. In addition, synergies between the extracted matrix from different types of vegetable oils and the carbamate pesticide, carbofuran, were observed, which led to significant deviations of the sensor's performance from its anticipated behavior in buffered solution. Taking the aforementioned into consideration, appropriate calibration curves for each type of vegetable oil were drafted, which allowed for the highly reproducible determination of different pesticide concentrations in pretreated real samples. Collectively, a better understanding of AChE inhibition by single or multiple contaminants present in vegetable oils was gained, which can find many applications in numerous fields, ranging from sensor development to the design of new pesticides and medicinal products.


Assuntos
Técnicas Biossensoriais , Carbofurano , Praguicidas , Praguicidas/química , Acetilcolinesterase/química , Enzimas Imobilizadas/química , Óleos de Plantas , Técnicas Biossensoriais/métodos
17.
Prep Biochem Biotechnol ; 53(7): 763-772, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36332158

RESUMO

India generates 126.6 and 42 million tons of Rice straw (RS) and Rice husk (RH) annually, respectively. These agro-processing wastes feedstock are dumped in landfills or burnt, releasing toxic gases and particulate matter into the environment. This paper explores the valorization of these wastes feedstock into sustainable, economic products. We compare these wastes as matrices for lipase immobilization. These matrices were characterized, different parameters (pH, temperature, ionic strength, and metal ion cofactors) were checked, and the selected matrix was analyzed for reusability and hydrolysis of vegetable oils. Lipase immobilized Rice straw (LIRS) showed the highest activity with 72.84% protein loading. Field emission scanning electron microscopy (FESEM) demonstrated morphological changes after enzyme immobilization. FTIR showed no new bond formation, and immobilization data was fitted to Freundlich adsorption isotherm (with K = 12.18 mg/g, nF = 4.5). The highest activity with protein loading, 91.05%, was observed at pH 8, 37 °C temperature, 50 mM ionic strength, and lipase activity doubled in the presence of Mg2+ ions. The LIRS retained 75% of its initial activity up to five cycles and efficiently hydrolyzed different oils. The results reflected that the LIRS system performs better and can be used to degrade oily waste.


Assuntos
Lipase , Oryza , Lipase/química , Oryza/metabolismo , Hidrólise , Enzimas Imobilizadas/química , Óleos de Plantas , Adsorção , Concentração de Íons de Hidrogênio
18.
Appl Biochem Biotechnol ; 195(1): 432-450, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36087232

RESUMO

The enzymatic production of biodiesel from waste cooking oils (WCOs) offers a green and sustainable solution for the liquid fuel manufacture as well as waste resource recovery. In present study, liquid lipase was used to simplify the catalysis process, thereby reducing biodiesel production costs. An engineered Escherichia coli expressing Geobacillus thermocatenulatus lipase 2 (GTL2) was screened at an enzyme activity of 6.96 U/mg, after evaluating the propagating stability of the recombinant plasmids exceeding 86.11%. Through the beneficial feeding strategy and effective pH control, high-level production of GTL2 by fed-batch fermentation was achieved with an enzyme activity of 434.32 U/mg, which was almost 62 times that of shake flask fermentation. In addition, liquid GTL2 was used to prepare fatty acid methyl esters (FAMEs) using WCOs. The effects of the reaction time, catalyst loading, temperature, and methanol-to-oil molar ratio on FAMEs production using WCOs were explored, and a maximum FAMEs yield of 96.62% was achieved under optimized conditions. These results indicate that liquid GTL2 is a promising biocatalyst for efficient utilization of WCOs in the synthesis of biodiesel and provide a novel enzymatic process for biodiesel reducing the cost of production.


Assuntos
Biocombustíveis , Lipase , Lipase/química , Fermentação , Escherichia coli/genética , Escherichia coli/metabolismo , Esterificação , Enzimas Imobilizadas/química , Óleos , Catálise , Culinária , Óleos de Plantas/química
19.
Enzyme Microb Technol ; 161: 110116, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36037553

RESUMO

Glutaraldehyde is usually used as a cross-linking agent in the immobilization of enzymes, but this will have a negative effect on the enzyme. Dialdehyde starch can effectively replace glutaraldehyde as a cross-linking agent, but the large particle size of dialdehyde starch affects the performance of immobilized enzyme. In this study, dialdehyde starch nanoparticles (DSNP) were combined with modified Fe3O4 to obtain magnetic carrier (MDSNP), and Candida Antarctica lipase B (CALB) was crosslinked to the carrier to obtain magnetic immobilized enzyme (MDSNPCALB). The characterization results show that the functional groups of each material have obvious characteristic absorption peaks, strong diffraction peaks and typical crystal structures, high magnetism, no coercivity, relatively good dispersion and nano particle size. MDSNPCALB was added to degummed rice bran oil (RBO) and ethanolamine was used as an acyl receptor for acylation and deacidification. After repeated use for 10 times, MDSNPCALB remained highly active, indicating that MDSNPCALB can be effectively used for the deacidification of RBO.


Assuntos
Enzimas Imobilizadas , Proteínas Fúngicas , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Glutaral , Fenômenos Magnéticos , Óleo de Farelo de Arroz , Amido/química
20.
J Sep Sci ; 45(18): 3635-3645, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35852941

RESUMO

In this study, tyrosinase was immobilized on carboxyl functionalized silica-coated magnetic nanoparticles for the first time to be used for fishing of tyrosinase's ligands present in complex plant extract. The immobilized tyrosinase was characterized by transmission electron microscopy, vibrating sample magnetometry, Fourier transform infrared spectroscopy, thermo-gravimetric analyzer, and atomic force microscopy. The reusability and thermostability of the immobilized tyrosinase were found significantly superior to its free counterpart. Two tyrosinase's ligands, that is, caffeic acid (1) and rosmarinic acid (2), were fished out from extract of the traditional Chinese medicine Prunellae Spica by the immobilized tyrosinase. Compound 1 was found to be an activator of the enzyme with the half maximal effective concentration value of 0.27 ± 0.06 mM, while compound 2 was an inhibitor with the half maximal inhibitory concentration value of 0.14 ± 0.03 mM. Taking advantage of the convenience of magnetic separation and specific extraction ability of ligand fishing, the proposed method exhibited great potential for screening of bioactive compounds from complex matrices.


Assuntos
Nanopartículas de Magnetita , Monofenol Mono-Oxigenase , Enzimas Imobilizadas/química , Ligantes , Nanopartículas de Magnetita/química , Monofenol Mono-Oxigenase/química , Extratos Vegetais/química , Dióxido de Silício
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