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1.
Bioresour Technol ; 331: 125063, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33813167

RESUMO

Enhanced covalent immobilization of xylanase from Chaetomium globosum (XylCg) onto SiO2 nanoparticles was achieved by the modification of surface residues. The mutation of surface residues to lysine by site-directed mutagenesis increased the immobilization efficiency (IE) and immobilization yield (IY). The immobilized mutant XylCg (N172K-H173K-S176K-K133A-K148A) exhibited an IY of 99.5% and IE of 135%, which were 1.8- and 4.3-fold higher than immobilized wildtype (WT). Regarding the catalytic properties, the kcat and kcat/Km values were 1850 s-1 and 2030 mL mg-1 s-1 for the immobilized mutant, and 331 s-1 and 404 mL mg-1 s-1 for the immobilized WT, respectively. Additionally, the immobilized mutant exhibited four times higher thermal stability than the immobilized WT at 60 °C. These results suggest that surface-mutated lysine residues confer good stability and orientation on the support matrix, thus improving the overall performance of xylanase.


Assuntos
Nanopartículas , Dióxido de Silício , Chaetomium , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Lisina , Temperatura
2.
Food Chem ; 354: 129511, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-33735695

RESUMO

Based on the successful synthesis of mercaptomethamidophos as a substrate, a novel nanogold/mercaptomethamidophos multi-residue electrochemical biosensor was designed and fabricated by combining nanoscale effect, strong Au-S bonds as well as interaction between acetylcholinesterase (AChE) and mercaptomethamidophos, which can simultaneously detect 11 kinds of organophosphorus pesticides (OPPs) and total amount of OPPs using indirect competitive method. Electrochemical behavior of the modified electrode was characterized by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The AChE concentration and incubation time were optimized at 37.4 °C to achieve the best detection effect. This biosensor exhibits excellent electrochemical properties with a wider linear range of 0.1 ~ 1500 ng·mL-1, lower detection limit of 0.019 ~ 0.077 ng·mL-1, better stability and repeatability, which realizes the rapid detection of total amount of OPPs, and can simultaneously detect a large class of OPPs rather than one kind of OPP. Two OPPs (trichlorfon, dichlorvos) were detected in actual samples of apple and cabbage and achieved satisfactory test results.


Assuntos
Técnicas Biossensoriais/métodos , Nanopartículas Metálicas/química , Compostos Organofosforados/química , Compostos Organotiofosforados/química , Praguicidas/análise , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Brassica/química , Brassica/metabolismo , Espectroscopia Dielétrica , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ouro/química , Limite de Detecção
3.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668968

RESUMO

The unique chemical, optical, and electrical characteristics of nanoparticles make their utilization highly successful in every field of biological sciences as compared to their bulk counterpart. These properties arise as a result of their miniature size, which provides them an excellent surface area-to-volume ratio, inner structure, and shape, and hence increases their surface characteristics. Therefore, this study was undertaken to engineer gold nanoparticles (AuNPs) for improving their catalytic activity and stability in biotechnological processes. The characterization of AuNPs was performed by XRD, UV spectra, and TEM. The synthesized AuNPs were surface-modified by polyvinyl alcohol (PVA) for binding the enzyme in excellent yield. The developed immobilized enzyme system (PVA-AuNPs-ß-galactosidase) displayed pH optima at pH 7.0 and temperature optima at 40 °C. Moreover, the stability of PVA-AuNPs-ß-galactosidase was significantly enhanced at wider pH and temperature ranges and at higher galactose concentrations, in contrast to the free enzyme. ß-galactosidase bound to PVA-modified AuNPs exhibited greater operational activity, even after its sixth reuse. The developed nanosystem may prove useful in producing lactose-free dairy products for lactose-intolerant patients.


Assuntos
Laticínios , Ouro/química , Lactose/química , Nanopartículas Metálicas/química , beta-Galactosidase/química , Laticínios/análise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ouro/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Kluyveromyces/enzimologia , Lactose/metabolismo , Intolerância à Lactose/metabolismo , Teste de Tolerância a Lactose , Tamanho da Partícula , Propriedades de Superfície , Temperatura , beta-Galactosidase/metabolismo
4.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669157

RESUMO

α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from Aspergillus niger L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%.


Assuntos
Galactose/biossíntese , Oligossacarídeos/biossíntese , Prebióticos/análise , alfa-Galactosidase/metabolismo , Aspergillus niger/enzimologia , Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Galactose/química , Oligossacarídeos/química , alfa-Galactosidase/química
5.
Molecules ; 26(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673063

RESUMO

This paper aims to investigate the effects of some salts (NaCl, (NH4)2SO4 and Na2SO4) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH4)2SO4 generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.


Assuntos
Estabilidade Enzimática/efeitos dos fármacos , Enzimas Imobilizadas/química , Sais/química , Catalase/química , Enzimas Imobilizadas/antagonistas & inibidores , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Cinética , Lacase/química , Lipase/química , Compostos Orgânicos/química , Penicilina Amidase/química , Peptídeo Hidrolases/química , Sais/farmacologia , Soluções/química , Soluções/farmacologia , Temperatura
6.
Food Chem ; 352: 129325, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33691212

RESUMO

The milk-coagulating enzyme, rennet, is widely used in cheese making. Recently stabilization of rennet, especially in accelerated cheese ripening, has received considerable interest. As we know encapsulation is one of the enzyme immobilization methods, which could increase enzyme stability. In this study, the effects of alginate, chitosan and, CaCl2 on rennet encapsulation were evaluated and optimized using RSM. Under the optimal conditions alginate, chitosan, and CaCl2 were 0.04%, 0.1%, and 0.1% respectively. At the optimum point, encapsulation efficiency, particle size, and zeta potential were evaluated to be 61.8%, 323 nm, and 25 mV, respectively. The effect of temperature and pH on the enzyme activity was evaluated, and the results showed that encapsulated enzyme had higher activity at various pH and temperature in comparison with the free enzyme. Also, the enzyme release data in all pH values were fitted to Korsmeyer-Peppas model and the n exponent indicated that the release mechanism was Fickian. The electrostatic interactions between enzyme, alginate, and chitosan were confirmed by infrared spectroscopy. No statistical difference was found between the Km and Vmax of encapsulated and free enzymes.


Assuntos
Alginatos/química , Quitosana/química , Quimosina/química , Enzimas Imobilizadas/química , Nanopartículas/química , Cloreto de Cálcio/química , Tamanho da Partícula
7.
Chem Commun (Camb) ; 57(30): 3704-3707, 2021 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-33729260

RESUMO

Accurate and rapid diagnostic tests are critical to reducing the impact of SARS-CoV-2. This study presents early, but promising measurements of SARS-CoV-2 using the ACE2 enzyme as the recognition element to achieve clinically relevant detection. The test provides a scalable route to sensitive, specific, rapid and low cost mass testing.


Assuntos
/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Enzimas Imobilizadas/química , /isolamento & purificação , /metabolismo , Técnicas Biossensoriais/instrumentação , /instrumentação , Técnicas Eletroquímicas/instrumentação , Eletrodos , Enzimas Imobilizadas/metabolismo , Fluorcarbonetos/química , Ouro/química , Humanos , Limite de Detecção , Glicoproteína da Espícula de Coronavírus/metabolismo
8.
Carbohydr Polym ; 260: 117795, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33712143

RESUMO

High-value utilization of cellulosic biomasses via the most promising enzymatic method is the key to solve a series of global strategic issues but its industrialization was seriously hindered by the high cost. Immobilization of enzyme to realize its recycling is one solution; however, how to capture and hydrolyze the insoluble cellulose effectively via the immobilization system remains challenging. Herein, inspired by the predation process of the sea anemone, a cost-effective biomimetic cellulase-loaded enzymatic film was constructed. The cellulase loaded on the film can adjust its spatial orientation freely, thus their catalytic centres can easily reach the surface of the cellulose to perform the "predation" process effectively. As a result, this immobilization system can largely increase the efficiency of the insoluble cellulose hydrolysis and can be recycled for at least 8 cycles without activities loss. Therefore, it can largely reduce the cost of the cellulose conversion in the industrial areas.


Assuntos
Anemone/química , Celulase/metabolismo , Celulose/metabolismo , Materiais Biomiméticos/química , Catálise , Celulase/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Hidrólise , Polietileno/química
9.
Water Sci Technol ; 83(4): 906-921, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33617497

RESUMO

Chitosan/organic rectorite (CTS/OREC) composites were prepared and characterized by Fourier transform infrared spectrometry and X-ray diffraction. Polyphenol oxidase (PPO) was immobilized on CTS/OREC by physical adsorption (APPO) and covalent binding (CPPO). Taguchi method was applied in the optimization of immobilization conditions resulting in the highest enzyme activity of 16.37 × 103 and 8.92 × 103U/g for APPO and CPPO, respectively. APPO enzyme activity was higher than that of CPPO, while CPPO showed the higher enzyme loading capacity than that of APPO. The removal percentage of phenolic compound, including phenol (PH), 4-chlorophenol (4-CP) and 2,4-dichlorophenol (2,4-DCP), by immobilized PPO was also explored. The results indicated that APPO was more efficient in phenolic compounds removal than CPPO. APPO contributed to a quick removal in the first hour, and the removal percentage of PH, 4-CP and 2,4-DCP could reach 69.3 ± 4.2%, 89.8 ± 2.5% and 93.8 ± 1.7% within 2 h, respectively. The order of removal percentage of phenolic compounds for both immobilized PPO was 2,4-DCP > 4-CP > PH. After 10 consecutive operations, the removal percentage of 2,4-DCP reached 73.2 ± 2.6% and 60.3 ± 1.5% for APPO and CPPO, respectively. The results introduced a novel support for PPO immobilization, and the immobilized PPO had great potential in wastewater treatment.


Assuntos
Quitosana , Silicatos de Alumínio , Catecol Oxidase , Enzimas Imobilizadas , Concentração de Íons de Hidrogênio , Minerais , Fenóis
10.
Sensors (Basel) ; 21(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572795

RESUMO

The integration of nanomaterials as electron mediators in electrochemical biosensors is taking on an essential role. Due to their high surface-to-volume ratio and high conductivity, metallic nanowires are an interesting option. In this paper, silver nanowires (AgNWs) were exploited to design a novel catechol electrochemical biosensor, and the benefits of increasing the aspect ratio of the electron mediator (nanowires vs. nanoparticles) were analyzed. Atomic force microscopy (AFM) studies have shown a homogeneous distribution of the enzyme along the silver nanowires, maximizing the contact surface. The large contact area promotes electron transfer between the enzyme and the electrode surface, resulting in a Limit of Detection (LOD) of 2.7 × 10-6 M for tyrosinase immobilized onto AgNWs (AgNWs-Tyr), which is one order of magnitude lower than the LOD of 3.2 × 10-5 M) obtained using tyrosinase immobilized onto silver nanoparticles (AgNPs-Tyr). The calculated KM constant was 122 mM. The simultaneous use of electrochemistry and AFM has demonstrated a limited electrochemical fouling that facilitates stable and reproducible detection. Finally, the biosensor showed excellent anti-interference characteristics toward the main phenols present in wines including vanillin, pyrogallol, quercetin and catechin. The biosensor was able to successfully detect the presence of catechol in real wine samples. These results make AgNWs promising elements in nanowired biosensors for the sensitive, stable and rapid voltammetric detection of phenols in real applications.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Nanofios , Catecóis , Elétrons , Enzimas Imobilizadas , Prata
11.
Food Chem ; 349: 129050, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33556730

RESUMO

The objective of this research was to evaluate the immobilization of the enzyme ß-galactosidase in a genipin-activated chitosan support. The influence of the number of spheres and substrate concentration on immobilization yield (IY) and enzyme activity (EA) was analyzed using experimental design. Thermal, operational and storage stabilities were assessed, and the enzymatic derivatives were characterized by thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The TGA showed that the enzymatic derivatives kept their thermal behavior, and the SEM images revealed smooth surfaces in all the spheres. The optimized conditions for the immobilization process were 4.57 mg·mL-1 of spheres and a substrate concentration of 10 mM (IY = 84.13%; EA = 24.97 U·g-1). Thermal stability was enhanced at 10 and 37 °C, enabling four successive cycles of lactose hydrolysis in diluted UHT milk. Therefore, the immobilized enzyme in genipin-activated chitosan has potential for lactose hydrolysis and applications in the food industry.


Assuntos
Quitosana/química , Enzimas Imobilizadas/química , Iridoides/química , Kluyveromyces/enzimologia , Leite/química , beta-Galactosidase/química , Animais , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrólise , Lactose/química , beta-Galactosidase/metabolismo
12.
Molecules ; 26(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525568

RESUMO

Cyclodextrins (CDs) and their derivatives have attracted significant attention in the pharmaceutical, food, and textile industries, which has led to an increased demand for their production. CD is typically produced by the action of cyclodextrin glycosyltransferase (CGTase) on starch. Owing to the relatively high cost of enzymes, the economic feasibility of the entire process strongly depends on the effective retention and recycling of CGTase in the reaction system, while maintaining its stability. CGTase enzymes immobilized on various supports such as porous glass beads or glyoxyl-agarose have been previously used to achieve this objective. Nevertheless, the attachment of biocatalysts on conventional supports is associated with numerous drawbacks, including enzyme leaching prominent in physical adsorption, reduced activity as a result of chemisorption, and increased mass transfer limitations. Recent reports on the successful utilization of metal-organic frameworks (MOFs) as supports for various enzymes suggest that CGTase could be immobilized for enhanced production of CDs. The three-dimensional microenvironment of MOFs could maintain the stability of CGTase while posing minimal diffusional limitations. Moreover, the presence of different functional groups on the surfaces of MOFs could provide multiple points for attachment of CGTase, thereby reducing enzyme loss through leaching. The present review focuses on the advantages MOFs can offer as support for CGTase immobilization as well as their potential for application in CD production.


Assuntos
Ciclodextrinas/química , Enzimas Imobilizadas/química , Glucosiltransferases/química , Estruturas Metalorgânicas/química , Glioxilatos/química , Sefarose/química , Amido/química
13.
J Dairy Sci ; 104(4): 3888-3898, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33589258

RESUMO

In this research, we explored various immobilized enzyme support materials, including the novel nylon-6 fiber membrane (NFM), and evaluated the increase in surface area and its effect on enzyme binding potential. We also manipulated incubation and reaction conditions and assessed the subsequent effects on activity and stability of ß-galactosidase, with comparisons between various solid support materials and free (dissolved) enzyme. Nylon-6 fiber membranes were created by electrospinning and were compared with other materials as solid supports for enzyme binding. The other materials included polyvinylidene fluoride 5-kDa nanofiltration dairy membranes, nylon-6 pellets, and silica glass beads. Scanning electron microscopy revealed the large surface area of NFM, which correlated with greater enzyme activity compared with the relatively flatter surfaces of the other solid support materials. Enzyme activity was measured spectrophotometrically with the color-changing substrate o-nitrophenyl-ß-d-galactopyranoside. Compared with the other solid supports, NFM had greater maximum enzyme binding potential. Across pH conditions ranging from 3.5 to 6.0 (including the optimal pH of 4.0-5.0), enzyme activity was maintained on the membrane-immobilized samples, whereas free enzyme did not maintain activity. Altering the storage temperature (4, 22, and 50°C) affected enzyme stability (i.e., the ability of the enzyme to maintain activity over time) of free and polyvinylidene fluoride membrane samples. However, NFM samples maintained stability across the varying storage temperatures. Increasing the immobilization solution enzyme concentration above the maximum enzyme binding capacity had no significant effect on enzyme stability for membrane-immobilized samples; however, both had lower mean stability than free enzyme by approximately 74%. With further development, ß-galactosidase immobilized on NFM or other membranes could be used in continuous processing in the dairy industry for a combination of filtration and lactose hydrolysis-creating products that are reduced in lactose and increased in sweetness, with no requirement for "added sugars" on the nutrition label and no enzyme listed as final product ingredient.


Assuntos
Enzimas Imobilizadas , Polímeros , Animais , Caprolactama/análogos & derivados , Concentração de Íons de Hidrogênio , Hidrólise , Lactose , Temperatura , beta-Galactosidase
14.
Food Chem ; 349: 129127, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33561794

RESUMO

Metal-organic structures (MOFs) have been designed for a wide range of applications due to their high porosity, large surface area, and flexibility. For the first time in this work, the successful immobilization of α-amylase is confirmed by the use of ZIF-8 as easy and good support. The morphology, functional groups, and chemical composition of the support and immobilized α-amylase were tested using different methods such as scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and thermal gravimetric analysis (TGA). The enzymatic activities of the immobilized olibanum-bovine serum albumin@zeolitic imidazolate frameworks nanocomposite (OLB/BSA@ZIF-8)-α-amylase were compared with the free one. The pH and thermal stability of the OLB/BSA@ZIF-8-α-amylase were significantly enhanced compared to the free enzyme. The OLB/BSA@ZIF-8-α-amylase displayed excellent long-term storage stability, which could protect more than 90% of the initial activity for 8 weeks. Besides, the OLB/BSA@ZIF-8-α-amylase had high reusability, which showed a high degree of activity (more than 81%) after 20 cycles. This is the first study that uses OLB/BSA@ZIF-8 nanocomposite as immobilizing support for the immobilization of α-amylase. Improved catalytic efficiency (Vmax/Km) values, reusability, and storage stability of immobilized α-amylase can make it suitable in industrial and biotechnological applications.


Assuntos
Enzimas Imobilizadas/química , Estruturas Metalorgânicas/química , Nanocompostos/química , Soroalbumina Bovina/química , alfa-Amilases/química , Animais , Biocatálise , Biotecnologia , Bovinos , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Porosidade , Zeolitas/química , alfa-Amilases/metabolismo
15.
Biosens Bioelectron ; 178: 112997, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33535157

RESUMO

Continuous monitoring of biological metabolites of interest necessitates sensors that are robust, versatile, miniaturizable, and reliable. Electrochemical biosensors have dominated the field of biosensors for decades due to their robust and inexpensive nature. Classically, these sensors use amperometric and voltammetric methods as the sensing modality. One of the greatest limitations with these methods is the dependence of the signal (current, i) on the electrode size, which can change with respect to time due to fouling. Here, we present open circuit potential, an electrochemical technique that is relatively insensitive to electrode size, as a reliable alternative to amperometric and voltammetric techniques for monitoring metabolites of interest. The sensor operates by trapping an oxidase enzyme in a chitosan hydrogel. The oxidase enzyme is required for metabolite specificity. When the oxidase enzyme meets its substrate, oxygen is consumed, and hydrogen peroxide is generated. Hydrogen peroxide generation dominates a half reaction at the platinum surface, resulting in a change in potential. Using the above criteria, we demonstrate the efficacy, long lifetime, sensitivity, and ease of fabrication of glucose sensors, and miniaturize the sensors from macro- to microelectrodes. Additionally, we demonstrate the ease with which this platform can be extended to detect other analytes in the form of a galactose sensor. Our results set a foundation for the generalized use of potentiometric sensors for a broad range of metabolites and applications.


Assuntos
Técnicas Biossensoriais , Glucose Oxidase , Hidrogéis , Técnicas Eletroquímicas , Enzimas Imobilizadas , Glucose , Oxirredutases
16.
Bioresour Technol ; 323: 124611, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33418354

RESUMO

α-L-Rhamnosidase (Rha) is a biotechnologically important enzyme that degrades biomass containing natural rhamnoside. Herein, the recombinant Rha was successfully immobilized on magnetic metal-organic frameworks (MOFs), and used to hydrolyze rutin. Magnetic MOFs were constructed by binding Cu2+ and PABA to the surface of Fe3O4 nanoparticles coated with a polydopamine film through coordinate covalent bonds, and the enzyme was attached to the MOFs using the cross-linking agents EDC/NHS. The immobilized enzyme Rha@MOF reached an activity of 25.09 U/g with a lower apparent Km value compared with the free enzyme. The conversion rate of 20 g/L rutin was 91.42%, corresponding to an isoquercitrin productivity of 12.78 g/L/h. Rha@MOF also exhibited significantly improved reusability; the conversion rate was still 73.55% after 30 cycles at 60 °C. These results indicated that the magnetic MOF-immobilized enzyme was a feasible biocatalyst for the conversion of flavonoids with low aqueous solubility.


Assuntos
Estruturas Metalorgânicas , Rutina , Enzimas Imobilizadas , Glicosídeo Hidrolases , Hidrólise , Fenômenos Magnéticos
17.
Nat Commun ; 12(1): 340, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436601

RESUMO

Multistep enzyme-catalyzed cascade reactions are highly efficient in nature due to the confinement and concentration of the enzymes within nanocompartments. In this way, rates are exceptionally high, and loss of intermediates minimised. Similarly, extended enzyme cascades trapped and crowded within the nanoconfined environment of a porous conducting metal oxide electrode material form the basis of a powerful way to study and exploit myriad complex biocatalytic reactions and pathways. One of the confined enzymes, ferredoxin-NADP+ reductase, serves as a transducer, rapidly and reversibly recycling nicotinamide cofactors electrochemically for immediate delivery to the next enzyme along the chain, thereby making it possible to energize, control and observe extended cascade reactions driven in either direction depending on the electrode potential that is applied. Here we show as proof of concept the synthesis of aspartic acid from pyruvic acid or its reverse oxidative decarboxylation/deamination, involving five nanoconfined enzymes.


Assuntos
Eletroquímica , Enzimas/metabolismo , Biocatálise , Eletrodos , Enzimas Imobilizadas/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Hidrodinâmica , Cinética , Nanopartículas/química , Nanoporos
18.
Bioresour Technol ; 324: 124689, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33450627

RESUMO

A method for specific immobilization of whole-cell with covalent bonds was developed through a click reaction between alkyne and azide groups. In this approach, magnetic nanoparticle Fe3O4@SiO2-NH2-alkyne was synthesized with Fe3O4 core preparation, SiO2 coating, and alkyne functionalization on the surface. The azides were successfully integrated onto the cell surface of the recombinant E. coli harboring glycerol dehydrogenase, which was employed as the model cell. The highest immobilization yield of 83% and activity recovery of 94% were obtained under the conditions of 0.67 mg mg-1 cell-support ratio, pH 6.0, temperature 45 °C, and 20 mM Cu2+ concentration. The immobilized cell showed good reusability, which remained over 50% of initial activity after 10 cycles of utilization. Its activity was 9.7-fold higher than that of the free cell at the condition of pH 8.0 and each optimal temperature. Furthermore, the immobilized cell showed significantly higher activity, operational stability, and reusability.


Assuntos
Enzimas Imobilizadas , Nanopartículas de Magnetita , Azidas , Química Click , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Polissacarídeos , Dióxido de Silício
19.
ACS Appl Mater Interfaces ; 13(2): 2179-2188, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33405501

RESUMO

The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance (T50, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.


Assuntos
Hidrolases de Éster Carboxílico/farmacologia , Erwinia amylovora/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Nanosferas/química , Doenças das Plantas/prevenção & controle , Percepção de Quorum/efeitos dos fármacos , Acil-Butirolactonas/metabolismo , Hidrolases de Éster Carboxílico/administração & dosagem , Hidrolases de Éster Carboxílico/genética , Evolução Molecular Direcionada/métodos , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/farmacologia , Erwinia amylovora/fisiologia , Modelos Moleculares , Peptídeos/química , Doenças das Plantas/microbiologia , Pyrus/microbiologia
20.
ACS Appl Mater Interfaces ; 13(5): 6168-6179, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33499600

RESUMO

Developing novel immobilization methods to maximize the catalytic performance of enzymes has been a permanent pursuit of scientific researchers. Engineered Escherichia coli biofilms have attracted great concern as surface display platforms for enzyme immobilization. However, current biological conjugation methods, such as the SpyTag/SpyCatcher tagging pair, that immobilize enzymes onto E. coli biofilms seriously hamper enzymatic performance. Through phage display screening of lipase-binding peptides (LBPs) and co-expression of CsgB (nucleation protein of curli nanofibers) and LBP2-modified CsgA (CsgALBP2, major structural subunit of curli nanofibers) proteins, we developed E. coli BL21::ΔCsgA-CsgB-CsgALBP2 (LBP2-functionalized) biofilms as surface display platforms to maximize the catalytic performance of lipase (Lip181). After immobilization onto LBP2-functionalized biofilm materials, Lip181 showed increased thermostability, pH, and storage stability. Surprisingly, the relative activity of immobilized Lip181 increased from 8.43 to 11.33 U/mg through this immobilization strategy. Furthermore, the highest loading of lipase on LBP2-functionalized biofilm materials reached up to 27.90 mg/g of wet biofilm materials, equivalent to 210.49 mg/g of dry biofilm materials, revealing their potential as a surface with high enzyme loading capacity. Additionally, immobilized Lip181 was used to hydrolyze phthalic acid esters, and the hydrolysis rate against dibutyl phthalate was up to 100%. Thus, LBP2-mediated immobilization of lipases was demonstrated to be far more advantageous than the traditional SpyTag/SpyCatcher strategy in maximizing enzymatic performance, thereby providing a better alternative for enzyme immobilization onto E. coli biofilms.


Assuntos
Biofilmes , Escherichia coli/metabolismo , Lipase/metabolismo , Peptídeos/metabolismo , Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lipase/química , Estrutura Molecular , Tamanho da Partícula , Peptídeos/química , Ligação Proteica , Propriedades de Superfície
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