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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
Int J Biol Macromol ; 172: 270-280, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33418049

RESUMO

Enzyme immobilization can increase enzyme reusability to reduce cost of industrial production. Ginkgo biloba leaf extract is commonly used for medical purposes, but it contains ginkgolic acid, which has negative effects on human health. Here, we report a novel approach to solve the problem by degrading the ginkgolic acid with immobilized-laccase, where core/shell composite nanoparticles prepared by coaxial electrospraying might be first applied to enzyme immobilization. The core/shell Fe3O4/nylon 6,6 composite nanoparticles (FNCNs) were prepared using one-step coaxial electrospraying and can be simply recovered by magnetic force. The glutaraldehyde-treated FNCNs (FNGCNs) were used to immobilize laccase. As a result, thermal stability of the free laccase was significantly improved in the range of 60-90 °C after immobilization. The laccase-immobilized FNGCNs (L-FNGCNs) were applied to degrade the ginkgolic acids, and the rate constants (k) and times (τ50) were ~0.02 min-1 and lower than 39 min, respectively, showing good catalytic performance. Furthermore, the L-FNGCNs exhibited a relative activity higher than 0.5 after being stored for 21 days or reused for 5 cycles, showing good storage stability and reusability. Therefore, the FNGCNs carrier was a promising enzyme immobilization system and its further development and applications were of interest.


Assuntos
Óxido Ferroso-Férrico/química , Proteínas Fúngicas/química , Ginkgo biloba/química , Lacase/química , Nanopartículas de Magnetita/química , Salicilatos/química , Reagentes para Ligações Cruzadas/química , Técnicas Eletroquímicas , Enzimas Imobilizadas/química , Enzimas Imobilizadas/isolamento & purificação , Reutilização de Equipamento , Proteínas Fúngicas/isolamento & purificação , Glutaral/química , Hidrólise , Cinética , Lacase/isolamento & purificação , Nanopartículas de Magnetita/ultraestrutura , Nylons/química , Extratos Vegetais/química , Folhas de Planta/química , Polyporaceae/química , Polyporaceae/enzimologia
11.
Molecules ; 26(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430075

RESUMO

Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (kcat/Km) and the Michaelis-Menten constant (Km) of immobilized lipoxygenase were 21% and 49% of kcat/Km and Km of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest.


Assuntos
Enzimas Imobilizadas/química , Lipoxigenase/química , Oryza/química , Dióxido de Silício/química , Proteínas de Soja/química , Soja/enzimologia , Catálise
12.
Int J Biol Macromol ; 170: 583-592, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385453

RESUMO

The present work pronounces the three phase partitioning (TPP)-facilitated preparation of porous cross-linked Candida antarctica lipase B (CaLB) aggregates (pCLEAs) for 5-Hydroxymethylfurfural (HMF) esters synthesis. CLEAs and pCLEAs of CaLB were prepared with eupergit as the support under the optimized conditions of pH 8.0, eupergit/protein ratio of 3.0:1.0, 50 mM cross-linker concentration and 3.3 mg/mL BSA concentration in 4 h. The optimum starch concentration for pCLEAs was 0.20%, m/v. The maximum biocatalytic load was 650 U/g (CLEAs) and 721 U/g (pCLEAs), and the immobilized biocatalysts were stable over a pH range of 6.0-9.0 and temperature range of (40-60)°C. The BET surface area of CLEAs and pCLEAs were 21.3 and 29.1 m2/g, respectively, and the catalytic efficiency of pCLEAs was 2.2-fold higher than that of CLEAs. Subsequently, the pCLEAs of CaLB were utilized for the manufacturing of industrially significant HMF esters. Under the optimized transesterification conditions, HMF conversion with pCLEAs CaLB was 1.41- and 1.25-fold higher than with free and CLEAs CaLB, respectively. The pCLEAs were reused upto 8 consecutive transesterification cycles and the produced HMF esters reduced the surface tension of water from 72 mN/m to 32.6 mN/m, proving its potential application as surface-active compounds.


Assuntos
Reagentes para Ligações Cruzadas/química , Esterificação/efeitos dos fármacos , Proteínas Fúngicas/química , Furaldeído/análogos & derivados , Lipase/química , Biocatálise/efeitos dos fármacos , Catálise/efeitos dos fármacos , Enzimas Imobilizadas/química , Ésteres/química , Furaldeído/química , Concentração de Íons de Hidrogênio , Porosidade , Amido/química , Tensoativos/química
13.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498198

RESUMO

Biocatalysts represent an efficient, highly selective and greener alternative to metal catalysts in both industry and academia. In the last two decades, the interest in biocatalytic transformations has increased due to an urgent need for more sustainable industrial processes that comply with the principles of green chemistry. Thanks to the recent advances in biotechnologies, protein engineering and the Nobel prize awarded concept of direct enzymatic evolution, the synthetic enzymatic toolbox has expanded significantly. In particular, the implementation of biocatalysts in continuous flow systems has attracted much attention, especially from industry. The advantages of flow chemistry enable biosynthesis to overcome well-known limitations of "classic" enzymatic catalysis, such as time-consuming work-ups and enzyme inhibition, as well as difficult scale-up and process intensifications. Moreover, continuous flow biocatalysis provides access to practical, economical and more sustainable synthetic pathways, an important aspect for the future of pharmaceutical companies if they want to compete in the market while complying with European Medicines Agency (EMA), Food and Drug Administration (FDA) and green chemistry requirements. This review focuses on the most recent advances in the use of flow biocatalysis for the synthesis of active pharmaceutical ingredients (APIs), pharmaceuticals and natural products, and the advantages and limitations are discussed.


Assuntos
Biocatálise , Química Verde/métodos , Compostos Fitoquímicos/síntese química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Química Verde/instrumentação
14.
Carbohydr Polym ; 256: 117511, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33483032

RESUMO

A combined enzymatic treatment/acid hydrolysis technique was utilized to synthesize cellulose nanocrystals (CNCs) from sugar beet pulp. CNCs were functionalized with magnetite nanoparticles and dopamine making a versatile nano-carrier (DA/Fe3O4NPs@CNCs) for covalent enzyme immobilization. Oxygene/amine functionalities, high magnetization value, and specific surface area of DA/Fe3O4NPs@CNCs made it a reusable and green candidate for conjugation to hydrolytic enzyme cocktails (three cellulases, two hemicellulases, and their combinations) to prepare an innovative and practical nano-biocatalyst for biomass conversion. The conjugated enzymes showed an enhanced optimum temperature (∼ 10 °C), improved thermal stability, and shifted optimum pH toward alkaline pHs. Covalent attachment could successfully suppress the enzyme leaching and provide easy recovery/reuse of the nano-biocatalyst up to 10 cycles, with > 50% of initial activity. Application of the nano-biocatalyst in hydrolysis of rice straw and sugar beet pulp showed an increase (20-76%) in the yield of fermentable sugars compared to the free enzyme cocktails.


Assuntos
Celulose/química , Dopamina/química , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Nanotecnologia/métodos , Açúcares/química , Beta vulgaris/química , Biomassa , Estabilidade Enzimática , Fermentação , Humanos , Concentração de Íons de Hidrogênio , Cinética , Nanopartículas de Magnetita/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Raízes de Plantas/química , Temperatura
15.
ACS Appl Mater Interfaces ; 13(3): 4146-4155, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33440928

RESUMO

Immobilization can be used to improve the stability of lipases and enhances lipase recovery and reusability, which increases its commercial value and industrial applications. Nevertheless, immobilization frequently causes conformational changes of the lipases, which decrease lipase catalytic activity. in the present work, we synthesized UIO-66 and grafted UIO-66 crystals with proline for immobilization of Candida rugosa lipase (CRL). As indicated by steady-state fluorescence microscopy, grafting of proline onto UIO-66 crystals induced beneficial conformational change in CRL. CRL immobilized on UIO-66/Pro (CRL@UIO-66/Pro) demonstrated higher enzyme activity and better recyclability than that immobilized on UIO-66 (CRL@UIO-66) in both hydrolysis (CRL@UIO-66/Pro: 0.34 U; CRL@UIO-66: 0.15 U) and transesterification (CRL@UIO-66/Pro: 0.93 U; CRL@UIO-66: 0.25 U) reactions. The higher values of kcat and kcat/Km of CRL@UIO-66/Pro also showed that it had better catalytic efficiency as compared to CRL@UIO-66. It is also worth noting that CRL@UIO-66/Pro (0.93 U) demonstrated a much higher transesterification activity as compared to free CRL (0.11 U), indicating that UIO-66/Pro has increased the solvent stability of CRL. Both CRL@UIO-66 and CRL@UIO-66/Pro were also used for the fabrication of biosensors for nitrofen with a wide linear range (0-100 µM), lower limit of detection, and good recovery rate.


Assuntos
Lipase/química , Compostos Organometálicos/química , Praguicidas/análise , Éteres Fenílicos/análise , Ácidos Ftálicos/química , Prolina/análogos & derivados , Saccharomycetales/enzimologia , Técnicas Biossensoriais/métodos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Limite de Detecção , Modelos Moleculares
16.
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
17.
ACS Appl Mater Interfaces ; 13(5): 6081-6090, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33504155

RESUMO

Matrix metalloproteinases (MMPs) play an important role in tumor progression. The study of dynamic MMPs activity at the single-cell level can dissect tumor heterogeneity in the time domain and facilitate finding out more efficient clinical solutions for tumor treatment. Due to the fluidity of the carrier oil, the existing droplet-based methods for single-cell MMP analysis rarely have the capability to track proteolytic assays in droplets continuously. Therefore, we describe a thermosetting oil for real-time monitoring of MMP assays in droplets, which can immobilize droplets by transforming into solid after droplet generation. The solidification of this oil can be accomplished in 33 min at 37 °C, basing on the hydrosilation of vinyl silicone oil and hydrosilicone oil without other inducers (e.g. UV, Ca2+). Through monitoring the MMP assays of single cells, the reaction rates can be calculated according to real-time fluorescent curves, showing significant cell heterogeneity in MMP activity. Moreover, the dynamic MMP activity reveals that some of the A549 cells transiently secrete MMP. In conclusion, the thermosetting oil enables immobilize droplets to achieve real-time monitoring of single-cell proteolytic activity without impairing the flexibility of droplet microfluidics and has a potential in other cell-based assays for providing dynamic information at high resolutions.


Assuntos
Metaloproteinases da Matriz/metabolismo , Técnicas Analíticas Microfluídicas , Óleos de Silicone/química , Análise de Célula Única , Temperatura , Células A549 , Sobrevivência Celular , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Metaloproteinases da Matriz/química , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , Proteólise , Análise de Célula Única/instrumentação , Propriedades de Superfície
18.
Int J Biol Macromol ; 169: 282-289, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33333097

RESUMO

Transphosphatidylation catalyzed by phospholipase D has gained increasing attention for producing phosphatidylserine (PS), which can be used in functional food and medicine. In this study, we investigated the effects of six signal peptides on the secretion of PLD (PLDsa) from Streptomyces antibioticus TCCC 21059 in the food-grade GRAS bacterium Bacillus subtilis. It indicated that the optimal signal peptide DacB with an Ala-X-Ala sequence motif at the C-terminus showed the highest secretory expression ability, resulting in increased production of 2.84 U/mL PLDsa. Then PLDsa was immobilized on the epoxy-based carriers, and one of these carriers allowed PLDsa loading of up to 2.7 mg/g. The immobilized PLDsa was more stable over a wide range of pH value (4.5-7.5) and temperature (16 °C-60 °C) than free PLDsa. Subsequently, the synthesis of PS from soybean phosphatidylcholine (PC) was carried out in purely aqueous solution using immobilized PLDsa, leading to a high yield of 65%. The immobilized PLDsa catalyst maintained a relative PS production of 60% after 5 recycles. Notably, the use of toxic solvent was completely eliminated in the whole process, which would be more profitable for the application of PS.


Assuntos
Bacillus subtilis/enzimologia , Fosfatidilserinas/biossíntese , Fosfolipase D/biossíntese , Bacillus subtilis/metabolismo , Secreções Corporais/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Fosfolipase D/química , Fosfolipase D/metabolismo , Sinais Direcionadores de Proteínas , Solventes , Temperatura , Água
19.
Int J Biol Macromol ; 169: 228-238, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33338531

RESUMO

Robust amylases with stability and catalysis at multitude of extremities are the need of an hour. Enzyme immobilization may prove beneficial at commercial scale to achieve such attributes. In the present study, a commercially available amylase was immobilized on graphene oxide (GO) - magnetite (Fe3O4) nanoparticles through covalent bonding. The structural and morphological characterizations were conducted by XRD, SEM and TEM. Further, FTIR and TGA confirmed the interaction between amylase, GO and nanoparticles. The variables, such as concentrations of GO (1.3 mg), Fe3O4 (58 µg), and amylase (4.5 mg) were optimized by the response surface methodology using central composite design. High loading capacity of 77.58 µg amylase over 1 µg GO-magnetite nanoparticles was achieved under optimum conditions. Biochemically, the pH optimum remained unaltered, i.e., pH 7, whereas, the alkalitolerance was increased by ~20% in relative activities upon immobilization. The half-life of soluble amylase was 13 h, which enhanced to 20 h upon immobilization in 20 mM phosphate buffer, pH 7 at 50 °C. Besides, the thermodynamic parameters supported the stability trends. The immobilized amylase could be used for 11 subsequent cycles. The mentioned attributes and the dextrose equivalent values during the production of high maltose containing syrup highlighted its commercialization.


Assuntos
Nanopartículas de Magnetita/química , Maltose/química , alfa-Amilases/isolamento & purificação , Amilases/química , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Grafite/química , Concentração de Íons de Hidrogênio , Cinética , Temperatura , Termodinâmica , alfa-Amilases/química , beta-Amilase/química
20.
Int J Biol Macromol ; 169: 239-250, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33345972

RESUMO

In this study, SBA-15 was modified by halogen & haloalkanes and later used to immobilize lipases. The hydrolysis activity and the glycerolysis performance of the immobilized lipases was carefully studied. Highest activity of the immobilized Candida antarctica lipase B (CALB), Lipase from Aspergillus oryzae (AOL), Lecitase® Ultra (LU) and lipase from Rhizomucor miehei (RML) was respectively at 5577, 12000, 2822 and 11,577 U/g; in addition, the highest activity was obtained from the lowest or moderate lipase loading, at 25.73, 90.72, 89.52 and 30.56 mg/g respectively. The mechanism of lipase immobilization was studied and it was through interfacial activation. The halogen & haloalkanes modification of SBA-15 afforded considerable glycerolysis activity for diacylglycerols (DAG) preparation. CALB@SBA-15-CH2CH2(CF2)5CF3 and CALB@SBA-15-CH2CH2(CF2)7CF3 were suitable for DAG production, they both exhibited good reusability in glycerolysis reaction, with 117.36% and 93.06% of their initial glycerolysis activity retained respectively after ten cycles of reuse. The relationships between temperature with triacylglycerols (TAG) conversion were lnV0 = 3.13-3.07/T and lnV0 = 7.90-4.64/T respectively for CALB@SBA-15-CH2CH2(CF2)5CF3 and CALB@SBA-15-CH2CH2(CF2)7CF3; in addition, their activation energy (Ea) was respectively at 25.50 and 38.54 kJ/mol.


Assuntos
Halogênios/química , Lipase/isolamento & purificação , Dióxido de Silício/química , Basidiomycota , Biocatálise , Candida , China , Diglicerídeos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Proteínas Fúngicas , Hidrólise , Lipase/química , Rhizomucor , Temperatura , Termodinâmica , Triglicerídeos
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