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 de 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/enzimologiaRESUMO
The design of interfaces that selectively react with molecules to transform them into compounds of industrial interest is an emerging area of research. An example of such reactions is the hydrolytic conversion of ester-based molecules to lipids and alcohols, which is of interest to the food, and pharmaceutical industries. In this study, a functional bio-interfaced layer was designed to hydrolyze 4-nitrophenyl acetate (pNPA) and Ricinus Communis (castor) oil rich in triglycerides using lipase b from Candida antarctica (CALB, EC 3.1.1.3). The attachment of CALB was performed via non-covalent immobilization over a polymer film of vertically aligned cylinders that resulted from the self-assembly of the di-block copolymer polystyrene-block-poly(4-vinyl pyridine) (PS-b-P4VP). This polymer-lipase model will serve as the groundwork for the design of further bioactive layers for separation applications requiring similar hydrolytic processes. Results from the fabricated functional bio-interfaced material include cylinders with featured pore size of 19â¯nm, d spacing of 34â¯nm, and ca. 40â¯nm of thickness. The polymer-enzyme layers were physically characterized using AFM, XPS, and FTIR. The immobilized enzyme was able to retain 91% of the initial enzymatic activity when using 4-nitrophenyl acetate (pNPA) and 78% when exposed to triglycerides from castor oil.
Assuntos
Poluentes Ambientais/química , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Lipase/química , Nitrofenóis/química , Poliestirenos/química , Polivinil/química , Triglicerídeos/química , Candida/química , Candida/enzimologia , Óleo de Rícino/química , Enzimas Imobilizadas/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Humanos , Hidrólise , Lipase/isolamento & purificação , Porosidade , Ricinus/químicaRESUMO
Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/análise , Avaliação Pré-Clínica de Medicamentos/métodos , Enzimas Imobilizadas/química , Peptidil Dipeptidase A/química , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Benzilisoquinolinas/análise , Benzilisoquinolinas/química , Benzilisoquinolinas/farmacologia , Berberina/análogos & derivados , Berberina/análise , Berberina/química , Berberina/farmacologia , Cromatografia de Afinidade/economia , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Análise Custo-Benefício , Avaliação Pré-Clínica de Medicamentos/economia , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Enzimas Imobilizadas/isolamento & purificação , Células HEK293 , Humanos , Oligopeptídeos/química , Peptidil Dipeptidase A/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em Tandem/economia , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodosRESUMO
Treated silica xerogel with protic ionic liquid (PIL) and bifunctional agents (glutaraldehyde and epichlorohydrin) is a novel support strategy used in the effective immobilization of lipase from Burkholderia cepacia (LBC) by covalent binding. As biocatalysts with the highest activity recovery yields, LBC immobilized by covalent binding with epichlorohydrin without (203%) and with PIL (250%), was assessed by the following the hydrolysis reaction of olive oil and characterized biochemically (Michaelisâ»Menten constant, optimum pH and temperature, and operational stability). Further, the potential transesterification activity for three substrates: sunflower, soybean, and colza oils, was also determined, achieving a conversion of ethyl esters between 70 and 98%. The supports and the immobilized lipase systems were characterized using Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), elemental analysis, and thermogravimetric (TG) analysis.
Assuntos
Proteínas de Bactérias/química , Enzimas Imobilizadas/química , Líquidos Iônicos/química , Lipase/química , Azeite de Oliva/química , Óleo de Soja/química , Óleo de Girassol/química , Proteínas de Bactérias/isolamento & purificação , Biocombustíveis/provisão & distribuição , Burkholderia cepacia/química , Burkholderia cepacia/enzimologia , Reagentes de Ligações Cruzadas/química , Enzimas Imobilizadas/isolamento & purificação , Epicloroidrina/química , Esterificação , Géis , Glutaral/química , Humanos , Concentração de Íons de Hidrogênio , Lipase/isolamento & purificação , Dióxido de Silício/química , TemperaturaRESUMO
Lipases are the most widely employed enzymes in commercial industries. The catalytic mechanism of most lipases involves a step called "interfacial activation". As interfacial activation can lead to a significant increase in catalytic activity, it is of profound importance in developing lipase immobilization methods. To obtain a potential biocatalyst for industrial biodiesel production, an effective strategy for enhancement of catalytic activity and stability of immobilized lipase was developed. This was performed through the combination of interfacial activation with hybrid magnetic cross-linked lipase aggregates. This biocatalyst was investigated for the immobilization of lipase from Rhizomucor miehei (RML). Under the optimal conditions, the activity recovery of the surfactant-activated magnetic RML cross-linked enzyme aggregates (CLEAs) was as high as 2058%, with a 20-fold improvement over the free RML. Moreover, the immobilized RML showed excellent catalytic performance for the biodiesel reaction at a yield of 93%, and more importantly, could be easily separated from the reaction mixture by simple magnetic decantation, and retained more than 84% of its initial activities after five instances of reuse. This study provides a new and versatile approach for designing and fabricating immobilized lipase with high activation and stability.
Assuntos
Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Jatropha/química , Lipase/química , Óleos de Plantas/química , Rhizomucor/química , Biocatálise , Biocombustíveis , Ativação Enzimática , Enzimas Imobilizadas/isolamento & purificação , Reutilização de Equipamento , Esterificação , Proteínas Fúngicas/isolamento & purificação , Lipase/isolamento & purificação , Imãs , Agregados Proteicos , Rhizomucor/enzimologia , Tensoativos/químicaRESUMO
Tannase (Tan410) from a soil metagenomic library was immobilized on different supports, including mesoporous silica SBA-15, chitosan, calcium alginate, and amberlite IRC 50. Entrapment in calcium alginate beads was comparatively found to be the best method and was further characterized. The optimum pH of the immobilized Tan410 was shifted toward neutrality compared with the free enzyme (from pH 6.4 to pH 7.0). The optimum temperature was determined to be 45°C for the immobilized enzyme and 30°C for the free enzyme, respectively. The immobilized enzyme had no loss of activity after 10 cycles, and retained more than 90% of its original activity after storage for 30 days. After immobilization, the enzyme activity was only slightly affected by Hg(2+), which completely inhibited the activity of the free enzyme. The immobilized tannase was used to remove 80% of tannins from a green tea infusion on the first treatment. The beads were used for six successive runs resulting in overall hydrolysis of 56% of the tannins.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Enzimas Imobilizadas/metabolismo , Biblioteca Gênica , Metagenoma , Taninos/metabolismo , Chá/química , Biotransformação , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Concentração de Íons de Hidrogênio , Mercúrio/metabolismo , Microesferas , Microbiologia do Solo , TemperaturaRESUMO
Ni-NTA functionalized iron oxide magnetic nanoparticle was synthesized and used to selectively immobilize a his-tagged enzyme from cell free extract as an active and recyclable nanobiocatalyst, where purification and immobilization of the target enzyme were accomplished in one pot.
Assuntos
Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Magnetismo , Nanopartículas/química , Níquel/química , Ácido Nitrilotriacético/química , Enzimas Imobilizadas/isolamento & purificação , Epóxido Hidrolases/química , Epóxido Hidrolases/isolamento & purificação , Epóxido Hidrolases/metabolismo , Modelos Moleculares , Conformação Proteica , Solanum tuberosum/enzimologiaRESUMO
A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (ß-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Ressonância de Plasmônio de Superfície/métodos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/isolamento & purificação , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/isolamento & purificação , Cálcio/metabolismo , Linhagem Celular , Clonagem Molecular , Inibidores Enzimáticos/química , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Enzimas Imobilizadas/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Modelos MolecularesRESUMO
The extracellular medium-chain-length polyhydroxyalkanoate (MCL-PHA) depolymerase of Pseudomonas fluorescens GK13 catalyzes the hydrolysis of poly(3-hydroxyoctanoic acid) [P(3HO)]. Based on the strong tendency of the enzyme to interact with hydrophobic materials, a low-cost method which allows the rapid and easy purification and immobilization of the enzyme has been developed. Thus, the extracellular P(3HO) depolymerase present in the culture broth of cells of P. fluorescens GK13 grown on mineral medium supplemented with P(3HO) as the sole carbon and energy source has been tightly adsorbed onto a commercially available polypropylene support (Accurel MP-1000) with high yield and specificity. The activity of the pure enzyme was enhanced by the presence of detergents and organic solvents, and it was retained after treatment with an SDS-denaturing cocktail under both reducing and nonreducing conditions. The time course of the P(3HO) hydrolysis catalyzed by the soluble and immobilized enzyme has been assessed, and the resulting products have been identified. After 24 h of hydrolysis, the dimeric ester of 3-HO [(R)-3-HO-HO] was obtained as the main product of the soluble enzyme. However, the immobilized enzyme catalyzes almost the complete hydrolysis of P(3HO) polymer to (R)-3-HO monomers under the same conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Caprilatos/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Pseudomonas fluorescens/enzimologia , Proteínas de Bactérias/isolamento & purificação , Hidrolases de Éster Carboxílico/isolamento & purificação , Meios de Cultura/química , Enzimas Imobilizadas/isolamento & purificação , Enzimas Imobilizadas/metabolismo , Fatores de TempoRESUMO
Insoluble concanavalin A-beta galactosidase complex was obtained by using jack bean extract and this complex was crosslinked with glutaraldehyde, in order to maintain the integrity of complex in the presence of its substrate or products. Concanavalin A-beta galactosidase complex retained 92% of the initial enzyme activity whereas crosslinked complex showed 88% activity. Entrapment of concanavalin A-beta galactosidase complex into calcium alginate beads provided suitability to use this preparation in reactors. Temperature- and pH-optima of the various immobilized beta galactosidase preparations were the same as its soluble counterpart. Entrapped crosslinked concanavalin A-beta galactosidase complex retained more than 50% activity after 1h exposure with 4.0 M urea at room temperature. Moreover, entrapped crosslinked concanavalin A-beta galactosidase complex retained 81 and 62% of the original enzymatic activity in the presence of 5% calcium chloride and 5% galactose, respectively. Entrapped crosslinked concanavalin A-beta galactosidase complex preparation was more superior in the continuous hydrolysis of lactose in a batch process as compared to the other entrapped preparations. This entrapped crosslinked concanavalin A-beta galactosidase complex retained 95% activity after seventh repeated use and 93% of its original activity even after 2 months storage at 4 degrees C.
Assuntos
Aspergillus oryzae/enzimologia , beta-Galactosidase/isolamento & purificação , Alginatos , Cloreto de Cálcio/farmacologia , Canavalia , Concanavalina A/química , Reagentes de Ligações Cruzadas , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/isolamento & purificação , Enzimas Imobilizadas/metabolismo , Galactose/farmacologia , Ácido Glucurônico , Glutaral , Ácidos Hexurônicos , Concentração de Íons de Hidrogênio , Hidrólise , Lactose/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/isolamento & purificação , Substâncias Macromoleculares/metabolismo , Extratos Vegetais/química , Desnaturação Proteica/efeitos dos fármacos , Temperatura , Ureia/farmacologia , beta-Galactosidase/química , beta-Galactosidase/metabolismoRESUMO
Polypropylene powders as the adsorbent for organic solution containing n-hexadecane and olive oil were employed as the carbon source for producing an alkaline lipase from Acinetobacter radioresistens. The best volumetric ratio of n-hexadecane to olive oil around 5 for lipase production was determined from shake-flask and fermentation cultivations. The existence of a maximum time course lipase activity of the aqueous phase was attributed to the compensation effects of olive oil on cell growth and lipase production, repression of lipase synthesis by oleic acid, and lipase adsorption on the supports. A linear relationship between the average cell growth rate in the exponential phase and the ratio of surface areas of the supports was found. The benefits of using the present fermentation process include less foaming and emulsion of the broth, less organic phase used, higher lipase production, and easy recovery of the lipase in the centrifugation step.
Assuntos
Acinetobacter/enzimologia , Acinetobacter/crescimento & desenvolvimento , Lipase/biossíntese , Lipase/isolamento & purificação , Acinetobacter/classificação , Alcanos/metabolismo , Carbono , Células Cultivadas , Ativação Enzimática , Enzimas Imobilizadas/biossíntese , Enzimas Imobilizadas/isolamento & purificação , Membranas Artificiais , Azeite de Oliva , Óleos de Plantas/metabolismo , Polipropilenos , Pós , Controle de QualidadeRESUMO
The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.
Assuntos
Enzimas Imobilizadas/isolamento & purificação , Mananas/isolamento & purificação , Marcadores de Afinidade , Biotecnologia , Precipitação Química , Concanavalina A , Fabaceae , Glucana 1,4-alfa-Glucosidase/isolamento & purificação , Glicosídeo Hidrolases/isolamento & purificação , Glicosilação , Lectinas , Mananas/química , Lectinas de Plantas , Plantas Medicinais , beta-FrutofuranosidaseRESUMO
A heterogeneous multienzyme preparation with the peptidase activity, isolated from the cells of Pseudomonadacea bacteria, was immobilized on alumina. The specific activity of the immobilized enzyme complex is not a simple function of the bound protein quantity, but depends on immobilization conditions. An additional glutaraldehyde treatment results in higher thermostability of the immobilized enzyme preparation. The substrate specificity of the preparation retains after immobilization, and it becomes less sensitive to pH changes.