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1.
Nat Commun ; 11(1): 1049, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103000

RESUMO

Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 µM myoglobin and 5 µg mL-1 trypsin is electrosonically sprayed (-3 kV) from a homemade setup to produce tiny (∼9 µm) microdroplets, we obtain 100% sequence coverage in less than 1 ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 °C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (∼60 µm) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (∼148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development.


Assuntos
Hormônio Adrenocorticotrópico/análise , Mioglobina/análise , Análise de Sequência de Proteína/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Trastuzumab/análise , Hormônio Adrenocorticotrópico/metabolismo , Enzimas Imobilizadas/química , Humanos , Mioglobina/metabolismo , Trastuzumab/metabolismo , Tripsina/metabolismo
2.
Chemosphere ; 246: 125676, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31918078

RESUMO

Covalent-immobilization of the laccase enzyme onto the iron oxide nanoparticles was achieved using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) as cross-linkers. The presence of sulphur moeity in the laccase immobilized nanoparticles (LNPs) observed through Scanning Electron Microscopy- Energy dispersive X-ray spectroscopy (SEM-EDS) spectra confirmed the immobilization of laccase enzyme. The TEM analysis of iron oxide nanoparticles (FNPs), chitosan coated iron nanoparticles (CNPs) and laccase immobilized nanoparticles (LNPs) confirmed their sizes around 12, 15 and 20 nm, respectively. The effect of LNPs in degrading chlorpyrifos under field conditions was studied by simulating the conditions in a column. Column A, which was used as control showed more leaching of chlorpyrifos as compared to column B containing LNPs. The sorption coefficient (Kd) value obtained for control (column A) and LNPs containing column B were 21.6 and 112.3 L/kg, respectively. LNPs altered the Kd values of soil thereby showing lesser leaching potential. Higher the Kd value, lesser will be the leaching potential in the ground water. Copper in laccase enzyme resulted in hydrolysis of chlorpyrifos. Chitosan used for coating on FNPs and soil organic matter resulted in the adsoption of chlorpyrifos. Current results will allow a better assessment of the role of LNPs as a competent deterrent in chlorpyrifos mobility and degradation.


Assuntos
Clorpirifos/análise , Lacase/química , Nanopartículas/química , Poluentes do Solo/química , Quitosana/química , Clorpirifos/metabolismo , Cobre , Enzimas Imobilizadas/química , Compostos Férricos , Lacase/metabolismo , Solo
3.
J Agric Food Chem ; 68(5): 1373-1381, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31927950

RESUMO

Most of the microorganisms can form biofilms, which makes biofilms an abundant bioresource to be exploited. Due to the limitations of the application of current immobilization methods for biofilms, we developed an immobilization method called the biofilm polysaccharide display (BPD) strategy while maintaining the native biofilm structure and catalytic microenvironment of Clostridium acetobutylicum B3. Lipase Lip181 showed significant improvements in stability after chemical immobilization. For example, immobilized Lip181 retained 74.23% of its original activity after incubation for 14 days, while free Lip181 was totally deactivated. In addition, immobilized Lip181 maintained high residual activity (pH 5.0-11.0), which showed improved resistance to pH changes. Notably, this method did not decrease but slightly increased the relative activity of Lip181 from 6.39 to 6.78 U/mg. Immobilized Lip181 was used to prepare cinnamyl acetate, and it showed a maximum yield of 85.09%. Overall, this biofilm immobilization method may promote the development of biocatalytic and biofilm materials.


Assuntos
Materiais Biocompatíveis/química , Biofilmes , Clostridium acetobutylicum/química , Lipase/metabolismo , Polissacarídeos/química , Biocatálise , Clostridium acetobutylicum/fisiologia , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lipase/química , Polissacarídeos/metabolismo
4.
Chem Commun (Camb) ; 56(13): 2004-2007, 2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-31960849

RESUMO

The operation of wearable epidermal biofuel cells is prone to rapid irreversible deactivation effects under dynamic sweat pH changes from neutral to acidic. We demonstrate that the encapsulation of lactate-oxidase (LOx) within a hydrophobic protective carbon-paste anode imparts unusually high stability during dynamically changing pH fluctuations and allows the BFC to continue harvesting the lactate bioenergy even after long exposures to acidic conditions. The unique power-recovery ability of the carbon-paste BFC after its failure in harsh pH is attributed to the protective action of the non-polar paste environment.


Assuntos
Fontes de Energia Bioelétrica , Técnicas Biossensoriais/métodos , Glucose Oxidase/metabolismo , Carbono/química , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ácido Láctico/química , Suor/química , Dispositivos Eletrônicos Vestíveis
5.
Food Chem ; 310: 125741, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31806389

RESUMO

Polyphenol oxidase (PPO), also known as tyrosinase and catechol oxidase, is the enzyme responsible for enzymatic browning in foods. It causes undesirable organoleptic, nutritional and colour changes. Here, we report the preparation of five nanomaterials and a study of their ability to modulate PPO enzyme activity. The materials consist of UVM-7 supports (a mesoporous silica material) modified with diverse functional groups (i.e. amine, carboxylic acid, isocyanate, alkane and pyridine). We also studied the PPO immobilisation capability of the materials. All the materials, except the carboxylic acid functionalised one, offer high PPO loading capabilities and the immobilisation speed increases with functionalisation. Nevertheless, only a minor effect of the inhibition of enzymatic browning was produced. Furthermore, the amine containing material was able to capture not only PPO, but also the oxidation products. Such behaviour was validated with fresh apple juice in which browning was avoided, even 90 min in the presence of oxygen at room temperature.


Assuntos
Catecol Oxidase/química , Catecol Oxidase/metabolismo , Sucos de Frutas e Vegetais , Nanoestruturas/química , Dióxido de Silício/química , Ácidos Carboxílicos/química , Ácido Edético/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Malus/química , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Piridinas/química , Propriedades de Superfície
6.
J Agric Food Chem ; 68(1): 242-249, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31668065

RESUMO

n-3 polyunsaturated fatty acid (PUFA)-rich lysophosphatidylcholine (LPC) with many beneficial effects was effectively synthesized by immobilized MAS1 lipase-catalyzed esterification of n-3 PUFA with sn-glycero-3-phosphatidylcholine (GPC) under vacuum in a solvent-free system. Immobilized MAS1 lipase was found to be a more suitable catalyst for the production of n-3 PUFA-rich LPC when compared with Novozym 435. The maximal GPC conversion and LPC content (93.12% and 90.77 mol %) were obtained under the optimized conditions (enzyme loading of 300 U/g substrate, temperature of 55 °C, and n-3 PUFA/GPC molar ratio of 20:1). Moreover, it was observed that 1-acyl-sn-glycero-3-lysophosphatidylcholine (sn-1 acyl LPC) was the main reaction product, as demonstrated by molecular docking. These results showed that immobilized MAS1 lipase had high phospholipase activity with a predominant specificity for the sn-1 hydroxyl position of GPC to efficiently synthesize highly pure n-3 PUFA-rich LPC from GPC for industrial application.


Assuntos
Proteínas de Bactérias/química , Ácidos Graxos Ômega-3/química , Lipase/química , Lisofosfatidilcolinas/química , Streptomyces/enzimologia , Biocatálise , Enzimas Imobilizadas/química , Esterificação
7.
J Photochem Photobiol B ; 202: 111675, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733612

RESUMO

The photofunctionalization of three different carbons with two proteins was studied at room temperature. Water solutions of bovine serum albumin, BSA, and α-amylase, AA, were photolyzed at 21 °C in the presence of graphite microparticles (6.20 µm), MPG, graphene oxide, MPGO, and graphene oxide modified with SO2, mMPGO. The insertion of BSA on carbon matrixes occurred with a deoxygenation reaction, most likely due to a dehydration step of a water molecule. XPS, TOC and TGA, showed that the BSA photo-insertion on MPG was highly efficient with 34.9% of the weight of MPG after photolysis, with an initial concentration of 1 g∙L-1 of BSA. A high yield of AA photoinsertion on the carbons was also obtained. The calculated weight of AA inserted on MPG and MPGO after photolysis was 22.30% and 18.08%, respectively, with respect to the initial weight of carbon, when the initial concentration of AA was 60 mg∙L-1. AA immobilized on MPG was active while the enzyme on MPGO showed a smaller activity, within the experimental error. Although a certain extent of denaturalization of both proteins was observed during photolysis, the molecular weight and composition changed very little during the photolysis, which would produce mainly conformational changes and isomerization reactions.


Assuntos
Carbono/química , Soroalbumina Bovina/química , alfa-Amilases/química , Animais , Bovinos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Grafite/química , Luz , Fotólise/efeitos da radiação , Dióxido de Silício/química , Água/química , alfa-Amilases/metabolismo
8.
J Sci Food Agric ; 100(4): 1426-1435, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31710696

RESUMO

BACKGROUND: In this study, SBA-15 was functionalized by silane coupling reagents, then lipase from Thermomyces lanuginosus (TLL) was immobilized onto the parent and the organically modified SBA-15 for diacylglycerol (DAG) production through glycerolysis. RESULTS: Diacylglycerol content of 54.77 ± 0.63%, and triacylglycerol (TAG) conversion of 77.75 ± 1.24%, were obtained from the parent SBA-15 supported TLL-mediated glycerolysis reaction in a solvent-free system. However, poor performance was unexpectedly observed when co-solvents were introduced into the reaction system. After organic modification, the functionalized SBA-15 supported TLL samples all exhibited reasonable performance, producing DAG content over 40 wt% and TAG conversion over 70 wt%. Higher DAG content, up to 59.19 ± 1.10%, was observed from the phenyl group-modified SBA-15 supported TLL. The operational stability of the immobilized TLL samples in glycerolysis was also improved after organic functionalization. The phenyl group-modified SBA-15 supported TLL showed good reusability in the present glycerolysis reaction, and 95.21 ± 4.87% of the initial glycerolysis activity remained after five cycles of reuse. CONCLUSION: The organic modification of SBA-15 improved the catalytic performance of its supported TLL in glycerolysis, in terms of TAG conversion, DAG content, and reusability. © 2019 Society of Chemical Industry.


Assuntos
Ascomicetos/enzimologia , Diglicerídeos/química , Proteínas Fúngicas/química , Lipase/química , Dióxido de Silício/química , Biocatálise , Enzimas Imobilizadas/química , Triglicerídeos/química
9.
Talanta ; 206: 120171, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514875

RESUMO

The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl®.


Assuntos
Cromatografia Líquida/métodos , Enzimas Imobilizadas/química , Glicopeptídeos/análise , Animais , Bovinos , Gonadotropina Coriônica/análise , Gonadotropina Coriônica/química , Cromatografia Líquida/instrumentação , Glicopeptídeos/química , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Pepsina A/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteólise , Sefarose/química , Suínos , Espectrometria de Massas em Tandem/métodos , Tripsina/química
10.
Talanta ; 206: 120180, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514886

RESUMO

A novel analytical approach is proposed to discriminate between solid biopsies of chromophobe renal cell carcinoma (chRCC) and renal oncocytoma (RO). The method comprises the following steps: (i) ultrasonic extraction of proteins from solid biopsies, (ii) protein depletion with acetonitrile, (iii) ultrasonic assisted in-solution digestion using magnetic nanoparticle with immobilized trypsin, (iv) C18 tip-based preconcentration of peptides, (v) sequential extraction of the peptides with ACN, (vi) MALDI-snapshot of the extracts and (vii) investigation of the extract containing the most discriminating features using high resolution mass spectrometry. With this approach we have been able to differentially cluster renal oncocytoma and chromophobe renal cell carcinoma and identified 18 proteins specific to chromophobe and seven unique to renal oncocytoma. Chromophobes express proteins associated with ATP function (ATP5I & 5E; VATE1 & G2; ADT2), glycolysis (PGK1) and neuromedin whilst oncocytomas express ATP5H, ATPA, DEPD7 and TRIPB thyroid receptor interacting protein.


Assuntos
Adenoma Oxífilo/diagnóstico , Biomarcadores Tumorais/análise , Carcinoma de Células Renais/diagnóstico , Neoplasias Renais/diagnóstico , Rim/química , Fragmentos de Peptídeos/análise , Proteínas/análise , Acetonitrilos/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/isolamento & purificação , Biópsia , Diagnóstico Diferencial , Enzimas Imobilizadas/química , Feminino , Humanos , Rim/patologia , Nanopartículas de Magnetita/química , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas/química , Proteínas/isolamento & purificação , Proteômica/métodos , Extração em Fase Sólida/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tripsina/química , Ondas Ultrassônicas
11.
Carbohydr Polym ; 229: 115471, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826427

RESUMO

Micro/nano celluloses (MC)/NC) were magnetized by nanoγ-Fe2O3 into the nanoγ-Fe2O3@MC (NMMC) and nanoγ-Fe2O3@NC (NMMC) which oxidized to NMMCD and NMNCD dialdehydes for Schiff-base immobilization of urease as NMMCD/urease and NMMCD/urease. The relative enzyme-activity of the immobilized ureases were comparable with the free-urease, although 75%-80% of the enzyme activity preserved for NMMCD/urease and NMNCD/urease after six cycles. The compared catalytic activities of the NMMCD/urease and NMMCD/urease in Biginelli/Hantzsch reactions in water at 60 °C surprised us by 100% selectivity for the Biginelli product 3,4-dihydropyrimidin-2(1H)-one (DHPM1). With the superiority of NMNCD/urease, this high selectivity using immobilized ureases is owing to the admirable urease inhibitory of the formed Biginelli product DHPM1 by urea condensation instead of urea hydrolysis. The robust enzyme inhibitory of the DHPM1 for free urease was also confirmed by phenol red test to show the deactivation of enzyme for enzymatic hydrolysis of urea and ammonia production in water.


Assuntos
Celulose/química , Enzimas Imobilizadas/química , Ureia/química , Urease/química , Aspergillus niger/enzimologia , Catálise , Celulose/isolamento & purificação , Inibidores Enzimáticos/química , Enzimas Imobilizadas/antagonistas & inibidores , Compostos Férricos/química , Gossypium/química , Concentração de Íons de Hidrogênio , Fenômenos Magnéticos , Estabilidade Proteica , Temperatura , Urease/antagonistas & inibidores
12.
Chemosphere ; 240: 124882, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31726609

RESUMO

Biomimetic dynamic membrane (BDM) has been employed as a promising membrane separation technology regarding water/wastewater treatment (Model pollutant is methylene blue). Given its catalytic function on micro-pollutant removal and fouling control, detailed mechanism for impacts of fabrication method, carriers (CNT and GO) and laccase on the construction of biomimetic layer and enzyme immobilization have not been clear so far. In this work, the BDM performance with various fabrication methods, carriers and laccase were investigated and verified. The BDM fabrication tests demonstrated that BDM with mixed filtration method had better filtration performance (up to 120 L m-2 h-1 flux and 80% removal rate) than BDM with stepwise filtration method. Moreover, the laccases immobilized on GO exhibited a stronger laccase activity than those on CNT. Increasing CNT or GO dosage strengthened removal rate, but lowered flux, meanwhile flux and removal rate exhibited a significant fluctuation with certain laccase dosage. At 25 g m-2 CNT or GO dosage and 50 g m-2 laccase dosage, the optimized flux and removal rate values were obtained. Further study investigated the surface morphology and property of BDM, showing that BDM with mixed filtration method turned out to be the optimized enzyme immobilization mechanism and fabrication method. In addition, during multiple filtration cycles, with the optimized conditions, the removal rate, flux and laccase activity of BDM could maintain at high levels. On account of the finding of the present study, selecting a suitable fabrication method, appropriate CNT or GO dosage and laccase dosage can indeed optimize the structure of biomimetic layer and enzyme immobilization, expanding its possibility on sustainable operation.


Assuntos
Biomimética/métodos , Enzimas Imobilizadas/química , Filtração/métodos , Lacase/química , Membranas Artificiais , Purificação da Água/métodos , Catálise , Concentração de Íons de Hidrogênio , Azul de Metileno/análise , Águas Residuárias/química , Poluentes Químicos da Água/análise
13.
Crit Rev Biotechnol ; 40(1): 1-14, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31640492

RESUMO

The present review emphasizes on the quantification of biogenic amines (BAs) which are regarded as a quality indicator of food freshness or spoilage and for evaluating microbial action while food processing. BAs have various potential adverse effects on human health and they are widely found in varying concentrations in different food stuffs. In the quest for a reliable method for their precise detection, BA biosensors have emerged as an efficient tool which enables rapid and accurate assessment in miniature form. Various combinations of amine oxidase enzymes have been used for the fabrication of biosensors in order to enhance specific biorecognition and signal transduction. This article also summarizes the widely employed components used in the construction of a pertinent biosensor and the research results conducted previously. The meticulous description regarding the choice of transducers and the significant role of mediators in a high response biosensor has been reviewed. Moreover, it also encompasses the utilization of highly attractive electrolytic characteristics of nanoparticles to enhance the specificity and accuracy of BA biosensors.


Assuntos
Aminas Biogênicas/análise , Técnicas Biossensoriais , Oxirredutases atuantes sobre Doadores de Grupo CH-NH2/química , Enzimas Imobilizadas/química , Nanopartículas/química
14.
Enzyme Microb Technol ; 132: 109384, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731948

RESUMO

Cellulose triacetate (CTAB) synthesized by cellulose extracted from sugarcane bagasse, and commercial cellulose acetate (CA) were used to produce nanofiber membranes contained bromelain by electrospinning technique. About 1.3 g of cellulose acetate per gram of bagasse were obtained, and both CTAB and CA was characterized by analysis of Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC). The nanofiber membranes were produced by electrospinning process testing the following conditions: voltage 25 kV, flow rate 4 mL/h and distance 10 cm, using acetone/ dimethylformamide (DMF) (85:15 m/ m) to 15% cellulose triacetate (70% CA + 30% CTAB) or CA solutions. Scanning Electron Microscopy (SEM) was used to nanofiber membranes characterization. Bromelain was immobilized on the nanofiber membranes by crosslinking with glutaraldehyde and directly in the electrospinning step, the highest activity recovery was about 675% and in vitro controlled release tests were performed to semi-quantitatively evaluate the release of the enzyme bromelain thus demonstrating complete release process in 3 days.


Assuntos
Bromelaínas/química , Celulose/análogos & derivados , Eletroquímica/métodos , Nanofibras/química , Saccharum/química , Varredura Diferencial de Calorimetria , Celulose/química , Enzimas Imobilizadas/química , Microscopia Eletrônica de Varredura , Espectroscopia de Infravermelho com Transformada de Fourier
15.
Enzyme Microb Technol ; 132: 109388, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731951

RESUMO

Conjugated linolenic acid (CLA) has important health effects, and their phospholipids forms (PL) are advantageous vehicles of this bioactive agent. Acidolysis of soybean phosphatidylcholine (PC) with conjugated linolenic acid (CLA) catalyzed by Lecitase® Ultra immobilized on Duolite A658 was studied. This reaction is typically limited by hydrolysis, producing 60-90 % of lyso- and glycero-PC and yielding low the process efficiency. Drying the amphiphilic PC material was found the key factor for maximal diacylglycerol phosphatidylcholines (PC) production and different drying approaches were studied to maximize the formation of PC rich in CLA in a solvent free process. PC lyophilization for 24 h getting dry solid appearance (PC; 783 ±â€¯11 mg water/Kg PC) or other standard protocols to reduce water content/activity of reaction medium, did not improve the reaction performance. However, adding 4 extra days to the second step of lyophilization at high vacuum (1 Pa) and moderate temperature (20 °C), followed by further PC dehydration with molecular sieves, drastically reduced the hydrolysis process by achieving a extensive PC dehydration (279 ±â€¯4 mg water/Kg PC), obtaining for the first time >99% molar yield of diacyl-PC product. After 24 h of reaction, a diacyl-PC product with 72.3% CLA content was obtained. PC molecules containing two CLA were the major species formed.


Assuntos
Enzimas Imobilizadas/química , Liofilização/métodos , Fosfolipases/química , Fosfolipídeos/química , Catálise , Hidrólise , Fosfatidilcolinas/química , Pró-Fármacos , Ácido alfa-Linoleico/química
16.
Enzyme Microb Technol ; 132: 109390, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731959

RESUMO

In this study, we presented a new approach for immobilizing JBU (Jack bean urease), by producing urease cross-linked enzyme lyophilisates (CLELs). Through the use of bovine serum albumin (BSA), lyophilisation, cross-linking with dextran polyaldehyde (DPA), and optimizing cross-linker pH, the urease-CLELs produced show an increase in relative catalytic activity that is 1.47 times higher than that of free urease, while remaining stable up to temperatures of 85 °C. Urease-CLEL activity increases in direct proportion with the increasing BSA content due to the offered additional lysine (Lys) groups which are potential cross-linking points providing better immobilization and retention of JBU, while lyophilisation also enables stabilization by eliminating solvating water molecules and intra-molecular reactions that may block the cross-linking residues. Two most commonly used cross-linkers that are reacting with the available Lys groups, i.e.glutaraldehyde (GA) and bulkier alternative DPA, have been selected for the immobilization of urease. The catalytic activity increase with DPA suggests an improved access to the active site through hindering blockage, while the increase with alkaline pH of the cross-linkers indicates decreased buffer inhibition. The long lifetime (113% residual activity after 4 weeks), recyclability (132% residual activity after 10 cycles) and thermal stability (276% relative activity at 85 °C) of these urease-CLELs demonstrate that they are technologically attractive as green biocatalysts, while our immobilization approach offers an alternative to conventional methods for proteins that are difficult to immobilise.


Assuntos
Reagentes para Ligações Cruzadas/química , Enzimas Imobilizadas/química , Urease/química , Domínio Catalítico , Liofilização , Cinética , Lisina/química , Soroalbumina Bovina/química , Vigna/enzimologia
17.
Biosens Bioelectron ; 151: 111971, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31868610

RESUMO

For D-amino acid (DAA) electrochemical biosensors, it is necessary to achieve chiral recognition in racemic solutions or mixtures. However, common chiral recognition is only performed in a single isomer solution. Here, D-amino acid oxidase (DAAO) was used as a chiral selector, and carbon nanotubes (CNTs) as a signal amplifier to construct a non-mediator-style DAA biosensor. The biosensor showed high performance against enantiomer interference: in alanine (Ala) enantiomer mixtures, accurate quantification of D-Ala was achieved when the concentration ratio of L-Ala to D-Ala was 100. In Ala racemic solutions, the linear equation slope was almost consistent with that of standard D-Ala. This high performance was due to the combination of stereoselectivity (enzyme protein) and a catalytic reaction (redox center). The mechanism for the electrical signal change of the biosensor was explored and verified by cyclic voltammetry (CV). The results showed that (i) flavin adenine dinucleotide (FAD, redox center of DAAO) was a direct electroactive substance that produced a reduction peak current; in the presence of O2, the amount of FAD increased leading to an increase of the reduction peak current. (ii) In the presence of DAA, the chemical reaction FAD+DAA â†’ imino acids+ FADH2 occurred and consumed FAD, which resulted in its decrease; thus, the reduction peak current also decreased. Under the same oxygen concentration, the linear decrease of the reduction peak current in the presence of DAA was due to FAD consumption. The biosensor was used for practical analyses in milk and urine samples with satisfactory results.


Assuntos
Alanina/análise , D-Aminoácido Oxidase/química , Enzimas Imobilizadas/química , Técnicas Biossensoriais , Catálise , Técnicas Eletroquímicas , Eletrodos , Flavina-Adenina Dinucleotídeo/química , Nanotubos de Carbono/química , Oxirredução , Estereoisomerismo , Propriedades de Superfície
18.
Food Chem ; 309: 125777, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31699560

RESUMO

For the first time, polyaldehyde kefiran was applied to immobilize the pectinase on the glass bead and the finding was compared with free enzyme. Polyaldehyde kefiran, produced by periodate oxidation of kefiran, showed an aldehyde content of 23.6 ±â€¯0.9% that was confirmed by FTIR. The results showed although the optimum temperature (50 °C) was not changed by immobilization, the optimum pH was shifted from 5.0 to 5.5. In optimal conditions, the specific activity of the free and immobilized pectinase was 2.93 ±â€¯0.13 and 1.87 ±â€¯0.24 unit/mg, respectively. Also, the immobilized pectinase had a higher thermal and pH stability than free enzyme. Km and Vmax of the immobilized pectinase were higher and lower than the free enzyme, respectively. In addition, SEM and AFM images illustrated a completely non-uniform surface after enzyme immobilization on the glass bead, which seemed to be related to the polyaldehyde kefiran strands.


Assuntos
Poligalacturonase/metabolismo , Polissacarídeos/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Oxirredução , Poligalacturonase/química , Temperatura
19.
Talanta ; 206: 120218, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514867

RESUMO

Proteinase K (ProK) is used for the degradation of proteins in cell lysates to isolate nucleic acids, and for the speciation of proteins for mass spectrometry analysis. In this work, a novel and sensitive immobilization process was developed for examination of protein mixtures by combining MALDI-ToF-MS and nLC-TIMS-ToF-MS/MS systems. To achieve these goals, magnetic nanoparticles (MPs) were prepared via thermal coprecipitation reaction under alkaline condition. The MPs were grafted with a silica layer (i.e., 3-(2,3-epoxypropoxy) propyltrimethoxysilane; EPTES) containing reactive epoxy groups. Then, the silica-grafted magnetic particles were coated with a long chain hydrophilic poly(ethylene glycol) diamine polymer (PEGDAP). The prepared materials were characterized by the Brunauer-Emmett-Teller (BET) method, X-ray diffraction (XRD), scanning electron microscopy (SEM) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The VSM data show that the MPs@EPTES@PEGDAP has paramagnetic performance with a saturation magnetization of approximately 32.3 emu g-1. Proteinase K (EC 3.4.21.64) was covalently immobilized on the MPs@EPTES by reaction of its epoxy groups with amine groups of the enzyme. On the other hand, the ProK was immobilized on the MPs@EPTES@PEGDAP after activation with glutaraldehyde and the immobilization reaction was realized by the coupling reaction between aldehyde groups of the support and amine groups of the enzyme. The amounts of immobilized ProK on the MPs@EPTES and MPs@EPTES@PEGDAP were found to be 27.4 and 19.6 mg g-1and the retained activities were determined to be 29 and 87%, respectively. For the first time, some important features such as thermal and storage stabilities, reusability and potential use in protein speciation for mass spectrometry-based techniques were also evaluated. For examples, after six weeks of storage at 4 °C, the immobilized ProK on the MPs@EPTES@PEGDAP-ProK still maintained 59% of its initial activity. However, at the end of the six-week storage period, its free counterpart had lost all of its initial activity. The immobilized ProK was also utilized for degradation and identification of model proteins (i.e., α-2-HS glycoprotein, ß-casein, bovine serum albumin and immunoglobulin). After enzymatic treatment, the digested peptides were analyzed and mapped by using nLC-TIMS-ToF-MS/MS systems.


Assuntos
Endopeptidase K/química , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Fragmentos de Peptídeos/análise , Proteínas/química , Cinética , Estrutura Molecular , Polietilenoglicóis/química , Proteólise , Dióxido de Silício/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos
20.
Food Chem ; 309: 125710, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31704076

RESUMO

The glucose isomerase GICA from Caldicoprobacter algeriensis was immobilized by ionic adsorption on polymethacrylate carriers (Sepabeads EC-EA and EC-HA) or covalent attachment to glyoxal agarose. The Sepabeads EC-HA yielded the highest recovery of activity (89%). The optimum temperature and pH of immobilized GICA were 90 °C and 7.0, respectively, similar to the corresponding values of free enzyme. Nevertheless, the adsorbed enzyme displayed higher relative activity at acidic pH, greater thermostability, and better storage stability, compared to the free form. Moreover, the immobilized enzyme showed an excellent operational stability, in 15 successive 3 h reaction cycles at 85 °C under a batch reactor, preserving 83% of its initial activity. Interestingly, a continuous process for High Fructose Syrup (HFS) production was established with the adsorbed GICA using a packed bed reactor during eleven days at 70 °C. HPAEC-PAD analysis showed a maximum bioconversion rate of 49% after 48 h of operation.


Assuntos
Aldose-Cetose Isomerases/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Clostridiales/enzimologia , Frutose/metabolismo , Aldose-Cetose Isomerases/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Sefarose/química , Temperatura
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