RESUMO
It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).
Assuntos
Aldeídos , Dextranos , Enzimas Imobilizadas , Ficina , Dextranos/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ficina/química , Ficina/metabolismo , Aldeídos/química , Hemoglobinas/química , Hemoglobinas/metabolismo , Biocatálise , Especificidade por Substrato , Caseínas/química , Caseínas/metabolismo , Estabilidade EnzimáticaRESUMO
This study investigates the features of interactions between cysteine proteases (bromelain, ficin, and papain) and a graft copolymer of carboxymethyl cellulose sodium salt with N-vinylimidazole. The objective is to understand the influence of this interactions on the proteolytic activity and stability of the enzymes. The enzymes were immobilized through complexation with the carrier. The interaction mechanism was examined using Fourier-transform infrared spectroscopy and flexible molecular docking simulations. The findings reveal that the enzymes interact with the functional groups of the carrier via amino acid residues, resulting in the formation of secondary structure elements and enzyme's active sites. These interactions induce modulation of active site of the enzymes, leading to an enhancement in their proteolytic activity. Furthermore, the immobilized enzymes demonstrate superior stability compared to their native counterparts. Notably, during a 21-day incubation period, no protein release from the conjugates was observed. These results suggest that the complexation of the enzymes with the graft copolymer has the potential to improve their performance as biocatalysts, with applications in various fields such as biomedicine, pharmaceutics, and biotechnology.
Assuntos
Bromelaínas , Papaína , Papaína/metabolismo , Ficina/química , Ficina/metabolismo , Carboximetilcelulose Sódica , Simulação de Acoplamento Molecular , Polímeros , Cloreto de Sódio , Cloreto de Sódio na Dieta , SódioRESUMO
BACKGROUND: The achenes/seeds of endemic jelly fig (Ficus pumila var. awkeotsang) fruit have been applied to prepare a traditional beverage in Taiwan. Upon fruit harvest, jelly fig latex exuded from stalks was discarded. Protease activity was monitored in its latex. Proteases capable of hydrolyzing proteins have many application aspects based on diverse characteristics. Commercial plant proteases are frequently from latex. RESULTS: The latex protease of jelly fig, termed FaFicin, was purified to homogeneity with a molecular mass of ~32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to liquid chromatographic-tandem mass spectrometric analyses, the expected protein band of protease was matched to ficin A, ficin B or chymopapain from common fig or papaya. Iodoacetamide, an inhibitor of cysteine protease, inhibited its protease activity completely. Hence FaFicin was identified as a papain-like cysteine protease (PLCP), exhibiting more than 80% and 70% activity as assayed at pH 5-8 and 40-70 °C, respectively. It maintained ~89% of initial activity after 120 min at 55 °C and pH 7. Moreover, FaFicin could degrade the myosin and actin of meat, and clot milk. CONCLUSION: The ficin FaFicin was obtained, purified and identified as a PLCP member from agricultural waste: jelly fig latex. It possessed activity under a wide range of pH values and temperature, and exhibited excellent thermostability. Based on its initial evaluation as a meat tenderizer and milk clotting reagent, the application of FaFicin was possible, which may extend utilization of jelly fig. © 2022 Society of Chemical Industry.
Assuntos
Cisteína Proteases , Ficus , Ficina/química , Ficina/metabolismo , Ficus/química , Látex/química , Alérgenos , Peptídeo HidrolasesRESUMO
BACKGROUND: The physiological role of fig latex is to protect the plant from pathogens. Latex is a rich source of proteases, predominantly ficin. Fig latex also contains collagenolytic protease and chitinolytic enzymes. Our aim was to investigate changes in protein composition, enzyme and antifungal activities of fig latex during fruit ripening. RESULTS: Comparison of latex samples in different time periods showed a uniform increase of protein concentration in chronological order. The content of collagenolytic protease did not differ significantly in the latex samples, while the content of ficin decreased. Ficin-specific activity towards casein was the highest at the beginning of fruit development (about 80 U mg(-1)). Specific milk clotting activity increased as well as the abundance of casein band in the clots. Specific chitinolytic activity at the beginning of flowering was 6.5 times higher than the activity in the period when fruits are ripe. Antifungal activity is the most extensive in spring. CONCLUSION: Ficin forms with different casein specificities are present in different proportions during fruit ripening, which is of importance for applications in the dairy industry. The protection mechanism against insects and fungi, which relies on chitinolytic activity, is the most important in the early phases of flowering and is replaced with other strategies over time.
Assuntos
Ficina/análise , Ficus/química , Frutas/crescimento & desenvolvimento , Fungicidas Industriais/análise , Látex/química , Peptídeo Hidrolases/análise , Animais , Caseínas/metabolismo , Quitina/metabolismo , Ficina/metabolismo , Frutas/química , Inseticidas , Látex/farmacologia , Leite/química , Leite/metabolismo , Proteínas de Plantas/análise , Saccharomyces cerevisiae/efeitos dos fármacos , Especificidade por SubstratoRESUMO
Oligopeptides are of high importance for various industrial applications, e.g. cosmetical or medical. Homooligomerizations and co-oligomerizations with anionic amino acid esters are well described but a successful synthesis of cationic heterooligopeptides has been missing so far. The present study reports the ficain-catalyzed heterooligomerizations of LysOEt with MetOEt, leading to cationic heterooligopeptides with a yield up to 49.5% (w/w). MALDI-ToF/ToF-MS analyses proved successful syntheses of cationic heterooligopeptides with a DP between 7 and 10 amino acid residues, with the enzyme exhibiting a clear preference for methionine.
Assuntos
Ficina/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Metionina/análogos & derivados , Oligopeptídeos/biossíntese , Cátions , Ésteres , Metionina/metabolismo , Oligopeptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Rh proteins and the Wr(b) antigen, which results from an interaction between Band 3 and glycophorin A, are the most common targets for warm immunoglobulin (Ig)G autoantibodies. Apart from autoanti-Di(b) , a scarce specificity, IgG warm autoantibodies specific for Band 3 have never been characterized by serologic methods. STUDY DESIGN AND METHODS: Blood samples from 120 patients with autoimmune hemolytic anemia (AIHA) and IgG-coated red blood cells (RBCs) were studied by serologic methods. Some autoantibodies were investigated by immunochemical methods. RESULTS: Autoantibodies against the third external loop of Band 3 have a distinctive pattern of reactivity in that they fail to react after RBC treatment with α-chymotrypsin and pronase, whereas papain, ficin, and trypsin have no effect. Eleven (9%) patients had pure anti-Band 3 autoantibodies. Autoanti-Band 3 antibodies were associated with other specificities in 66 (55%) patients. Immunoprecipitation and rare RBCs from a Wu+ homozygote, known to have an unusual pattern of reactivity after protease treatment, were used to confirm the Band 3 specificity. Treatment with sodium hypochlorite, believed to oxidize the Met residue at Position 559 in the third loop, showed that these autoantibodies were heterogeneous. Most antibodies reacted optimally at 37 °C, but two patients had incomplete cold IgG autoantibodies. Unlike autoantibodies to Rh proteins, warm autoanti-Band 3 activate complement and are almost totally bound to autologous RBCs. CONCLUSION: We describe the first cases of warm IgG autoantibodies specific for the third loop of Band 3. This external loop also appears as a major target in patients with warm antibody AIHA.
Assuntos
Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Anemia Hemolítica Autoimune/imunologia , Quimotripsina/metabolismo , Feminino , Ficina/metabolismo , Humanos , Imunoprecipitação , Masculino , Pronase/metabolismoRESUMO
Patients demonstrating warm autoantibody specificity present serologic challenges for laboratory staff performing antibody identification in the blood bank. Autoantibody can be removed from plasma or serum by adsorption onto autologous red blood cells (RBCs) provided the patient has not been transfused in the previous 3 months. The adsorption process can be enhanced by enzyme pretreatment of autologous RBCs.
Assuntos
Autoanticorpos/isolamento & purificação , Eritrócitos/química , Ficina/metabolismo , Papaína/metabolismo , Adsorção , Especificidade de Anticorpos , Autoanticorpos/imunologia , Bancos de Sangue , Transfusão de Sangue , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Ficina/farmacologia , Humanos , Papaína/farmacologia , TemperaturaRESUMO
Ficin has dual enzyme activity, i.e., protease and peroxidase-like activity. In some respects, its application is limited by the protease activity of ficin. Herein, we used tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to break the three pairs of disulfide bonds of ficin, and then blocked the free thiol groups with N-ethylmaleimide (NEM) to synthesize ficin-TN. The results showed that ficin-TN had increased peroxidase-like activity and reduced protease activity. According to this phenomenon, we have exploited a colorimetric method with high sensitivity and selectivity for the one-step detection of glucose. Comparing with ficin, ficin-TN has wider detection range (0.1-300 µM) and lower detection limit (88 nM), and our method is simpler and more timesaving than other two-step methods. Furthermore, the actual appliances of ficin-TN for glucose detection in human serum have been illustrated with satisfied result, suggesting that its promising utilization in various fields.
Assuntos
Colorimetria , Ficina , Peroxidase , Ficina/metabolismo , Glucose , Humanos , Peróxido de Hidrogênio , Oxirredução , Peroxidase/metabolismo , PeroxidasesRESUMO
Bromelain, papain, and ficin are studied the most for meat tenderization, but have limited application due to their short lifetime. The aim of this work is to identify the adsorption mechanisms of these cysteine proteases on chitosan to improve the enzymes' stability. It is known that immobilization can lead to a significant loss of enzyme activity, which we observed during the sorption of bromelain (protease activity compared to soluble enzyme is 49% for medium and 64% for high molecular weight chitosan), papain (34 and 28% respectively) and ficin (69 and 70% respectively). Immobilization on the chitosan matrix leads to a partial destruction of protein helical structure (from 5 to 19%). Using computer modelling, we have shown that the sorption of cysteine proteases on chitosan is carried out by molecule regions located on the border of domains L and R, including active cites of the enzymes, which explains the decrease in their catalytic activity upon immobilization. The immobilization on chitosan does not shift the optimal range of pH (7.5) and temperature values (60 °C for bromelain and papain, 37-60 °C for ficin), but significantly increases the stability of biocatalysts (from 5.8 times for bromelain to 7.6 times for papain).
Assuntos
Bromelaínas/química , Bromelaínas/metabolismo , Quitosana/metabolismo , Composição de Medicamentos/métodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ficina/química , Ficina/metabolismo , Papaína/química , Papaína/metabolismo , Adsorção , Ananas/enzimologia , Biocatálise , Biotecnologia/métodos , Carica/enzimologia , Domínio Catalítico , Estabilidade Enzimática , Ficus/enzimologia , Concentração de Íons de Hidrogênio , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Estrutura Secundária de Proteína , TemperaturaRESUMO
Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100.
Assuntos
Ficus/enzimologia , Microbiologia de Alimentos/métodos , Látex/metabolismo , Listeria monocytogenes/isolamento & purificação , Papaína/metabolismo , Carica/enzimologia , Colágeno/metabolismo , Contagem de Colônia Microbiana , Ficina/química , Ficina/metabolismo , Látex/química , Produtos da Carne/microbiologia , Especificidade por SubstratoRESUMO
Due to the problems raised by the use of animal or microbial recombinant proteases, the use of proteases from vegetable origin is becoming increasingly popular.. Among them, sulfidryl proteases have a special interest. Ficin is an outstanding example of this kind of proteases. This paper aims to be to make a comprehensive review of the recent uses of this enzyme, including for example protein hydrolysis, the production of bioactive peptides and antibodies fragments (researchers point that ficin results are more reproducible than using other proteases), meat tenderization, milk coagulations in cheese making or peptide synthesis. Efforts to get industrial immobilized biocatalysts of the enzyme will be also described. The review shows the huge potential and brilliant prospect that this enzyme can have in the near future.
Assuntos
Biotecnologia/métodos , Enzimas Imobilizadas/metabolismo , Ficina/metabolismo , Leite/efeitos dos fármacos , Papaína/efeitos dos fármacos , Extratos Vegetais/farmacologia , Plantas/enzimologia , Animais , Antiparasitários/farmacologia , Biocatálise , Queijo , Combinação de Medicamentos , Ficina/farmacologia , Hemostáticos/farmacologia , Hidrólise , Leite/metabolismo , Peptídeo Hidrolases/metabolismo , Extratos Vegetais/química , Sódio na DietaRESUMO
2-[(1-methylpropyl)dithio]-1H-imidazole (IV-2) is a known inhibitor of the thioredoxin system. It causes the oxidation of cysteine residues from both thioredoxin reductase and thioredoxin, with only the latter leading to irreversible inhibition of protein function. Although IV-2 is considered to be the first specific inhibitor of thioredoxin to undergo evaluation in cancer patients (under the name PX-12), it is unclear whether the oxidative ability of IV-2 is limited to proteins of the thioredoxin family. The current study investigated the specificity of IV-2 by examining its interaction with tubulin, a protein in which cysteine oxidation causes loss of polymerization competence. The cellular effects of IV-2 were examined in MCF-7 breast cancer and endothelial cells (human umbilical vein endothelial cells). Immunocytochemistry revealed a loss of microtubule structure with Western blot analysis confirming that treated cells contained a higher proportion of unpolymerized tubulin. Cell-free tubulin polymerization assays showed a dose-dependent inhibition of tubulin polymerization and depolymerization of preformed microtubules, confirming a direct interaction between IV-2 and tubulin. Further investigation of the tubulin interaction, through analysis of sulfhydryl reactivity and disulfide bond formation, suggested that IV-2 acts through the oxidation of cysteines in tubulin. Biochemical assays indicated that the oxidative properties of IV-2 are not limited to thioredoxin and tubulin, as cysteine-dependent proteases were also inhibited. Breast cancer cells with thioredoxin silenced by short interfering RNA remained sensitive to IV-2, albeit at higher antiproliferative GI50 values than in cells with normal thioredoxin function. These findings show that modulation of targets other than thioredoxin contribute to the effects of IV-2 on proliferating cells.
Assuntos
Cisteína/metabolismo , Dissulfetos/farmacologia , Imidazóis/farmacologia , Tubulina (Proteína)/metabolismo , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dissulfetos/química , Ficina/antagonistas & inibidores , Ficina/metabolismo , Humanos , Imidazóis/química , Microtúbulos/efeitos dos fármacos , Estrutura Molecular , Oxirredução/efeitos dos fármacos , Papaína/antagonistas & inibidores , Papaína/metabolismo , Inibidores de Proteases/farmacologia , RNA Interferente Pequeno/genética , Sensibilidade e Especificidade , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Dopamine (DA), a catecholamine neurotransmitter, is considered to be an important indicator for mental diseases detection in the clinic. In this study, a novel fluorescent sensing platform consisting of the ficin-H2O2-tyramine system for determining DA in cerebrospinal fluids (CSF) was established. The proposed method is based on the fact that ficin, a mimetic peroxidase, can catalyze H2O2 decomposition into OH radicals, which can oxidize non-fluorescent tyramine into fluorescent dityramine. When DA was introduced, DA can compete with tyramine for OH and resulting in the oxidation reaction of tyramine inhibited along with the fluorescence intensity of the system decreased, which provides a unique strategy for fluorescence detection of DA. Under optimal conditions, the fluorescence intensity decreased linearly with the DA level over a wide concentration range from 0.05 to 12.0⯵M (R2â¯=â¯0.995) with a detection limit of 46â¯nM (3σ/k). More importantly, the proposed sensing approach exhibits high sensitivity, good selectivity and has been successfully applied to DA sensing in complex biological samples, which made it hold great potential for DA determination in chemical and biological analytical applications.
Assuntos
Encefalopatias/líquido cefalorraquidiano , Encéfalo/metabolismo , Dopamina/líquido cefalorraquidiano , Ficina/metabolismo , Fluorescência , Peroxidase/metabolismo , Catálise , Humanos , Limite de Detecção , Oxirredução , Espectrometria de FluorescênciaRESUMO
Ficin extract has been aminated using ethylenediamine and carbodiimide to transform all exposed carboxylic groups into amino groups, retaining around 80% of activity versus benzoyl-d,l-arginine p-nitroanilide hydrochloride (BANA) and 90% versus casein. This aminated enzyme was then immobilized on glyoxyl agarose beads. After optimization of the immobilization protocol (immobilization at pHâ¯10 for just 1â¯h), the new biocatalyst was compared to that obtained using the non-aminated enzyme. Activity versus BANA was lower, but was higher versus casein. The new biocatalyst was more stable than the reference mainly at pHâ¯7. The new biocatalyst permitted to have a more linear course and a higher hydrolysis yield of casein at 75⯰C. Moreover, the activity of the new preparations was significantly higher than the reference or the free enzyme in 8â¯M urea, at pHâ¯7 and 55⯰C. The enzyme in an overloaded biocatalyst exhibited a much higher specific activity versus casein (75% of the low loaded biocatalysts) than the non-aminated enzyme (only 30%), suggesting a more appropriate enzyme orientation that decreased steric hindrances. Finally, the enzyme was reused for 5â¯cycles of casein hydrolysis at 40⯰C and pHâ¯7 without any decrease in enzyme activity.
Assuntos
Caseínas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ficina/química , Ficina/metabolismo , Glioxilatos/química , Sefarose/química , Aminação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , TemperaturaRESUMO
A droplet-based microfluidic synthesis approach for preparation of ficin capped gold nano clusters (AuNCs) was developed. Well dispersed AuNCs could be procured within 8â¯min. Upon excitation wavelength at 340â¯nm, the resultant AuNCs exhibited a strong blue fluorescence with the maximum emission at 450â¯nm. Due to the aggregation-induced "turn-off" fluorescence mechanism, the synthesized AuNCs as a fluorescent probe displayed high sensitivity and good selectivity for sensing ferric ions. The relative fluorescence intensity versus ferric ions concentration yielded a good linear calibration in the range of 10.0-1000.0⯵M (R2 = 0.998) and the limit of detection was 4.1⯵M. Moreover, the possible mechanism for abated fluorescence intensity of AuNCs by adding ferric ions was discussed briefly. Further, the as-prepared fluorescent AuNCs was successfully applied for the detection of serum ferric ions. The results indicated that the droplet-based microfluidic synthesis system could provide a new way for the rapid preparation of AuNCs with good polydispersity and have potential as the sensing probes for the analysis of ferric ions in real biological samples.
Assuntos
Compostos Férricos/sangue , Ficina/química , Corantes Fluorescentes/química , Ouro/química , Nanopartículas Metálicas/química , Ficina/metabolismo , Humanos , Íons/sangue , Técnicas Analíticas Microfluídicas , Tamanho da Partícula , Espectrometria de Fluorescência , Propriedades de SuperfícieRESUMO
Our research has shown that the degree of photosensitivity of the cysteine proteases can be arranged in the following order: bromelain â ficin â papain. After the UV irradiation with 151â¯J·m-2 intensity of a bromelain solution, the enzyme activity has increased. No decrease in the catalytic capacity and the change in the size of the molecule was recorded in the 151-6040â¯J·m-2 range of irradiation intensities. A decrease in the catalytic capacity of ficin and the increase of its globule size occurred after exposure to a radiation of 3020â¯J·m-2 intensity. The decrease in papain activity was observed at the UV irradiation intensity of 453â¯J·m-2, and an increase of the papain globule size was detected at 755â¯J·m-2. Immobilization on chitosan matrix leads to the increase in the stability of heterogeneous biocatalysts with respect to UV irradiation in comparison with free enzymes. The changes in IR spectra of immobilized cysteine proteases practically do not affect the bands due to the protein component of the system: amide I, amide II, amide III. Therefore, it can be postulated that the chitosan matrix acts as photoprotector for immobilized ficin, bromelain and papain. The obtained results can be helpful for development of drugs based on chitosan and cysteine proteases in combination with phototherapy, as well as for choosing their sterilization conditions.
Assuntos
Bromelaínas/metabolismo , Ficina/metabolismo , Papaína/metabolismo , Raios Ultravioleta , Biocatálise/efeitos da radiação , Bromelaínas/química , Quitosana/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ficina/química , Cinética , Papaína/química , Estrutura Terciária de ProteínaRESUMO
The capability of ficin, a cystine protease, to form peptide bonds was investigated using several types of N-Boc-amino acid phenyl and naphthyl esters as acyl donor components. Enzyme-catalyzed peptide synthesis was carried out under optimized reaction conditions of pH, acyl acceptor concentration and selection of the best yield organic solvent. It used a condensation of N-Boc-Ala-OpGu and Ala-pNA as a model reaction. The products were obtained in 72-96% yield using 10 different substrates, within a few minutes of reaction time.
Assuntos
Ficina/metabolismo , Peptídeos/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Catálise , Bovinos , Ficus/enzimologia , Concentração de Íons de Hidrogênio , Estrutura Molecular , Especificidade por SubstratoRESUMO
In this work, ficin extract (3.4.22.3, >1.0â¯u/mg) was immobilized for the first time in poly (vinyl alcohol) (PVA) electrospun nanofibers in order to preserve its tridimensional molecular structure. The analysis of the morphology of the electrospun nanofibers was carried out using scanning electron microscopy (SEM) showing a diameter in the range of 124-194â¯nm. The interaction of the ficin extract with the nanofibers structure was analyzed by infrared spectroscopy (FTIR) analysis. The immobilization step was achieved through crosslinking involving the exposure to glutaraldehyde (GA) vapor. The enzyme catalytic behavior was followed by ultraviolet spectroscopy (UV) using the Earlanger method using Nα-benzoyl-l-arginine-4-nitroanilide hydrochloride (BAPA) as substrate. The maximum catalytic activity was reached with 1â¯h of crosslinking, 20% of enzyme loading and pHâ¯8. The immobilized ficin extract showed 92% of the enzyme activity of the crude ficin extract. The enzymatic activity of the immobilized ficin extract was conserved after a total of nine reuse cycles and maintained after being stored for 25â¯days. Finally, both the glass transition (Tg) and heat of fusion (Hf) were affected by number of enzyme molecules inside the polymeric nanofibers matrix according to the study of the thermal properties by differential scanning calorimetry (DSC).
Assuntos
Enzimas Imobilizadas/metabolismo , Ficina/metabolismo , Nanofibras/química , Polímeros/química , Glutaral/química , Concentração de Íons de Hidrogênio , Nanofibras/ultraestrutura , Tamanho da Partícula , Álcool de Polivinil/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Fatores de TempoRESUMO
Ficin, a classical sulfhydryl protease, was found to possess intrinsic peroxidase-like activity. In this paper, we have put forward a novel strategy to improving the peroxidase-like activity of ficin through binding heme. Heme-ficin complexes were successfully obtained by simple one-step syntheticism. The results demonstrated that the catalytic activity and efficiency of heme-ficin complexes were about 1.7 times and 3 times higher than those of native ficin, respectively. Taking advantages of the high peroxidase-like activity, the heme-ficin complexes were used for colorimetric determination of uric acid with a low detection limit of 0.25⯵M. Based on the excellent selectivity and sensitivity, we detected the concentration of uric acid in human serum successfully. On the basis of these findings, the heme-ficin complexes are promising for wide applications in various fields. Thus we not only optimized the peroxidase-like activity of the ficin, but also established a new strategy for development of artificial enzyme mimics by mimicking the architecture of the active site in horseradish peroxidase.
Assuntos
Colorimetria , Ficina/metabolismo , Heme/metabolismo , Ácido Úrico/análise , Sítios de Ligação , Ficina/química , Voluntários Saudáveis , Heme/química , Humanos , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Modelos Moleculares , Estrutura Molecular , Ácido Úrico/metabolismoRESUMO
Ficin is classified as a sulfhydryl protease isolated from the latex of fig trees. In most cases, a particular enzyme fits a few types of substrate and catalyzes one type of reaction. In this investigation, we found sufficient proofs for the intrinsic peroxidase-like activity of ficin and designed experiments to examine its effectiveness in a variety of scenarios. Ficin can transform peroxidase substrates to colored products in the existence of H2O2. Our results also indicate that the active sites of peroxidase-like activity of ficin are different from that of protease, which reveals that one enzyme may catalyze more than one kind of substrate to perform different types of reactions. On the basis of these findings, H2O2 releasing from MCF-7 cells was detected successfully. Our findings support a wider application of ficin in biochemistry and open up the possibility of utilizing ficin as enzymatic mimics in biotechnology and environmental monitoring.