RESUMO
SCOPE: Turmeric curcuminoids mainly consist of curcumin (CUR), demethoxycurcumin (dCUR), and bisdemethoxycurcumin (bdCUR). CUR displays low bioavailability, partly due to poor solubilization in the intestinal lumen during digestion, while data for dCUR and bdCUR are scarce. The study aims to investigate the bioaccessibility of curcuminoids from turmeric extracts or from gamma-cyclodextrins, considering potential interactions with food. METHODS AND RESULTS: Using an in vitro digestion model (correlation with CUR bioavailability: r = 0.99), the study shows that curcuminoid bioaccessibility from turmeric extract without food is low: bdCUR (11.5 ± 0.6%) > dCUR (1.8 ± 0.1%) > CUR (0.8 ± 0.1%). Curcuminoids incorporated into gamma-cyclodextrins display higher bioaccessibilities (bdCUR: 21.1 ± 1.6%; dCUR: 14.3 ± 0.9%; CUR: 11.9 ± 0.7%). Curcuminoid bioaccessibility is highest without food (turmeric extract: 2.0 ± 0.1%; gamma-cyclodextrins: 12.4 ± 0.8%) and decreases with a meat- and potato-based meal (turmeric extract: 1.1 ± 0.2%; gamma-cyclodextrins: 2.4 ± 0.3%) or a wheat-based meal (turmeric extract: 0.1 ± 0.0%; gamma-cyclodextrins: 0.3 ± 0.1%). Curcuminoids exhibit low (<10%) incorporation efficiencies into synthetic mixed micelles (bdCUR > dCUR > CUR). CONCLUSIONS: bdCUR and dCUR show greater bioaccessibilities versus CUR. Food diminishes curcuminoid bioaccessibility, likely by adsorption mechanisms. Gamma-cyclodextrins improve curcuminoid bioaccessibility.
Assuntos
Curcumina , gama-Ciclodextrinas , Diarileptanoides , gama-Ciclodextrinas/metabolismo , Micelas , Adsorção , Curcumina/metabolismo , Curcuma/metabolismoRESUMO
The γ-cyclodextrin glycosyltransferase (γ-CGTase) from Evansella caseinilytica was expressed on the cell surface of E. coli using pAIDA-I autotransporter and was further utilized in the conversion of starch to γ-cyclodextrins (CDs). Maximum cyclization activity of 2.28 ± 0.46 U/g biomass was achieved after 3 h of induction using 0.1 mM IPTG at 37 ºC. Surface expression of γ-CGTase was confirmed using flow cytometry employing a FITC-conjugated anti-HIS antibody. Biochemical characterization of surface-displayed γ-CGTase revealed optima at pH 10.0 and 40 ºC along with a t1/2 of 24.75 min at 50ºC. The Km and Vmax values on soluble potato starch were 10.94 mg/ml and 4.33 µmoles min-1 g-1 DCW respectively, and the activation energy was calculated to be 89.8 kJ/mol. The surface displayed γ-CGTase was further utilized for CD production and specifically, γ-CD conversion was obtained. The maximum conversion was achieved at 50 ºC, pH 9.0 using soluble potato starch (2.5%; w/v) taking a final enzyme concentration of 0.6 U/g starch. The surface-displayed γ-CGTase was able to convert soluble potato starch (2.5%) into γ-CDs with a 72.7% specific yield and no other peaks corresponding to α- and ß-CDs were observed on HPLC. The enzyme was found to be ~100% operationally stable for up to 2 consecutive cycles of 24 h, with > 75% storage stability at - 20 ºC even after 7 days.
Assuntos
Ciclodextrinas , gama-Ciclodextrinas , Bacillus , Ciclodextrinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosiltransferases/metabolismo , Amido/metabolismo , gama-Ciclodextrinas/metabolismoRESUMO
Cyclodextrins (CDs) are doughnut-shaped cyclic oligosaccharides having a cavity and two rims. Inclusion binding in the cavity has long served as a classic model of molecular recognition, and rim binding has been neglected. We found that CDs recognize guests by size-sensitive binding using the two rims in addition to the cavity, using single-molecule electron microscopy and a library of graphitic cones as a solid-state substrate for complexation. For example, with its cavity and rim binding ability combined, γ-CD can recognize a guest of radius between 4 and 9 Å with a size-recognition precision of better than 1 Å, as shown by structural analysis of thousands of individual specimens and statistical analysis of the data thereof. A 2.5 ms resolution electron microscopic video provided direct evidence of the process of size recognition. The data suggest the occurrence of the rim binding mode for guests larger than the size of the CD cavity and illustrate a unique application of dynamic molecular electron microscopy for deciphering the spatiotemporal details of supramolecular events.
Assuntos
Ciclodextrinas/química , Ciclodextrinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Cinética , Microscopia Eletrônica de Transmissão , Nanotubos/química , Tamanho da Partícula , Termodinâmica , alfa-Ciclodextrinas/química , alfa-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismo , gama-Ciclodextrinas/química , gama-Ciclodextrinas/metabolismoRESUMO
Per(6-O-tert-butyldimethylsilyl)-α-, ß- and γ-cyclodextrin derivatives are well-known as synthetic intermediates that enable the selective mono-, partial, or perfunctionalization of the secondary face of the macrocycles. Although silylation of the primary rim is readily achieved by treatment with tert-butyldimethylsilyl chloride in the presence of pyridine (either alone or mixed with a co-solvent), the reaction typically results in a mixture containing both under- and oversilylated byproducts that are difficult to remove. To address this challenge in preparing a pure product in high yield, we describe an approach that centers on the addition of a controlled excess of silylating agent to avoid the presence of undersilylated species, followed by the removal of oversilylated species by column chromatography elution with carefully designed solvent mixtures. This methodology works well for 6-, 7-, and 8-member rings (α-, ß-, and γ-cyclodextrins, respectively) and has enabled us to repeatedly prepare up to â35 g of ≥98% pure product (as determined by HPLC) in 3 d. We also provide procedures for lower-scale reactions, as well as an example of how the ß-cyclodextrin derivative can be used for functionalization of the secondary face of the molecule.
Assuntos
Ciclodextrinas/síntese química , Silício/química , Ciclodextrinas/metabolismo , Estrutura Molecular , Compostos de Organossilício , Silício/metabolismo , Estereoisomerismo , beta-Ciclodextrinas , gama-Ciclodextrinas/síntese química , gama-Ciclodextrinas/metabolismoRESUMO
BACKGROUND: Gracilibacillus alcaliphilus SK51.001, a strain that produces ß-CGTase (ß-cyclodextrin glucanotransferase) (EC 2.4.1.19), was screened and isolated from Sudanese soil. The objective of this study was to sequence and characterize the ß-CGTase gene from G. alcaliphilus SK51.001. RESULTS: According to 16S rRNA analysis of the strain and its morphological shape, it was identified as G. alcaliphilus. The ß-CGTase gene was successfully cloned, sequenced, and expressed in Escherichia coli BL21. This gene showed 706 amino acid residues including 33 amino acids as a signal peptide. The active site residues of G. alcaliphilus SK51.001CGTase were described using enzyme modeling and docking with the products. The estimated molecular mass of G. alcaliphilus SK51.001CGTase was approximately 74 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the evaluation of the gel filtration showed approximately 85 kDa, which means G. alcaliphilus SK51.001CGTase is a monomer. The optimum temperature and pH of G. alcaliphilus SK51.001CGTase were 60 °C and 7.0 respectively. Gracilibacillus alcaliphilus SK51.001CGTase was comparatively stable at a pH levels between 6.0 and 9.0 and temperatures of 30-50 °C. The activity of G. alcaliphilus SK51.001CGTase was increased by Ni2+ , and Co2+ but inhibited by Al3+ and Fe3+ . The kinetic parameters of Km and Vmax were 2068.52 µg mL-1 and 0.13 µmol mL-1 min-1 , respectively. CONCLUSION: Gracilibacillus alcaliphilus SK51.001CGTase could hydrolyze soluble starch into α-, ß-, and γ-cyclodextrin in a ratio of 2: 83: 15% respectively. This high ratio production of ß-CD could allow the enzyme to be used in ß-CD production. © 2020 Society of Chemical Industry.
Assuntos
Bacillaceae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glucosiltransferases/química , Glucosiltransferases/genética , Bacillaceae/química , Bacillaceae/genética , Bacillaceae/isolamento & purificação , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Glucosiltransferases/metabolismo , Temperatura Alta , Cinética , Peso Molecular , Microbiologia do Solo , Amido/metabolismo , Especificidade por Substrato , gama-Ciclodextrinas/metabolismoRESUMO
γ-Cyclodextrin (γ-CD), a cyclic oligosaccharide containing eight glucose units linked by α-1,4-glycosidic bonds, can be produced from starch using cyclodextrin glycosyltransferase (CGTase). Unfortunately, this enzymatic process produces mixtures of α-, ß-, and γ-CD. In this study, amino acid residues in the subsite -3 (T47 and F91) and the central subsite (Y186) of Bacillus clarkii γ-CGTase were modified to improve the γ-CD production. The cyclization activities and product specificities of mutants T47H and F91W were similar to those of the wild-type. The cyclization activities of mutants F91N and F91L were significantly greater than those of the wild-type but their γ-CD product specificities were lower. Finally, the central subsite mutant Y186W displayed a γ-CD specificity (94.6%) significantly greater than that of the wild-type (77.1%). To maximize the γ-CD yield, the effects of added complexing agents were investigated. Among the cyclic complexing agents tested, low-boiling cyclododecanone was the smallest that precipitated with γ-CD. When cyclododecanone was used with Y186W, the total CD yield reached 72.6%, and 96.6% of the product was γ-CD. These results, which represent the highest γ-CD yield ever reported, may provide a way to improve large-scale γ-CD preparation and expand the uses of γ-CD in the future.
Assuntos
Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Glucosiltransferases/metabolismo , gama-Ciclodextrinas/metabolismo , Sequência de Aminoácidos , Bacillus/química , Bacillus/enzimologia , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glucosiltransferases/química , Glucosiltransferases/genética , Modelos Moleculares , Alinhamento de Sequência , Amido/metabolismo , Especificidade por SubstratoRESUMO
This work investigated the interaction between cyclodextrins and pullulanase to provide insight into the production and application of cyclodextrins. Enzyme activity and kinetic assays showed that α-cyclodextrin (α-CD), ß-cyclodextrin (ß-CD) and γ-cyclodextrin (γ-CD) inhibited pullulanase in a competitive manner. Circular dichroism spectra and fluorescence spectroscopy suggested the formation of cyclodextrin and pullulanase complexes. According to ITC assays and molecular docking results, compared with α-CD and γ-CD, ß-CD had the strongest affinity for pullulanase because of its appropriate cavity geometric dimensions. In addition, cyclodextrins interacted with pullulanase through hydrogen bonds, van der Waals force and hydrophobic interactions, the latter of which were verified as the major driving force. Phenylalanine 476 was the key amino acid residue in pullulanase for cyclodextrin recognition and binding.
Assuntos
Ciclodextrinas/química , Ciclodextrinas/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Calorimetria/métodos , Dicroísmo Circular , Glicosídeo Hidrolases/antagonistas & inibidores , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Fenilalanina/metabolismo , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , alfa-Ciclodextrinas/química , alfa-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismo , gama-Ciclodextrinas/química , gama-Ciclodextrinas/metabolismoRESUMO
Gingerols from the rhizome of fresh ginger (Zingiber officinale) were obtained by a simple extraction, followed by purification. The gingerols extract was composed of 6-gingerol (54%), 8-gingerol (20%), and 10-gingerol (26%). It was included into γ-cyclodextrin by classic co-dissolution procedures. Solid-state characterisation of γ-cyclodextrin·gingerols shows that this inclusion compound features 1:1 host-to-guest stoichiometry and that it is a microcrystalline powder with a crystalline cell that belongs to the tetragonal space group 4212, having the host molecules stacked in infinite channels where the gingerols are accommodated. In chimico studies with ABTSâ¢+ scavenging, NO⢠scavenging, ß-carotene peroxidation, and 5-LOX inhibition show that γ-cyclodextrin is a suitable carrier for gingerols, because it does not alter their reactivity towards these substances. Yogurt was tested as a matrix for the incorporation of gingerols and γ-cyclodextrin·gingerols into foodstuff. The colour of the fortified yogurt suffered little alterations. In the case of yogurt with the inclusion compound, γ-cyclodextrin·gingerols, as fortificant, these alterations were not perceptible to the naked eye. Moreover, yogurt with γ-cyclodextrin·gingerols showed a good antioxidant activity, thus being suitable for use in nutraceutical applications.
Assuntos
Catecóis/química , Álcoois Graxos/química , gama-Ciclodextrinas/isolamento & purificação , Antioxidantes/metabolismo , Catecóis/isolamento & purificação , Suplementos Nutricionais , Álcoois Graxos/isolamento & purificação , Zingiber officinale/química , Extratos Vegetais/química , Rizoma/química , Rizoma/efeitos dos fármacos , Iogurte , gama-Ciclodextrinas/análise , gama-Ciclodextrinas/metabolismoRESUMO
OBJECTIVES: Grapefruit (Citrus paradisi) juice enhances the oral bioavailability of drugs that are metabolized by intestinal cytochrome P450 3A4 (CYP3A4). Patients are advised to avoid drinking grapefruit juice to prevent this drug-grapefruit juice interaction. The aim of this study was to investigate whether processing grapefruit juice with cyclodextrins (CDs) would result in preventing or inhibiting this interaction. METHODS: Grapefruit juice and the major furanocoumarins found in grapefruit, bergamottin (BG) and 6', 7'-dihydroxy bergamottin (DHBG) were mixed with α, ß and γCDs. The effects of these processed juice samples and furanocoumarins on CYP3A activity were compared with the corresponding values for unprocessed juices and furanocoumarins. Interactions between CDs and these furanocoumarins were also investigated by phase solubility and 1 H NMR studies. KEY FINDINGS: The inhibition of CYP3A by grapefruit juice was significantly attenuated by processing particularly with γCD. Similar attenuation effects by γCD were observed in the cases of BG and DHBG. Furthermore, BG and DHBG were suggested to be strongly encapsulated in the cavity of γCD. CONCLUSION: The encapsulation of BG and DHBG by γCD and the resulting attenuation of the inhibition of CYP3A activity by grapefruit juice may be applicable to juice processing for preventing drug-grapefruit juice interactions.
Assuntos
Citrus paradisi/química , Citocromo P-450 CYP3A/metabolismo , Furocumarinas/farmacologia , gama-Ciclodextrinas/metabolismo , BebidasRESUMO
The production of γ-cyclodextrin usually includes the utilization of organic complexants. However, the non-complexant production of γ-cyclodextrin is always being explored due to the defects of organic complexants. However, in non-complexant production, the separation of γ-cyclodextrin from α- and ß-cyclodextrin is still a challenge. Here, the selective hydrolysis ability of a cyclodextrinase designated PpCD (cyclodextrinase from Palaeococcus pacificus) on α-cyclodextrin, ß-cyclodextrin, and γ-cyclodextrin was proved. The kcat /Km values of PpCD for α-cyclodextrin and ß-cyclodextrin were roughly 12-fold and 5-fold higher than that of γ-cyclodextrin. It was proved that PpCD had selective hydrolysis ability and its γ-cyclodextrin purification performance was apparent on various simulated cyclodextrin mixtures with reported proportions derived from different CGTases. Besides, the hydrolysis temperature was optimized and it could be seen that 85°C was appropriate for the production of γ-cyclodextrin. In addition, the production of γ-cyclodextrin was achieved by using PpCD in the γ-CGTase reaction products.
Assuntos
Glicosídeo Hidrolases/metabolismo , gama-Ciclodextrinas/metabolismo , Bacillus/enzimologia , Colorimetria , Hidrólise , Temperatura , gama-Ciclodextrinas/químicaRESUMO
The complexation between (2-hydroxypropyl)-γ-cyclodextrin (HP-γ-CD) and water-soluble betulin derivatives, betulin 3,28-disulfate (DSB) and betulin 3-acetate-28-sulfate (ASB), belonging to the class of pentacyclic lupane triterpenoids, was studied using mobility shift ACE (ms ACE). It was found that the complexation is a high-affinity interaction. In this case, a very low amount of HP-γ-CD should be added to the BGE, and triangular peaks are observed as a result of ligand deficiency in the sample zone. Le Saux et al. showed in 2005 that using the parameter a1 of the Haarhoff-Van der Linde (HVL) function instead of the migration time measured at the peak apex eliminates the effect of ligand deficiency on effective electrophoretic mobility. Therefore, the electrophoretic mobilities of asymmetrical peaks of DSB and ASB were calculated in this way. The obtained experimental data correspond to 1:1 complexes. The calculated values of binding constants logarithms at 25°C are 6.70 ± 0.05 and 7.03 ± 0.10 for the HP-γ-CD complexes of DSB and ASB, respectively.
Assuntos
Eletroforese Capilar/métodos , Triterpenos , gama-Ciclodextrinas , Sulfatos/química , Sulfatos/metabolismo , Triterpenos/química , Triterpenos/metabolismo , gama-Ciclodextrinas/química , gama-Ciclodextrinas/metabolismoRESUMO
Oxysterols, oxidization products of cholesterol, are regarded as bioactive lipids affecting various physiological functions. However, little is known of their effects on ion channels. Using inside-out patch clamp recording, we found that naturally occurring side-chain oxidized oxysterols, 20Shydroxycholesterol, 22Rhydroxycholesterol, 24Shydroxycholestero, 25hydroxycholesterol, and 27hydroxycholesterol, induced current reduction of large-conductance Ca2+- and voltage-activated K+ (slo1 BK) channels heterologously expressed in HEK293T cells. In contrast with side-chain oxidized oxysterols, naturally occurring ring oxidized ones, 7αhydroxycholesterol and 7ketocholesterol were without effect. By using 24Shydroxycholesterol (24SHC), the major brain oxysterol, we explored the inhibition mechanism. 24SHC inhibited Slo1 BK channels with an IC50 of ~2⯵M, and decreased macroscopic current by ~60%. This marked current decrease was accompanied by a rightward shift in the conductance-voltage relationship and a slowed activation kinetics, with the deactivation kinetics unaltered. Furthermore, the membrane sterol scavenger γcyclodextrin was found to rescue slo1 BK channels from the inhibition, implicating that 24S-HC may be intercalated into the plasma membrane to affect the channel. These findings unveil a novel physiological importance of oxysterols from a new angle that involves ion channel regulation.
Assuntos
Membrana Celular/metabolismo , Hidroxicolesteróis/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Células HEK293 , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Ligação Proteica , gama-Ciclodextrinas/metabolismoRESUMO
Perilla oil is abundant in α-linolenic acid, which is metabolized to long-chain n-3 fatty acids. This study aimed to determine thermal stability and bioavailability of perilla oil that was powdered by inclusion complexation with γ-cyclodextrin. Fatty acid analysis revealed that the relative abundance of α-linolenic and linoleic acids in the complexes was not affected by heating at 40⯰C for six days but decreased after heating at 60⯰C for three days. No adverse events occurred in rats fed with an experimental diet containing the complexes for two weeks. Plasma α-linolenic and eicosapentaenoic acids in rats fed with diets containing complexes and liquid perilla oil were equally high, indicating the preserved bioavailability of perilla oil in the complexes. Plasma arachidonic acid decreased only in rats fed with a diet containing the complexes. Results suggest that the complexes have potential as a useful source of α-linolenic acid to increase plasma n-3 fatty acids.
Assuntos
Ácido alfa-Linolênico/química , gama-Ciclodextrinas/química , Animais , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Dieta , Ácidos Graxos/sangue , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Linoleicos/sangue , Masculino , Óleos de Plantas/química , Óleos de Plantas/metabolismo , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Ácido alfa-Linolênico/metabolismo , gama-Ciclodextrinas/metabolismoRESUMO
The gene encoding the novel amylolytic enzyme designated CoMA was cloned from Corallococcus sp. strain EGB. The deduced amino acid sequence contained a predicted lipoprotein signal peptide (residues 1 to 18) and a conserved glycoside hydrolase family 13 (GH13) module. The amino acid sequence of CoMA exhibits low sequence identity (10 to 19%) with cyclodextrin-hydrolyzing enzymes (GH13_20) and is assigned to GH13_36. The most outstanding feature of CoMA is its ability to catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. Moreover, it can hydrolyze γ-cyclodextrin and starch to maltose and hydrolyze pullulan exclusively to panose with relative activities of 0.2, 1, and 0.14, respectively. CoMA showed both hydrolysis and transglycosylation activities toward α-1,4-glycosidic bonds but not to α-1,6-linkages. Moreover, glucosyl transfer was postulated to be the major transglycosidation reaction for producing a high level of maltose without the attendant production of glucose. These results indicated that CoMA possesses some unusual properties that distinguish it from maltogenic amylases and typical α-amylases. Its physicochemical properties suggested that it has potential for commercial development.IMPORTANCE The α-amylase from Corallococcus sp. EGB, which was classified to the GH13_36 subfamily, can catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. An action mechanism for producing a high level of maltose without the attendant production of glucose has been proposed. Moreover, it also can hydrolyze γ-cyclodextrin and pullulan. Its biochemical characterization suggested that CoMA may be involved the accumulation of maltose in Corallococcus media.
Assuntos
DNA Bacteriano/isolamento & purificação , Glicosídeo Hidrolases/genética , Myxococcales/genética , Oligossacarídeos/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Maltose/metabolismo , Myxococcales/metabolismo , Análise de Sequência de DNA , Amido/metabolismo , Temperatura , alfa-Amilases/genética , alfa-Amilases/metabolismo , gama-Ciclodextrinas/metabolismoRESUMO
One pertinent complication in bacterial infection is the growth of biofilms, that is, communities of surface-adhered bacteria resilient to antibiotics. Photodynamic inactivation (PDI) has been proposed as an alternative to antibiotic treatment; however, novel techniques complementing standard efficacy measures are required. Herein, we present an approach employing multiphoton microscopy complemented with Airyscan super-resolution microscopy, to visualize the distribution of curcumin in Staphylococcus epidermidis biofilms. The effects of complexation of curcumin with hydroxypropyl-γ-cyclodextrin (HPγCD) were studied. It was shown that HPγCD curcumin demonstrated higher bioavailability in the biofilms compared to curcumin, without affecting the subcellular uptake. Spectral quantification following PDI demonstrates a method for monitoring elimination of biofilms in real time using noninvasive 3D imaging. Additionally, spatially confined 2-photon inactivation was demonstrated for the first time in biofilms. These results support the feasibility of advanced optical microscopy as a sensitive tool for evaluating treatment efficacy in biofilms toward improved mechanistic studies of PDI.
Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/efeitos da radiação , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Microscopia Confocal , Fótons , Staphylococcus epidermidis/fisiologia , Curcumina/química , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Espaço Intracelular/efeitos da radiação , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Staphylococcus epidermidis/citologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/efeitos da radiação , gama-Ciclodextrinas/química , gama-Ciclodextrinas/metabolismo , gama-Ciclodextrinas/farmacologiaRESUMO
In comparison with natural α- and ß-cyclodextrin (CD), γ-CD has attracted much attention due to their large hydrophobic cavities, high water solubility, and bioavailability. However, the production of γ-CD is still rather expensive and time-consuming. To overcome the high cost and long induction time, pUC119 was selected as the gene expression vector, and the recombinant enzyme production time was reduced to 8h from 72h. Furthermore, for the first time, we have successfully produced γ-CD using ß-CD by cyclodextrin opening reactions through the recombinant CGTase in the presence of maltose. The kinetic mechanism of the coupling reaction was investigated. Moreover, the production of γ-CD could be affected by several key parameters, such as solvent type, reaction time, pH, and temperature. A maximum γ-CD yield of 32.9% was achieved by recombinant CGTase in the presence of 5-cyclohexadecen-1-one. This could be a promising method for the industrial production of γ-CD.
Assuntos
Glucosiltransferases/metabolismo , beta-Ciclodextrinas/metabolismo , gama-Ciclodextrinas/metabolismo , Glucosiltransferases/química , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , beta-Ciclodextrinas/química , gama-Ciclodextrinas/químicaRESUMO
Cyclodextrins (CDs) are produced from starch by cyclodextrin glucanotransferase (CGTase), which has cyclization activity. Specifically, α-CD is an important biomolecule, as it is a molecular carrier and soluble dietary fiber used in the food industry. Upon inspection of the conserved regions of the glycoside hydrolase (GH) 13 family amylases, the amino acids K232 and H233 of CGTase were identified as playing an important role in enzyme reaction specificity. A novel CD hydrolyzing enzyme, cyclodextrin glycosyl transferase (CGTase)-alpha, was developed using site-directed mutagenesis at these positions. Action pattern analysis using various substrates revealed that CGTase-alpha was able to hydrolyze ß- and γ-CD, but not α-CD. This selective CD hydrolyzing property was employed to purify α-CD from a CD mixture solution. The α-CD that remained after treatment with CGTase-alpha and exotype glucoamylase was purified using hydrophobic interaction chromatography with 99% purity.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glucosiltransferases/química , Glucosiltransferases/genética , alfa-Ciclodextrinas/química , gama-Ciclodextrinas/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Glucosiltransferases/metabolismo , Hidrólise , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Alinhamento de Sequência , Especificidade por Substrato , alfa-Ciclodextrinas/metabolismo , gama-Ciclodextrinas/metabolismoRESUMO
Curcumin has been successfully encapsulated in cyclodextrin-metal organic frameworks (CD-MOFs) without altering their crystallinity. The interaction between curcumin and CD-MOFs is strong through hydrogen bond type interaction between the OH group of cyclodextrin of CD-MOFs and the phenolic hydroxyl group of the curcumin. Interestingly, dissolving the curcumin loaded CD-MOFs crystals in water results in formation of a unique complex between curcumin, γCD and potassium cations. In fact, the initial interaction between curcumin and CD-MOF is crucial for the formation of the latter. This new complex formed in alkaline media at pH 11.5 has maximum absorbance at 520nm and emittance at 600nm. Most importantly, the stability of curcumin in this complex was enhanced by at least 3 orders of magnitude compared to free curcumin and curcumin:γ-CD at pH 11.5. These results suggest a promising benign system of CD-MOFs, which can be used to store and stabilize curcumin for food applications.
Assuntos
Curcumina/química , Ciclodextrinas/química , Metais/química , Curcumina/metabolismo , Ciclodextrinas/metabolismo , Metais/metabolismo , Água/química , Água/metabolismo , Difração de Raios X , gama-Ciclodextrinas/química , gama-Ciclodextrinas/metabolismoRESUMO
The composite material formed by phytosterol ester (PSE) and γ-cyclodextrin (γ-CD) disperses readily in water and has been used to mask undesirable flavours. This paper elucidates the structure of the PSE/γ-CD particle. Cryogenic scanning electron microscopy and contact angle measurements showed that the PSE/γ-CD particles formed a capsule-like structure with a hydrophilic surface. A phase-solubility study using cholesteryl oleate (ChO), one of the components of PSE, showed that ChO formed a hydrophilic and stoichiometric inclusion complex with γ-CD at a molar ratio of 2:5. The structure of the PSE/γ-CD inclusion complex was similar to that of ChO/γ-CD, based on differential scanning calorimetry and powder X-ray diffractometry results. Thus, we propose that the PSE/γ-CD particle has a capsule-like structure wherein a hydrophobic PSE droplet is surrounded by an outer layer of the hydrophilic PSE/γ-CD inclusion complex.
Assuntos
Fitosteróis/metabolismo , gama-Ciclodextrinas/metabolismo , Varredura Diferencial de Calorimetria , Ésteres do Colesterol/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Solubilidade , Água , Difração de Raios XRESUMO
An important excipient used to overcome poor solubility is cyclodextrin. However, data in the literature suggest that excessive overdosing of cyclodextrins can decrease the absorption of compounds administered with cyclodextrins, due to their lack of release from the complex. γ-Cyclodextrin is digestible in contrast to ß-cyclodextrins. This could potentially limit the sensitivity toward overdose, which was evaluated using benzo[a]pyrene in this study, in which rats were administered benzo[a]pyrene and different doses of the 2 cyclodextrins. Both cyclodextrins lowered the area under the curve and therefore the absorption of benzo[a]pyrene by up to a factor of 2 when dosed in high concentrations, thus indicating that overdosing of cyclodextrins may limit the oral absorption of a compound. This limitation may be artificial because the molar ratio of benzo[a]pyrene:cyclodextrin was >1:50,000 at the concentration where a significant decrease in the absorption was observed. No difference was observed between the 2 cyclodextrins, so digestibility seemed less important. More interesting was that the decrease in absorption was relatively small when compared with literature values, suggesting that the effect of overdosing a compound with cyclodextrins was lower than anticipated.