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1.
Food Chem ; 416: 135816, 2023 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-36893634

RESUMO

Sweet cherry (Prunus avium L.) fruits are prone to quality and quantity loss in shelf-life conditions and cold storage due to their short post-harvest life. Until now efforts have been made to extend the shelf life of the sweet cherry. However, an efficient and commercially scalable process remains elusive. To contribute to this challenge, here in this study, biobased composite coatings consisting of chitosan, mucilage, and levan, were applied on sweet cherry fruits and tested for postharvest parameters in both market and cold storage conditions. Results demonstrated that the shelf life of sweet cherries can be extended until the 30th day while retaining important post-harvest properties like decreased weight loss, fungal deterioration, increased stem removal force, total flavonoid, l-ascorbic acid, and oxalic acid. Given the cost-effectiveness of the polymers used, the findings of this study indicate the feasibility of extending the shelf-life of sweet cherries on a larger scale.


Assuntos
Quitosana , Prunus avium , Quitosana/farmacologia , Ácido Ascórbico/farmacologia , Antioxidantes/farmacologia , Polissacarídeos/farmacologia , Frutas , Frutanos
2.
Appl Microbiol Biotechnol ; 106(17): 5525-5538, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35896838

RESUMO

The rhizosphere context of inulin-accumulating plants, such as Jerusalem artichoke (Helianthus tuberosus), is an ideal starting basis for the discovery of inulolytic enzymes with potential for bio fructose production. We isolated a Glutamicibacter mishrai NJAU-1 strain from this context, showing exo-inulinase activity, releasing fructose from fructans. The growth conditions (pH 9.0; 15 °C) were adjusted, and the production of inulinase by Glutamicibacter mishrai NJAU-1 increased by 90% (0.32 U/mL). Intriguingly, both levan and inulin, but not fructose and sucrose, induced the production of exo-inulinase activity. Two exo-inulinase genes (inu1 and inu2) were cloned and heterologously expressed in Pichia pastoris. While INU2 preferentially hydrolyzed longer inulins, the smallest fructan 1-kestose appeared as the preferred substrate for INU1, also efficiently degrading nystose and sucrose. Active site docking studies with GFn- and Fn-type small inulins (G is glucose, F is fructose, and n is the number of ß (2-1) bound fructose moieties) revealed subtle substrate differences between INU1 and INU2. A possible explanation about substrate specificity and INU's protein structure is then suggested. KEY POINTS: • A Glutamicibacter mishrai strain harbored exo-inulinase activity. • Fructans induced the inulolytic activity in G. mishrai while the inulolytic activity was optimized at pH 9.0 and 15 °C. • Two exo-inulinases with differential substrate specificity were characterized.


Assuntos
Helianthus , Frutanos , Frutose , Glicosídeo Hidrolases , Inulina , Sacarose
3.
Int J Biol Macromol ; 213: 70-82, 2022 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-35644316

RESUMO

A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (-7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (-3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20-30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts.


Assuntos
Enzimas Imobilizadas , Engenharia de Proteínas , Aminoácidos , Reagentes de Ligações Cruzadas , Estabilidade Enzimática , Enzimas Imobilizadas/química , Glicosídeo Hidrolases , Temperatura
4.
J Agric Food Chem ; 69(44): 13125-13134, 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34618455

RESUMO

Thermostability and enzymatic activity are two vital indexes determining the application of an enzyme on an industrial scale. A truncated inulosucrase, Laga-ISΔ138-702, from Lactobacillus gasseri showed high catalysis activity. To further enhance its thermostability and activity, multiple sequence alignment (MSA) and rational design based on the modeled structure were performed. Variants A446E, S482A, I614M, and A627S were identified with an improved denaturation temperature (Tm) of more than 1 °C. A combinational mutation method was further carried out to explore the synergistic promotion effects of single-point mutants. Additionally, 33 residues at the N-terminus were truncated to construct mutant M4N-33. The half-life of M4N-33 at 55 °C increased by 120 times compared to that of Laga-ISΔ138-702, and the relative activity of M4N-33 increased up to 152% at the optimal pH and temperature (pH 5.5 and 60 °C). Molecular dynamics (MD) simulations illustrated the decreased b-factor of the surface loop of M4N-33.


Assuntos
Inulina , Estabilidade Enzimática , Cinética , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Temperatura
5.
Int J Biol Macromol ; 189: 223-231, 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34419542

RESUMO

Chemoresistance (CR) is one of the reasons why chemotherapy agents like Gemcitabine (GMC) remain insufficient in healing breast cancer. Activation of Nuclear Factor-kappa B (NF-κB) during chemotherapy is known as an important factor in the development of CR. The hydrophobic polyphenol curcumin is shown to inhibit NF-κB and hence CR. The aim of this work was to increase the poor bioavailability of curcumin by loading it into the nano-micelles made of Poly (Lactide-co-Glycolide) (PLGA) and levan, where levan as a natural fructose homopolymer makes the nano-micelle more stable and increases its uptake using the fructose moieties. In this study, a PLGA-levan-curcumin formulation (PLC) was designed and characterized. The size was measured as 154.16 ± 1.45 nm with a 67.68% encapsulation efficiency (EE%). The incorporation between the components was approved. Levan made the nano-micelles stable for at least three months, increased their uptake, and led to a 10,000-fold increase in the solubility of curcumin. The enhanced bioavailability of curcumin reduced the NF-κB levels elevated by GMC, both in vitro and in vivo. The PLC showed a complete tumor treatment, while GMC only showed a rate of 52%. These point to the great potential of the PLC to be used simultaneously with chemotherapy.


Assuntos
Curcumina/administração & dosagem , Curcumina/uso terapêutico , Frutanos/química , NF-kappa B/metabolismo , Nanopartículas/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Administração Oral , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Disponibilidade Biológica , Neoplasias da Mama/tratamento farmacológico , Varredura Diferencial de Calorimetria , Morte Celular/efeitos dos fármacos , Curcumina/farmacologia , Composição de Medicamentos , Difusão Dinâmica da Luz , Feminino , Fluorescência , Humanos , Células MCF-7 , Micelas , Nanopartículas/ultraestrutura , Tamanho da Partícula , Ratos , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
6.
Enzyme Microb Technol ; 149: 109857, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311894

RESUMO

Fructansucrases (FSs), including inulosucrase (IS) and levansucrase (LS), are the members of the Glycoside Hydrolase family 68 (GH68) enzymes. IS and LS catalyze the polymerization of the fructosyl moiety from sucrose to inulin- and levan-type fructans, respectively. Lactobacillus-derived FSs have relatively extended N- and C-terminal sequences. However, the functional roles of these sequences in their enzymatic properties and fructan biosynthesis remain largely unknown. Limosilactobacillus reuteri (basionym: Lactobacillus reuteri) 121 could produce both IS and LS, abbreviated as Lare121-IS and Lare121-LS, respectively. In this study, it was found that the terminal truncation displayed an obvious effect on their activities and the N-terminal truncated variants, Lare121-ISΔ177-701 and Lare121-LSΔ154-686, displayed the highest activities. Melting temperature (Tm) and the thermostability at 50 °C were measured to evaluate the stability of various truncated versions, revealing the different effects of N-terminal on the stability. The average molecular weight and polymerization degree of the fructans produced by different truncated variants did not change considerably, indicating that N-terminal truncation had low influence on fructan biosynthesis. In addition, it was found that N-terminal truncation could also improve the activity of other reported FSs from Lactobacillus species.


Assuntos
Lactobacillus , Limosilactobacillus reuteri , Catálise , Lactobacillus/genética , Limosilactobacillus reuteri/genética , Peso Molecular , Sacarose
7.
Biochem Biophys Rep ; 25: 100886, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33490643

RESUMO

Yeasts are important microorganisms used for ethanol production; however, they are not equally efficient in the amount of ethanol production under different environmental conditions. It is, therefore, necessary to screen for elite strains to utilize them for commercial production of these commodities. In this study, yeasts were isolated from different Ethiopian traditional fermented alcoholic beverages (teji, tella, shamiata and areqe tinisis), milk and ergo, teff and maize dough, soil and compost, flowers, and fruits to evaluate their potential use for ethanol fermentation process. Isolates were screened for efficient ethanol production and the selected ones were identified using phenotypic and genetic characters using D1/D2 region of LSU rDNA sequence analysis. The yeast isolates were evaluated based on their growth and fermentation of different carbon sources. Response surface methodology (RSM) was applied to optimize temperature, pH and incubation time using central composite design (CCD) in Design-Expert 7.0.0. A total of 211 yeasts colonies were isolated of which 60% were ethanologenic yeasts (ethanol producers) and 40% were non-ethanol producers. The yeast population detected from various sources was in the range of 10 5 CFU from traditional foods and beverages to that of 10 3 CFU from fruits and soil samples. The data also showed that the number of colony types (diversity) did not correlate with population density. The highly fermentative isolates were taxonomically characterized into four genera, of which 65% of the isolates (ETP37, ETP50; ETP53, ETP89, ETP94) were categorized under Saccharomyces cerevisiae, and the remaining were Pichia fermentans ETP22, Kluyveromyces marxianus ETP87, and Candida humilis ETP122. The S. cerevisiae isolates produced ethanol (7.6-9.0 g/L) similar with K. marxianus ETP87 producing 7.97 g/L; comparable to the ethanol produced from commercial baker's yeast (8.43 g/L) from 20 g/L dextrose; whereas C. humilis ETP122 and P. fermentans ETP22 produced 5.37 g/L and 6.43 g/L ethanol, respectively. S. cerevisiae ETP53, K. marxianus ETP87, P. fermentans ETP22 and C. humilis ETP122 tolerated 10% extraneous ethanol but the percentage of ethanol tolerance considerably decreased upon 15%. S. cerevisiae ETP53 produced ethanol optimally at pH 5.0, 60 h, and 34 o C. pH 4.8, temperature 36 o C, and 65 h of time were optimal growth conditions of ethanol fermentation by K. marxianus ETP87. The ethanol fermentation conditions of P. fermentans ETP22 was similar to S. cerevisiae ETP53 though the ethanol titer of S. cerevisiae ETP53 was higher than P. fermentans ETP22. Therefore, S. cerevisiae ETP53, K. marxianus and P. fermentans ETP22 are good candidates for ethanol production.

8.
Microorganisms ; 8(12)2020 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-33291251

RESUMO

Several exopolymers with different chemical composition and correspondingly variety in their physico-chemical properties from halophilic microorganisms have still been described, however, with a low production yield. Chromohalobacter canadensis 28 isolated from Pomorie saltern synthesized an unusual exopolymer (EP) containing 72% γ-polyglutamic acid (PGA), an essential cosmeceutical additive. Current work suggests a novel approach for effective EP synthesis by C. canadensis 28 using continuous cultures. Highest production was observed at low dilution rates reaching a level of 2.1 mg/mL at D = 0.035, similar to those in batch cultures (2.34 mg/mL), however avoiding all disadvantages of discontinuous fermentation processes. At steady state, the total quantities of the synthesized EP after 48 h cultivation for the given equipment volume in D = 0.035 h-1 and D = 0.075 h-1 were 8.67 and 12 g, correspondingly, while it was 2.9 g for batch culture. Process parameters did not change after a ten-day run at D = 0.35 h-1. A degree of purity of EP fraction received from continuous cultures was significantly increased up to 93-96%. A lack of cytotoxicity and high cell viability were observed for human dermal fibroblast cells after 24 h incubation with crude EP from C. canadensis 28 and purified PGA fraction that could suggest its high potential for cosmetic applications.

9.
Eng Life Sci ; 20(8): 357-367, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32774208

RESUMO

Unusual composition of an exopolymer (EP) from an obligate halophilic bacterium Chromohalobacter canadensis 28 has triggered an interest in development of an effective bioreactor process for its production. Its synthesis was investigated in 2-L bioreactor at agitation speeds at interval 600-1000 rpm, at a constant air flow rate of 0.5 vvm; aeration rates of 0.5, 1.0, and 1.5 vvm were tested at constant agitation rate of 900 rpm. EP production was affected by both, agitation and aeration. As a result twofold increase of EP yield was observed and additionally increased up to 3.08 mg/mL in a presence of surfactants. For effective scale-up of bioreactors mass transfer parameters were estimated and lowest values of KLa obtained for the highest productivity fermentation was established. Emulsification activity of EP exceeded that of trade hydrocolloids xanthan, guar gum, and cellulose. A good synergism between EP and commercial cellulose proved its potential exploration as an enhancer of emulsifying properties of trade emulsions. A pronounced lipophilic effect of EP was established toward olive oil and liquid paraffin. Cultivation of human keratinocyte cells (HaCaT) with crude EP and purified γ-polyglutamic acid (PGA) showed higher viability than control group.

10.
Eur J Pharm Sci ; 138: 105037, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31394257

RESUMO

This study aimed to develop a PLGA, Levan-based drug delivery system (DDS) of Curcumin using a quality-by-design (QbD) approach to reveal how formulation parameters affect the critical quality attributes (CQAs) of this DDS and to present an optimal design. First, a risk assessment was conducted to determine the impact of various process parameters on the CQAs of the DDS (i.e., average particle size, ZP, encapsulation efficiency and polydispersity index). Plackett-Burman design revealed that potential risk factors were Levan molecular weight, PLGA amount and acetone amount. Then, the optimization of the DDS was achieved through a Box-Behnken Design. The optimum formulation was prepared using low molecular weight Levan (134 kDa), 51.51 mg PLGA and 10 ml acetone. The model was validated and the optimized formulation was further characterized using different physic-chemical methods. The study resulted in the most stable NP with a spherical and uniform shape and physical stability tests indicated its stability for at least 60 days at room temperature. In conclusion, this study was an effort for developing a DDS which solubilizes Curcumin in clinically applicable concentrations.


Assuntos
Curcumina/química , Frutanos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Acetona/química , Química Farmacêutica/métodos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Peso Molecular , Nanopartículas/química , Tamanho da Partícula , Medição de Risco/métodos , Temperatura
11.
J Hazard Mater ; 374: 43-49, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30978629

RESUMO

In this study, the potential use of phosphonated Halomonas Levan (PhHL) as a natural and cost effective adsorbent for Bisphenol A (BPA), was systematically investigated via the study of the adsorption equilibrium, kinetics, and reuse potential as well as the interpretation of adsorption mechanism. The effects of pH and temperature on the adsorption were also evaluated. The maximum amount of BPA adsorbed on the unit weight of PhHL was determined as 104.8 (∓5.02) mg/g (at 298 K) and the maximum adsorption capacity was calculated as 126.6 mg/g by Sips model. FTIR and XPS studies were conducted to elucidate the adsorption mechanism. Based on the obtained results OH-pi and CH-pi interactions were found to be effective in the adsorption mechanism. The reuse ability was studied with three cycles of adsorption-desorption, and the results showed that the BPA adsorbed per gram of the PhHL decreased 28.6% after the third cycle. This study has shown that PhHL can be used as an effective adsorbent for the removal of BPA from aqueous solutions. The obtained results may be useful in the development of PhHL based adsorption systems for the removal of EDCs with similar chemical properties to BPA.


Assuntos
Compostos Benzidrílicos/análise , Frutanos/química , Fenóis/análise , Purificação da Água/métodos , Adsorção , Análise Custo-Benefício , Halomonas/química , Concentração de Íons de Hidrogênio , Cinética , Microscopia Eletrônica de Varredura , Ácidos Fosforosos/química , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Água , Poluentes Químicos da Água/análise , Purificação da Água/instrumentação
12.
Mater Sci Eng C Mater Biol Appl ; 100: 798-808, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948117

RESUMO

Electrospraying assures many advantages with taking less time and costing less relatively to the other conventional particle production methods. In this research, we investigated the encapsulation of melatonin (MEL) hormone in polycaprolactone (PCL) microparticles by using electrospraying method. Morphology analysis of the produced particles completed with Scanning Electron Microscopy (SEM). SEM images demonstrated that micro-particles of 3 wt% PCL solution has the most suitable particle diameter size (2.3 ±â€¯0.64 µm) for melatonin encapsulation. According to the characterization of the particles, electrospraying parameters like optimal collecting distance, the flow rate of the solution and voltage of the system detected as 8 cm, 0.5 ml/h, and 10 kV respectively. For determining the chemical bonds of scaffold Fourier-Transform Infrared Spectroscopy (FTIR) were used and FTIR results showed that melatonin successfully loaded into PCL micro-particles. Drug release kinetics of the melatonin loaded particles indicated that melatonin released with a burst at the beginning and release behavior became sustainable over a period of 8 h with the encapsulation efficiency of about 73%. In addition, both in-vitro and in-vivo studies of the graft materials also completed. Primary human osteoblasts (HOB) cells and female Sprague Dawley rats were used in in-vitro and in-vivo studies. Test results demonstrate cell population, and bone volume of the rats grafted with composites has remarkably increased, this caused remodelling in bone structure. Overall, these findings indicate that encapsulation of melatonin in the PCL particles with electrospray method is optimum for new synthetic graft material.


Assuntos
Melatonina/farmacologia , Microesferas , Poliésteres/química , Alicerces Teciduais/química , Animais , Varredura Diferencial de Calorimetria , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Feminino , Humanos , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Ratos Sprague-Dawley , Crânio/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura
13.
Food Chem ; 276: 174-179, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30409581

RESUMO

Preconcentrations of Cu(II), Ni(II) and Pb(II) ions by using Coprinus silvaticus immobilized multiwalled carbon nanotube (MWCNT) were investigated. Effects of important parameters on preconcentration procedure were examined. The best pH values of for Cu(II), Ni(II) and Pb(II) were found to be 6.0, 6.0 and 4.0, respectively. Flow rate of sample solution was 2.0 mL min-1, while desorption was achieved at 1.0 mL min-1 flow rate. Preconcentration factors were achieved as 60 for Cu(II), Ni(II) and 70 for Pb(II) (by dividing initial sample volume to final volume). LODs were calculated as 0.014, 0.016 and 0.093 ng mL-1, respectively for Cu(II), Ni(II) and Pb(II). Accuracy of the method was checked by applying to certified reference samples. Inductively coupled plasma optical emission spectrometer (ICP OES) was employed for measurements of Cu(II), Ni(II) and Pb(II) in digested baby food samples.


Assuntos
Cobre/análise , Coprinus/metabolismo , Alimentos Infantis/análise , Chumbo/análise , Nanotubos de Carbono/química , Níquel/análise , Adsorção , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Cobre/isolamento & purificação , Coprinus/química , Análise de Alimentos , Humanos , Concentração de Íons de Hidrogênio , Lactente , Íons/química , Chumbo/isolamento & purificação , Limite de Detecção , Níquel/isolamento & purificação , Extração em Fase Sólida , Espectrofotometria Atômica
14.
Bioinspir Biomim ; 14(1): 011001, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30457113

RESUMO

Levan is a fructose homopolysaccharide which gained attention recently for its unusual combination of properties distinguishing it from other natural biodegradable polysaccharides like chitosan, cellulose or starch. Among the strongest bioadhesives, film-forming levan is garnering interest for its role in some simple solutions to difficult problems. One of these is illustrated by the elegant research using laser-based techniques to construct levan films for healing wounds and burned tissue. Another is the development of bioresorbable electronic implants. Levan has been found in habitats as diverse as salterns and thermal waters to tropical plants and sugar factories. This review of the low viscosity, levan adhesive describes the mechanisms by which it forms bonds and the reasons behind some of its practical and industrial applications. Here we present descriptions from the literature for feasible approaches ready to transition from the laboratory to those searching for answers in fields as varied as medicine, packaging and furniture assembly.


Assuntos
Adesivos/farmacologia , Frutanos/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Humanos , Marketing/métodos , Viscosidade
15.
Carbohydr Polym ; 193: 316-325, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29773387

RESUMO

This represents the first systematic study where levan polysaccharide was used to fabricate fibrous matrices by co-axial and single-needle electrospinning techniques. For this, hydrolyzed (hHL) and sulfated hydrolyzed (ShHL) Halomonas levan were chemically synthesized and used together with polycaprolactone (PCL) and polyethyleneoxide (PEO) for the spinning process. In co-axially spun matrices, ultimate tensile strength (UTS) were found to increase with increasing ShHL concentration and elongation at break of PCL + ShHL matrices increased up to ten-fold when compared to PCL matrices. Similarly, in single-needle spun matrices, higher elongation at break values were obtained by blending HL and ShHL with PEO pointing to the effective energy absorbing features. Dense and fine fibers were characterized by FTIR and SEM. Cell viability and fluorescence imaging of L929 fibroblasts and HUVECs as well as heparin mimetic activity of the matrices pointed to their high potential to be used in decreasing neointimal proliferation and thrombogenicity of grafts and prosthesis.

16.
OMICS ; 22(3): 198-209, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29649388

RESUMO

Metabolic systems engineering is being used to redirect microbial metabolism for the overproduction of chemicals of interest with the aim of transforming microbial hosts into cellular factories. In this study, a genome-based metabolic systems engineering approach was designed and performed to improve biopolymer biosynthesis capability of a moderately halophilic bacterium Halomonas smyrnensis AAD6T producing levan, which is a fructose homopolymer with many potential uses in various industries and medicine. For this purpose, the genome-scale metabolic model for AAD6T was used to characterize the metabolic resource allocation, specifically to design metabolic engineering strategies for engineered bacteria with enhanced levan production capability. Simulations were performed in silico to determine optimal gene knockout strategies to develop new strains with enhanced levan production capability. The majority of the gene knockout strategies emphasized the vital role of the fructose uptake mechanism, and pointed out the fructose-specific phosphotransferase system (PTSfru) as the most promising target for further metabolic engineering studies. Therefore, the PTSfru of AAD6T was restructured with insertional mutagenesis and triparental mating techniques to construct a novel, engineered H. smyrnensis strain, BMA14. Fermentation experiments were carried out to demonstrate the high efficiency of the mutant strain BMA14 in terms of final levan concentration, sucrose consumption rate, and sucrose conversion efficiency, when compared to the AAD6T. The genome-based metabolic systems engineering approach presented in this study might be considered an efficient framework to redirect microbial metabolism for the overproduction of chemicals of interest, and the novel strain BMA14 might be considered a potential microbial cell factory for further studies aimed to design levan production processes with lower production costs.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Frutanos/biossíntese , Engenharia Genética , Genoma Bacteriano , Genômica , Modelos Biológicos , Fenômenos Fisiológicos Bacterianos , Biologia Computacional/métodos , Fermentação , Técnicas de Inativação de Genes , Engenharia Genética/métodos , Genômica/métodos , Engenharia Metabólica/métodos , Anotação de Sequência Molecular , Mutação
17.
Int J Biol Macromol ; 107(Pt B): 2312-2319, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29055698

RESUMO

Levan is a high molecular weight fructose-based biotechnologically available polysaccharide with a range of interesting properties qualifying this molecule for applications in biomedicine. In this study, new levan derivatives containing methacrylate groups attached either via ester or urethane linkages to the fructan backbone could be synthesized and structurally characterized by conventional analytical techniques. The photochemical crosslinking of these substances applying different photoinitiator systems and reaction conditions resulted in hydrogels of diverse properties which were investigated with regard to mechanical behaviour, hydrolytic degradability, and cytocompatibility. It was found that crosslinkable levan derivatives represent a new class of promising biopolymer-based macromers broadening the spectrum of available biomaterials to facilitate the adaption to the requirements of specific applications.


Assuntos
Frutanos/química , Hidrogéis/química , Metacrilatos/química , Polissacarídeos/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Reagentes de Ligações Cruzadas/química , Frutose/química , Hidrogéis/síntese química , Processos Fotoquímicos , Polietilenoglicóis/química , Polissacarídeos/síntese química
18.
Carbohydr Polym ; 165: 61-70, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28363576

RESUMO

Levan based cross-linker was successfully synthesized and used to prepare a series of more biocompatible and temperature responsive levan/N-isopropyl acrylamide (levan/pNIPA) hydrogels by redox polymerization at room temperature. Volume phase transition temperature (VPTT) of the hydrogels were precisely determined by derivative differential scanning calorimetry (DDSC). Incorporation of levan into the pNIPA hydrogel increased the VPTT from 32.8°C to 35.09°C, approaching to body temperature. Swelling behavior and 5-aminosalicylic acid (5-ASA) release of the hydrogels were found to vary significantly with temperature and composition. Moreover, a remarkable increase in thermal stability of levan within hydrogel with increase of pNIPA content was recorded. The biocompatibility of the hydrogels were tested against mouse fibroblast L929 cell line in phosphate buffer saline (PBS, pH 7.4). The hydrogels showed increasing biocompatibility with increasing levan ratio, indicating levan enhanced the hydrogel surface during swelling.

19.
Int J Biol Macromol ; 102: 565-570, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28412340

RESUMO

Exopolysaccharides are high-valued bio-products produced by various microbial species and have been described to possess biological response modifying activities. These bio-products have been effective as therapeutic agents in various human disease conditions. The objective of this study was to examine the effects of levan (a (2→6)-ß-d-fructan) produced on sucrose by the halophilic bacterium, Halomonas smyrnensis AAD6T, in human breast cancer MCF-7 cells. MCF-7 cells were exposed to levan for 24 and 48h. The antiproliferative activity was analyzed by the MTT assay. Oxidative stress was measured by the CM-H2DCFDA assay, and cell apoptosis was analyzed by the caspase-3/7 assay. Cell cycle was analyzed by flow cytometry and gene expression was determined by RT-PCR. Levan showed a time- and concentration-dependent antiproliferative activity, and this effect was associated with an increase in cell apoptosis and oxidative stress. In addition, levan increased the gene expression of p53 and p27. Here we demonstrated that levan exhibited an antiproliferative effect that was mediated by an increase in apoptosis and oxidative stress.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Frutanos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Antígeno Nuclear de Célula em Proliferação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética
20.
Pharm Dev Technol ; 22(5): 627-634, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26607946

RESUMO

Encapsulation of vancomycin (VANCO) into biodegradable levan microparticles was achieved using a simple preparation technique. Microparticles were prepared by using levan polysaccharide produced by a halophilic bacterium Halomonas smyrnensis AAD6T. To optimize efficiency of encapsulation process by precipitation method, three parameters were studied: drug and polymer concentrations and preparation rotating speed. The particles were characterized in vitro. The size of levan microparticles was changed between 0.404 µm and 1.276 µm. The surface charge was detected between +4.1 mV and +6.5 mV. The highest drug encapsulation capacity of the system was 74.7% and was depending on the polymer concentration. In dissolution studies, initial burst effect around 10-20% from all the formulations was observed and then the release was slowed down and continued at a constant level. In vitro antibiotic release from the microparticles was controlled with the drug carrier system and release fit to Higuchi kinetic model. All the released samples collected at different time intervals during dissolution studies have exhibited intrinsic bactericidal activity against Bacillus subtilis ATCC 6633. WST-1 cell proliferation and viability studies showed that VANCO-loaded levan microparticles at concentrations between 100 µg/mL and 1000 µg/mL were nontoxic to L929 cells. As conclusion, levan microparticulate system could be a potential carrier of antibiotic drugs such as VANCO.


Assuntos
Antibacterianos , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Vancomicina , Frutanos , Tamanho da Partícula
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