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1.
ACS Omega ; 9(6): 6578-6587, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38371800

RESUMO

Polymer blending offers an effective and economical approach to overcome the performance limitations of poly(lactic acid) (PLA). In this study, a series of copolymers poly(ethylene succinate-co-lactic acid) (PESL) were synthesized, featuring lactic acid (LA) contents that ranged from 20 to 86 wt %. This synthesis involved a one-pot industrial melt polycondensation process using succinic acid (SA), ethylene glycol (EG), and LA, catalyzed by titanium tetraisopropoxide (TTP). The goal was to produce a fully biobased copolymer expected to exhibit partial miscibility with pure poly(lactic acid) (PLA). To assess the capability of PESL copolymers in toughening PLA, we conducted tensile testing on PLA/PESL blends containing 15 wt % PESL. As a result, an elongation at break for the blends with 15 wt % loading of the copolymer PESL72 was directly enhanced to 250% with an ultimate strength of 35 MPa, compared to brittle PLA with less 10% tensile length. The morphological features of interfacial adhesion before and after tensile failure were measured by scanning electron microscopy (SEM). A significant enhancement in the chain mobility of the PLA/PESL blends was further evidenced by differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). These findings hold promise for the development of functional packaging materials based on PLA. The proposed copolymer design, which boasts strong industrial feasibility, can serve as a valuable guide for enhancing the toughness of PLA.

2.
J Control Release ; 367: 93-106, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38237690

RESUMO

The administration of protein therapeutics through oral means is seen as a convenient and painless experience for patients, making it a significant consideration in the field of drug delivery. Nevertheless, the challenging conditions within the gastrointestinal tract, along with the obstacles to absorption, impede the efficient transportation of proteins. Here, we successfully implemented post-synthetic modifications to attach medium-chain lipids (C10) onto the surface of zeolitic imidazole framework-90 (ZIF-90), then encapsulated the nanoparticles with sodium alginate, resulting in a potential platform for the oral administration of proteins. By means of biomimetic mineralization, ZIF-90 achieves a simple and efficient encapsulation of proteins of varying sizes, while shielding them against degradation by digestive enzymes. Sodium alginate hydrogel protects proteins against gastric acid and helps the cargo to rapidly penetrate the mucus layer. Through a mixed mechanism dominated by micropinocytosis, the C10-conjugated ZIF-90 (ZIF-90-C10) can be uptake by Caco-2 cells with a 200-400% increase and transported through the Golgi apparatus after escaping from lysosomes, exhibiting enhanced uptake in the overall gastrointestinal tract. Furthermore, ZIF-90-C10 retains its adenosine triphosphate-responsive release, which drastically lowers the likelihood of accumulation in vivo and allows targeted delivery for disease cells. Our work highlights mid-chain lipid conjugation as a potent approach to enhancing nanoparticle delivery efficiency and a potential strategy for oral delivery of biomacromolecules when combined with pH-responsive gels.


Assuntos
Estruturas Metalorgânicas , Nanopartículas , Zeolitas , Humanos , Hidrogéis , Células CACO-2 , Sistemas de Liberação de Medicamentos/métodos , Imidazóis , Alginatos , Lipídeos
3.
Biotechnol Bioeng ; 120(10): 3039-3056, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37309999

RESUMO

ß-elemene is one of the most commonly used antineoplastic drugs in cancer treatment. As a plant-derived natural chemical, biologically engineering microorganisms to produce germacrene A to be converted to ß-elemene harbors great expectations since chemical synthesis and plant isolation methods come with their production deficiencies. In this study, we report the design of an Escherichia coli cell factory for the de novo production of germacrene A to be converted to ß-elemene from a simple carbon source. A series of systematic approaches of engineering the isoprenoid and central carbon pathways, translational and protein engineering of the sesquiterpene synthase, and exporter engineering yielded high-efficient ß-elemene production. Specifically, deleting competing pathways in the central carbon pathway ensured the availability of acetyl-coA, pyruvate, and glyceraldehyde-3-phosphate for the isoprenoid pathways. Adopting lycopene color as a high throughput screening method, an optimized NSY305N was obtained via error-prone polymerase chain reaction mutagenesis. Further overexpression of key pathway enzymes, exporter genes, and translational engineering produced 1161.09 mg/L of ß-elemene in a shake flask. Finally, we detected the highest reported titer of 3.52 g/L of ß-elemene and 2.13 g/L germacrene A produced by an E. coli cell factory in a 4-L fed-batch fermentation. The systematic engineering reported here generally applies to microbial production of a broader range of chemicals. This illustrates that rewiring E. coli central metabolism is viable for producing acetyl-coA-derived and pyruvate-derived molecules cost-effectively.


Assuntos
Escherichia coli , Sesquiterpenos , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Acetilcoenzima A/metabolismo , Sesquiterpenos/metabolismo , Carbono/metabolismo
4.
Macromol Rapid Commun ; 43(15): e2100887, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35279032

RESUMO

The covalent cross-linking is an essential prerequisite for achieving the unique entropic elasticity of rubber products; however, the formation of a 3D cross-linked network and permanent cross-links makes thermosetting rubbers difficult to be recycled, causing serious environmental pollution at the end of their life. Herein, a facile, green, and promising strategy to introduce the exchangeable and cleavable acetal bonds into the chemically cross-linked networks of diene-typed rubbers is reported. For the first time, the hydroxyl-functionalized styrene-butadiene rubber (ESBR-HEMA) is prepared by introducing 2-hydroxyethyl methacrylate (HEMA) during the emulsion polymerization of styrene-butadiene rubber (ESBR). Then, based on hydroxyl-vinyl ether addition reactions, divinyl ether (DVE) could serve as a cross-linking agent to facilely and effectively cross-link hydroxyl-functionalized rubbers without additional additives, producing exchangeable and hydrolyzable acetal linkages. What's more, the acetal-containing cross-linked network in ESBR-HEMA vulcanizates could rearrange their topologies at elevated temperatures, endowing them with malleable and thermal reprocessing abilities. Moreover, the hydrolyzable acetal bonds could be selectively cleaved into hydroxyl and aldehyde groups in acidic conditions, resulting in a closed-loop chemical recycling of the ESBR-HEMA rubber. Hence, this work provides a facile and green cross-linking strategy for hydroxyl-functionalized rubbers to address the inherent problems brought from the covalent cross-linking of rubbers.


Assuntos
Acetais , Borracha , Butadienos/química , Elastômeros , Borracha/química , Estirenos
5.
Environ Sci Eur ; 33(1): 99, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34458054

RESUMO

This article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics wastes combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Known plastic-degrading enzymes will be optimised by integrated protein engineering to achieve high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt and temperature conditions. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will formulate enzyme cocktails tailored to specific waste streams and strives to enhance enzyme production significantly. In vivo and in vitro application of these cocktails enable stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any remaining material recalcitrant to the enzymatic activities will be recirculated into the process by physicochemical treatment. The Chinese-European MIX-UP consortium is multidisciplinary and industry-participating to address the market need for novel sustainable routes to valorise plastic waste streams. The project's new workflow realises a circular (bio)plastic economy and adds value to present poorly recycled plastic wastes where mechanical and chemical plastic recycling show limits.

6.
ACS Biomater Sci Eng ; 7(7): 3190-3200, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34152745

RESUMO

As the most representative family of proteinases related to tumorigenesis, matrix metalloproteinase-9 (MMP-9) represents a key player in cancer cell migration and regulation of the tumor microenvironment. The inhibition of MMP-9 activity has been pursued as a target for anticancer therapy. However, most synthetic MMP-9 inhibitors have failed in clinical trials because of their lack of selectivity. Here, an abiotic mimic based on molecularly imprinted nanoparticles has been designed as an inhibitor for MMP-9. To attain fast mass transfer and facilitate multifunctional roles, we synthesized the imprinted polymer thin layer on the surface of gold nanorods by reversible addition-fragmentation chain transfer polymerization using MMP-9 as the template, which captures MMP-9 selectively and inhibits its activity by providing steric hindrance to the activity-related domain of MMP-9. In vitro cell experiments and in vivo studies in mice demonstrate that the imprinted artificial antibody suppresses the migration and growth of metastatic tumors. The tumor growth inhibition rate reaches up to 54 ± 15%. Compared with the typical photothermal therapy induced by gold nanorods, the use of MMP-9-imprinted synthetic antibody could better inhibit the lung tumor metastasis by quenching the enzyme activity of MMP-9. This study offers a new paradigm in the engineering of imprinted nanoparticles as inhibitors for cancer therapy.


Assuntos
Neoplasias Pulmonares , Inibidores de Metaloproteinases de Matriz , Animais , Movimento Celular , Neoplasias Pulmonares/tratamento farmacológico , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Microambiente Tumoral
7.
J Air Waste Manag Assoc ; 70(2): 219-227, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31971493

RESUMO

The "green" production of bitumen has raised increasing interest in recent years to reduce the environmental, energy-related and petro-based concerns. Bio-oil, prepared by biomass pyrolysis, can be used as a substitute for petro-based bitumen in bitumen or bitumen-based coatings, for its similar properties of good adhesion and anti-corrosion characteristics as bitumen. Although biomass is a renewable and widespread chemical resource, its high-valued utilization is still difficult. Several studies have qualitatively demonstrated the use of bio-bitumen in practical applications. The present study investigates that adding some bio-oil to traditional bitumen to form a bio-bitumen could help improve the properties of traditional bitumen. Bio-bitumen was prepared from biomass pyrolysis oil and applied to self-adhesive and doped hot-melt sheets. Results of physical properties demonstrate that bio-bitumen is a potential substitute in bitumen coating sheet.Implications: This paper is intended to verify the effect of pyrolyzed bio-oil from wheat straw on the performance of bitumen, as well as the feasibility of application in the coating sheet. Up to now, the research on bio-bitumen is mainly in pavement bitumen. In the present research, bio-bitumen was applied to the coating sheet in different proportions. Interestingly, the prepared coating sheet exhibited higher adhesion. Other performances, such as temperature stability, mechanical strength and temperature flexibility of coating sheet showed improvement in the presence of bio-oil, which indicated the suitability of bio-oil in coating sheet bitumen.


Assuntos
Hidrocarbonetos , Óleos de Plantas , Polifenóis , Biomassa , Temperatura Alta , Pirólise , Triticum
8.
Chem Asian J ; 14(20): 3502-3514, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31339213

RESUMO

Traditional films cannot fully adapt to industrial applications and to intensified processes. Advanced mixed-matrix membranes comprising metal-organic frameworks (MOF) embedded in a polymer matrix have been developed with the goal of breaking the trade-off effect of traditional polymer membranes and achieving separation performance beyond Robeson's upper limit. The key challenges in the fabrication of MOF-based mixed-matrix membranes are an enhancement in compatibility between the inorganic filler and the polymer matrix, elimination of the irregular morphology and non-selective interfacial defects, and further improvement in the gas-separation performance. This review summarizes the recent advances in protocols and strategies in terms of designing interfacial interactions to enhance the MOF/polymer interface compatibility. This review aims at providing some meaningful insights into preparing MOF-based mixed-matrix membranes targeting ideal interfacial morphology and leading to excellent gas-separation performance.

9.
Biotechnol Biofuels ; 11: 134, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29760776

RESUMO

BACKGROUND: The production of biobutanol from renewable biomass resources is attractive. The energy-intensive separation process and low-titer solvents production are the key constraints on the economy-feasible acetone-butanol-ethanol (ABE) production by fermentation. To decrease energy consumption and increase the solvents concentration, a novel two-stage gas stripping-salting-out system was established for effective ABE separation from the fermentation broth using sweet sorghum bagasse as feedstock. RESULTS: The ABE condensate (143.6 g/L) after gas stripping, the first-stage separation, was recovered and introduced to salting-out process as the second-stage. K4P2O7 and K2HPO4 were used, respectively. The effect of saturated salt solution temperature on final ABE concentration was also investigated. The results showed high ABE recovery (99.32%) and ABE concentration (747.58 g/L) when adding saturated K4P2O7 solution at 323.15 K and 3.0 of salting-out factor. On this condition, the energy requirement of the downstream distillation process was 3.72 MJ/kg of ABE. CONCLUSIONS: High-titer cellulosic ABE production was separated from the fermentation broth by the novel two-stage gas stripping-salting-out process. The process was effective, which reduced the downstream process energy requirement significantly.

10.
J Agric Food Chem ; 66(20): 5200-5209, 2018 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-29722539

RESUMO

In the biosynthesis of S-adenosylmethionine (SAM) in baker's yeast ( Saccharomyces cerevisiae), ATP functions as both a precursor and a driving force. However, few published reports have dealt with the control of ATP concentration using genetic design. In this study we have adopted a new ATP regulation strategy in yeast for enhancing SAM biosynthesis, including altering NADH availability and regulating the oxygen supply. Different ATP regulation systems were designed based on the introduction of water-forming NADH oxidase, Vitreoscilla hemoglobin, and phosphite dehydrogenase in combination with overexpression of the gene SAM2. Via application of this strategy, after 28 h cultivation, the SAM titer in the yeast strain ABYSM-2 reached a maximum level close to 55 mg/L, an increase of 67% compared to the control strain. The results show that the ATP regulation strategy is a valuable tool for SAM production and might further enhance the synthesis of other ATP-driven metabolites in yeast.


Assuntos
Trifosfato de Adenosina/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Vias Biossintéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/economia , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Bioprocess Biosyst Eng ; 41(9): 1329-1336, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29846810

RESUMO

In this study, aiming to improve the economic feasibility of acetone-butanol-ethanol (ABE) fermentation process, generate valuable products and extend the product chain, esterification catalyzed by Candida sp. 99-125 cells was hybrid with the ABE fermentation-gas-stripping integration system. The gas-stripping condensate that contained concentrated ABE products was directly used for esterification without the participation of toxic organic solvents. Full-cell catalysis temperature and the cell dosage rate on oleate production were evaluated and optimized in the esterification process. Under the optimized conditions (35 °C, 8% of cells), ~ 68% of butyl oleate and ~ 12% of ethyl oleate were obtained after 4 h of esterification. The Candida sp. 99-125 cells were able to be reused for at least four cycles. The novel cascade process showed environmental benefits, which also showed promising in improving the economic feasibility of the conventional ABE fermentation process.


Assuntos
Acetona/metabolismo , Biocatálise , Butanóis/metabolismo , Candida/crescimento & desenvolvimento , Etanol/metabolismo , Ácidos Oleicos/biossíntese , Esterificação
12.
J Environ Manage ; 217: 196-206, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29604413

RESUMO

The extraction of vegetable oils is an energy-intensive process. It has moreover a significant environmental impact through hexane emissions and through the production of organic-loaded wastewater. A rice bran oil process was selected as the basis, since full data were available. By using Aspen Plus v8.2 simulation, with additional scripts, several improvements were examined, such as using heat exchanger networks, integrating a Vapor Recompression Heat Pump after the evaporation and stripping, and examining a nitrogen stripping of hexane in the rice bran meal desolventizing unit followed by a gas membrane to recover hexane. Energy savings by the different individual and combined improvements are calculated, and result in a 94.2% gain in steam consumption and a 73.8% overall energy saving. The power consumption of the membrane unit reduces the overall energy savings by about 5%. Hexane separation and enrichment by gas membranes facilitates its condensation and re-use, while achieving a reduction of hexane emissions by over 50%. Through the considerable reduction of required steam flow rates, 61% of waste water is eliminated, mostly as organic-loaded steam condensate. Through overall energy savings, 52% of related CO2 emissions are eliminated.


Assuntos
Óleos de Plantas , Dióxido de Carbono , Temperatura Alta , Oryza , Óleo de Farelo de Arroz , Solventes
13.
Biotechnol Biofuels ; 11: 30, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29445419

RESUMO

BACKGROUND: The production of fuels and chemicals from renewable resources is increasingly important due to the environmental concern and depletion of fossil fuel. Despite the fast technical development in the production of aviation fuels, there are still several shortcomings such as a high cost of raw materials, a low yield of aviation fuels, and poor process techno-economic consideration. In recent years, olefin metathesis has become a powerful and versatile tool for generating new carbon-carbon bonds. The cross-metathesis reaction, one kind of metathesis reaction, has a high potential to efficiently convert plant oil into valuable chemicals, such as α-olefin and bio-aviation fuel by combining with a hydrotreatment process. RESULTS: In this research, an efficient, four-step conversion of plant oil into bio-aviation fuel and valuable chemicals was developed by the combination of enzymatic transesterification, olefin cross-metathesis, and hydrotreating. Firstly, plant oil including oil with poor properties was esterified to fatty acid methyl esters by an enzyme-catalyzed process. Secondly, the fatty acid methyl esters were partially hydrotreated catalytically to transform poly-unsaturated fatty acid such as linoleic acid into oleic acid. The olefin cross-metathesis then transformed the oleic acid methyl ester (OAME) into 1-decene and 1-decenoic acid methyl ester (DAME). The catalysts used in this process were prepared/selected in function of the catalytic reaction and the reaction conditions were optimized. The carbon efficiency analysis of the new process illustrated that it was more economically feasible than the traditional hydrotreatment process. CONCLUSIONS: A highly efficient conversion process of plant oil into bio-aviation fuel and valuable chemicals by the combination of enzymatic transesterification, olefin cross-metathesis, and hydrotreatment with prepared and selected catalysts was designed. The reaction conditions were optimized. Plant oil was transformed into bio-aviation fuel and a high value α-olefin product with high carbon utilization.

14.
Appl Biochem Biotechnol ; 185(2): 385-395, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29164506

RESUMO

Glutathione (GSH) is an important reducing agent in the living cells. It is synthesized by a two-step reaction and requires two molecules of adenosine triphosphate (ATP) for one molecule GSH. The enzymatic cascade reaction in vitro is a promising approach to achieve a high titer and limit side reactions; although, a cost-effective phosphate donor for ATP regeneration is required. Triphosphate (PolyP(3)), tetraphosphate (PolyP(4)), and hexametaphosphate (PolyP(6)) were investigated in this study. Triphosphate inhibited the bifunctional GSH synthetase (GshF) from Streptococcus agalactiae, while no significant inhibition was observed by adding hexametaphosphate. The polyphosphate kinase from Corynebacterium glutamicum was hence investigated to use hexametaphosphate for regeneration of ATP. Further, the orthogonal experiment, which includes seven factors (buffer concentration, pH value, ADP concentration, GshF dosage, polyphosphate kinase (PPK) dosage, reaction temperature, substrate ratio of amino acid, and reaction times), indicated that the capacity of buffer is the most significant factor of the reaction conditions for enzymatic production of glutathione coupling with a PPK-based ATP regeneration system. After optimizing the Mg2+ concentration, the reaction was scaled up to 250 mL in a stirred reactor with pH feedback control to stabilize the pH value of reaction system and nitrogen protection to avoid the oxidation of product. A yield of 12.32 g/L was achieved. This work provided a potential GshF-based enzymatic way coupling the PPK-based ATP regeneration to product GSH in the optimal conditions towards cost-effectiveness at the industrial scale.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Corynebacterium glutamicum/enzimologia , Glutationa Sintase/química , Glutationa/química , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Polifosfatos/química , Streptococcus agalactiae/enzimologia
15.
Anal Chem ; 89(23): 12896-12901, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29120617

RESUMO

HIV latency is one of the major problems in HIV/AIDS cure. Imaging single-copy integrated proviral HIV DNA in host cell has both virology and clinical significance but remains technical challenge. Here, we developed a dual-color labeled CRISPR system to image the HIV-1 integrated proviral DNA in latently infected cells. The pair of CRISPRs was fluorescently labeled with two different color QDs using two alternative bioorthogonal ligation reactions. Integrated HIV-sequences are successfully mapped based on the colocalized signals of QDs in living cells. Compared to the existing zinc finger proteins and TALENs, the CRISPR system is much easier to operate and more efficient in imaging of internal genomic loci. Therefore, the proposed method could be not only a powerful tool for imaging proviral HIV-1, but also a versatile platform to image single genomic loci in living cells.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA Viral/genética , Infecções por HIV/diagnóstico por imagem , HIV-1/genética , Provírus/genética , Linhagem Celular Tumoral , Cor , Humanos , Pontos Quânticos/química
16.
Enzyme Microb Technol ; 104: 16-21, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28648175

RESUMO

Mannose-6-phosphate is an important phosphor-sugar, which is involved in many physiological functions and it is used to treat many diseases. Its production is however expensive since it requires costly substrate ATP as phosphorylation agent. This study has focused upon the direct synthesis of M6P by glucomannokinase using inorganic polyphosphate without involvement of ATP. The gene cloned for glucomannokinase has been sequenced from Mycobacterium phlei and it is transformed into Escherichia coli for expression. After purification involving affinity chromatography, a band of 30kDa corresponding to the enzyme has been isolated from induced crude supernatant. A total amount of 0.69mg/ml of enzyme has been successively obtained and the purity exceeds 90%. The kinetic assay studies show that this enzyme has more affinity towards polyphosphate and glucose than ATP and mannose respectively. The KM values of the enzyme for glucose, mannose, ATP and hexametaphosphate derived from experiments are 9.5, 203.7, 4.6, 1.7µM, respectively. The enzyme has shown a maximum production of mannose-6-phosphate at optimized conditions of pH 8.5, 25°C, poly(P)/mannose ratio 3:1 and in the presence of bivalent ion Mg2+. The results reveal that the glucomannokinase from Mycobacterium phlei suitable for further production of mannose-6-phosphate.


Assuntos
Manose/metabolismo , Mycobacterium phlei/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotecnologia , Glucose/metabolismo , Microbiologia Industrial , Cinética , Manosefosfatos/biossíntese , Mycobacterium phlei/genética , Fosfatos/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polifosfatos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
17.
Appl Microbiol Biotechnol ; 101(13): 5325-5332, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28417169

RESUMO

Adenosine-5'-triphosphate (ATP) is the energy equivalent of the living system. Polyphosphate (polyP) is the ancient energy storage equivalent of organisms. Polyphosphate kinases (PPKs) catalyze the polyP formation or ATP formation, to store energy or to regenerate ATP, respectively. However, most PPKs are active only in the presence of long polyPs, which are more difficult and more expensive to generate than the short polyPs. We investigated the PPK preference towards polyPs by site-directed mutagenesis and computational simulation, to understand the mechanism and further design enzymes for effective ATP regeneration using short polyPs for in vitro cascade reactions, which are highly desired for research and applications. The results suggest that the short polyPs inhibit PPK by blocking the ADP-binding pocket. Structural comparison between PPK (Corynebacterium glutamicum) and PPK (Sinorhizobium meliloti) indicates that three amino acid residues, i.e., lysine, glutamate, and threonine, are involved in the activity towards short polyP by fixing the adenosine group of ADP in between the subunits of the dimer, while the terminal phosphate group of ADP still offers an active site, which presents a binding pocket for ADP. A proposed triple mutant PPK (SMc02148-KET) demonstrates significant activity towards short polyP to form ATP from ADP. The obtained high glutathione titer (38.79 mM) and glucose-6-phosphate titer (87.35 mM) in cascade reactions with ATP regeneration using the triple mutant PPK (SMc02148-KET) reveal that the tailored PPK establishes the effective ATP regeneration system for ATP-dependent reactions.


Assuntos
Trifosfato de Adenosina/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Polifosfatos/metabolismo , Aminoácidos/metabolismo , Produtos Biológicos/metabolismo , Catálise , Domínio Catalítico , Química Bioinorgânica/economia , Química Bioinorgânica/métodos , Simulação por Computador , Corynebacterium glutamicum/química , Corynebacterium glutamicum/enzimologia , Escherichia coli/genética , Glucose-6-Fosfato/biossíntese , Glutationa/análise , Glutationa/biossíntese , Mutagênese Sítio-Dirigida , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Sinorhizobium meliloti/química , Sinorhizobium meliloti/enzimologia
18.
Angew Chem Int Ed Engl ; 56(4): 966-971, 2017 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-27918129

RESUMO

The use of biomacromolecular therapeutics has revolutionized disease treatment, but frequent injections are required owing to their short half-life in vivo. Thus there is a need for a drug delivery system that acts as a reservoir and releases the drug remotely "on demand". Here we demonstrate a simple light-triggered local drug delivery system through photo-thermal interactions of polymer-coated gold nanoparticles (AuNPs) inside an agarose hydrogel as therapeutic depot. Localized temperature increase induced by the visible light exposure caused reversible softening of the hydrogel matrix to release the pre-loaded therapeutics. The release profile can be adjusted by AuNPs and agarose concentrations, light intensity and exposure time. Importantly, the biological activity of the released bevacizumab was highly retained. In this study we demonstrate the potential application of this facile AuNPs/hydrogel system for ocular therapeutics delivery through its versatility to release multiple biologics, compatibility to ocular cells and spatiotemporal control using visible light.


Assuntos
Sistemas de Liberação de Medicamentos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Luz , Fotoquimioterapia , Proteínas/química , Ouro/química , Humanos , Nanopartículas Metálicas/química , Tamanho da Partícula , Polímeros/química , Propriedades de Superfície
19.
Anal Chem ; 88(21): 10675-10679, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27700062

RESUMO

Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the single-molecule/particle level. Here, we report a strategy to delay photobleaching of a light-switch complex, [Ru(phen)2dppx]2+, by insertion of a six-cysteine peptide into virus particles. The six-cysteine peptide was inserted into viral protein R of HIV-1 and assembled into infectious HIV-1 viral particles, where it effectively delayed the photobleaching of the [Ru(phen)2dppx]2+ complex used to label viral genomic RNAs. This delay in photobleaching allowed for a monofluorescent assay to be constructed for the real-time monitoring of viral uncoating, a poorly understood process. This novel strategy to delay photobleaching in infectious viral particles provides a powerful method to analyze viral uncoating at the single-particle level in real time.


Assuntos
Complexos de Coordenação/efeitos da radiação , Corantes Fluorescentes/efeitos da radiação , HIV-1/metabolismo , Fotodegradação , Desenvelopamento do Vírus , Complexos de Coordenação/química , Cisteína/genética , Corantes Fluorescentes/química , Células HEK293 , HIV-1/genética , Células HeLa , Humanos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Peptídeos/genética , RNA Viral/química , Proteínas Recombinantes de Fusão/genética , Rutênio/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética
20.
Bioresour Technol ; 211: 704-10, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27060246

RESUMO

In this study, sweet sorghum juice (SSJ) was used as the substrate in a simplified ABE fermentation-gas stripping integration process without nutrients supplementation. The sweet sorghum bagasse (SSB) after squeezing the fermentable juice was used as the immobilized carrier. The results indicated that the productivity of ABE fermentation process was improved by gas stripping integration. A total 24g/L of ABE solvents was obtained from 59.6g/L of initial sugar after 80h of fermentation with gas stripping. Then, long-term of fed-batch fermentation with continuous gas stripping was further performed. 112.9g/L of butanol, 44.1g/L of acetone, 9.5g/L of ethanol (total 166.5g/L of ABE) was produced in overall 312h of fermentation. At the same time, concentrated ABE product was obtained in the condensate of gas stripping.


Assuntos
Acetona/metabolismo , Bebidas , Biotecnologia/métodos , Butanóis/metabolismo , Etanol/metabolismo , Fermentação , Sorghum/química , Técnicas de Cultura Celular por Lotes , Gases , Cinética
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