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1.
Bone Joint J ; 101-B(10): 1238-1247, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31564153

RESUMO

AIMS: Options for the treatment of intra-articular ligament injuries are limited, and insufficient ligament reconstruction can cause painful joint instability, loss of function, and progressive development of degenerative arthritis. This study aimed to assess the capability of a biologically enhanced matrix material for ligament reconstruction to withstand tensile forces within the joint and enhance ligament regeneration needed to regain joint function. MATERIALS AND METHODS: A total of 18 New Zealand rabbits underwent bilateral anterior cruciate ligament reconstruction by autograft, FiberTape, or FiberTape-augmented autograft. Primary outcomes were biomechanical assessment (n = 17), microCT (µCT) assessment (n = 12), histological evaluation (n = 12), and quantitative polymerase chain reaction (qPCR) analysis (n = 6). RESULTS: At eight weeks, FiberTape alone or FiberTape-augmented autograft demonstrated increased biomechanical stability compared with autograft regarding ultimate load to failure (p = 0.035), elongation (p = 0.006), and energy absorption (p = 0.022). FiberTape-grafted samples also demonstrated increased bone mineral density in the bone tunnel (p = 0.039). Histological evaluation showed integration of all grafts in the bone tunnels by new bone formation, and limited signs of inflammation overall. A lack of prolonged inflammation in all samples was confirmed by quantification of inflammation biomarkers. However, no regeneration of ligament-like tissue was observed along the suture tape materials. Except for one autograft failure, no adverse events were detected. CONCLUSION: Our results indicate that FiberTape increases the biomechanical performance of intra-articular ligament reconstructions in a verified rabbit model at eight weeks. Within this period, FiberTape did not adversely affect bone tunnel healing or invoke a prolonged elevation in inflammation. Cite this article: Bone Joint J 2019;101-B:1238-1247.


Assuntos
Lesões do Ligamento Cruzado Anterior/cirurgia , Reconstrução do Ligamento Cruzado Anterior/métodos , Polietilenos/química , Tendões/transplante , Resistência à Tração/fisiologia , Análise de Variância , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Feminino , Humanos , Peso Molecular , Coelhos , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Técnicas de Sutura , Suturas , Transplante Autólogo
2.
World J Microbiol Biotechnol ; 35(9): 133, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432254

RESUMO

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.


Assuntos
Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Virgibacillus/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Genoma Bacteriano , Peso Molecular , Família Multigênica , Peptídeos/química , Peptídeos/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Virgibacillus/classificação , Virgibacillus/isolamento & purificação
3.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
4.
J Agric Food Chem ; 67(37): 10373-10379, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453692

RESUMO

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase, which is an important enzyme for efficient saccharification of agarose or preparation of bioactive oligosaccharides from agarose. Although many ß-agarases have been reported and characterized, there are only a few studies on α-agarases. Here, we cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately 200 kDa belonging to glycoside hydrolase family 96 from Catenovulum agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37 °C and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as agarobiose (A2), agarotetraose (A4), and agarohexaose (A6), in which A4 is the dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose (A3) and A6 into A2 and A4 but cannot act on A2, A3, or A4. This is the first report to characterize the α-agarase action on AOSs in detail. Therefore, CaLJ96 has potential for the manufacture of bioactive AOSs.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Alteromonadaceae/química , Alteromonadaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sefarose/química , Sefarose/metabolismo , Especificidade por Substrato
5.
J Med Microbiol ; 68(9): 1359-1366, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31364964

RESUMO

Methodology. Biochemical and molecular methods were used to identify 100 lactobacilli isolated from rectal swabs. Among these, L. paracasei ssp. paracasei LP5 and L. brevis LP9 showed significant antibacterial activity against S. agalactiae and L. monocytogenes. Accordingly, characterization of their bacteriocins, BacLP5 and BacLP9, was conducted to obtain information on their kinetic production, sensitivity to chemico-physical parameters and molecular weight. To investigate the possible use of the two Lactobacillus strains as probiotics, their gastrointestinal resistance, cellular adhesiveness and sensitivity to antibiotics were also studied.Results. The obtained data show that BacLP5 and BacLP9 most likely belong to class II bacteriocins and both have a molecular weight of approximately 3 kDa. The production of BacLP5 and BacLP9 started after 4 h (40 and 80 AU ml-1), respectively. Both of the Lactobacillus strains survived gastric and intestinal juices well and showed adhesive capability on HEp-2 cells.Conclusion. Due to their peculiar antimicrobial characteristics, L. paracasei ssp. paracasei LP5 and L. brevis LP9 are suitable for use in the treatment of vaginal disorders, through both oral and transvaginal administration.


Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Lactobacillus brevis/metabolismo , Lactobacillus paracasei/metabolismo , Antibacterianos/química , Antibacterianos/isolamento & purificação , Aderência Bacteriana , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Ácidos e Sais Biliares/farmacologia , Linhagem Celular , Fenômenos Químicos , Suco Gástrico/metabolismo , Humanos , Lactobacillus brevis/classificação , Lactobacillus brevis/isolamento & purificação , Lactobacillus paracasei/classificação , Lactobacillus paracasei/isolamento & purificação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Peso Molecular , Probióticos , Reto/microbiologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/crescimento & desenvolvimento
6.
Bioresour Technol ; 292: 121940, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31419707

RESUMO

Spent coffee grounds are a huge residual stream from instant coffee makers. The production of spent coffee oil and molecular weight fractionated hemicellulose hydrolysates via supercritical CO2 and a hydrothermal treatment followed by concentration, separation, and purification through cascade ultrafiltration/diafiltration (30-10-5 kDa) was studied. Hemicelluloses extraction yield reached 3.49 g/100 g of dry defatted spent coffee after 40 min at 160 °C. The ultrafiltration system allowed concentrating up to 5-fold certain groups of hemicellulose, being most of them retained in the first membrane. Hemicellulose concentration and molecular weight of the feed exerted a great influence on the mass transfer through the membrane due to the formation of aggregates. However, purification through diafiltration allowed both to decrease by-products retentions from 45.6% to 8.7%, increasing the molecular weight of each fraction. Six hemicellulose products were obtained with purities between 83.7 and 97.8 wt% and weight-average molecular weights between 1641 and 49,733 Da.


Assuntos
Café , Ultrafiltração , Peso Molecular , Polissacarídeos
7.
Waste Manag ; 97: 1-9, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31447015

RESUMO

In this study, refractory organics in a membrane bioreactor (MBR) effluent were investigated following the treatment of landfill leachate by the ozone combined hydrogen peroxide (O3/H2O2) and microwave-activated persulfate (MW/PS) processes. The treatment efficiency and the transformation characteristics of refractory organics and reactive oxygen species were determined. It was found that an acidic environment and an increase in the O3 dosage improved the organic removal efficiency in the O3/H2O2 process, and the use of H2O2 improved the treatment efficiency, while excessive H2O2 inhibited it. In the MW/PS process, an increase in the PS dosage and MW power greatly improved the treatment efficiency, while an alkaline environment inhibited it. Under the optimized reaction parameters, the O3/H2O2 and MW/PS processes effectively degraded refractory organics (i.e., humic acid and fulvic acid) into components with a smaller molecular weight and simpler structure. The humification, aromaticity, and conjugation of organics in wastewater were greatly reduced. Compared to the O3/H2O2 process, the MW/PS process had a better treatment effect on refractory organics, and there were more low molecular weight organics (<1 kDa) in the treated wastewater. Because O3 is the main selective oxidant in the O3/H2O2 process, a large amount of organic acids were accumulated. A large amount of hydroxyl radicals and sulfate radicals with strong oxidation ability were produced in the MW/PS process, and therefore the combined action of hydroxyl and sulfate radicals can efficiently decompose humus and intermediate organics. Overall, the MW/PS process was more effective in treating the MBR effluent than the O3/H2O2 process. The results of this study provide a reference for the selection of an advanced oxidation process to eliminate refractory organics in landfill leachate.


Assuntos
Ozônio , Poluentes Químicos da Água , Reatores Biológicos , Peróxido de Hidrogênio , Peso Molecular , Oxirredução
8.
Waste Manag ; 97: 88-96, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31447031

RESUMO

The creation of a circular economy for cellulose based textile waste is supported by the development of an upcycling method for cotton polyester blended waste garments. We present a separation procedure for cotton and polyester using [DBNH] [OAc], a superbase based ionic liquid, which allows the selective dissolution of the cellulose component. After the removal of PET, the resulting solution could be employed to dry-jet wet spin textile grade cellulose fibers down to the microfiber range (0.75-2.95 dtex) with breaking tenacities (27-48 cN/tex) and elongations (7-9%) comparable to commercial Lyocell fibers made from high-purity dissolving pulp. The treatment time in [DBNH] [OAc] was found to reduce the tensile properties (<52%) and the molar mass distribution (<51%) of PET under certain processing conditions.


Assuntos
Celulose , Poliésteres , Fibra de Algodão , Peso Molecular , Têxteis
9.
Int J Nanomedicine ; 14: 5449-5475, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31409998

RESUMO

Purpose: We created and evaluated an enhanced topical delivery system featuring a combination of highly skin-permeable growth factors (GFs), quercetin (QCN), and oxygen; these synergistically accelerated re-epithelialization and granulation tissue formation of/in diabetic wounds by increasing the levels of GFs and antioxidants, and the oxygen partial pressure, at the wound site. Methods: To enhance the therapeutic effects of exogenous administration of GFs for the treatment of diabetic wounds, we prepared highly skin-permeable GF complexes comprised of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), platelet-derived growth factor-A (PDGF-A), and basic fibroblast growth factor (bFGF), genetically attached, via the N-termini, to a low-molecular-weight protamine (LMWP) to form LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, and LMWP-bFGF, respectively. Furthermore, quercetin (QCN)- and oxygen-carrying 1-bromoperfluorooctane (PFOB)-loaded nanoemulsions (QCN-NE and OXY-PFOB-NE) were developed to improve the topical delivery of QCN and oxygen, respectively. After confirming the enhanced penetration of LMWP-GFs, QCN-NE, and oxygen delivered from OXY-PFOB-NE across human epidermis, we evaluated the effects of combining LMWP-GFs, QCN-NE, and OXY-PFOB-NE on proliferation of keratinocytes and fibroblasts, and the chronic wound closure rate of a diabetic mouse model. Results: The optimal ratios of LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, LMWP-bFGF, QCN-NE, and OXY-PFOB-NE were 1, 1, 0.02, 0.02, 0.2, and 60, respectively. Moreover, a Carbopol hydrogel containing LMWP-GFs, QCN-NE, and OXY-PFOB-NE (LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL) significantly improved scratch-wound recovery of keratinocytes and fibroblasts in vitro compared to that afforded by hydrogels containing each component alone. LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL significantly accelerated wound-healing in a diabetic mouse model, decreasing wound size by 54 and 35% compared to the vehicle and LMWP-GFs, respectively. Conclusion: LMWP-GFs/QCN-NE/OXY-PFOB-NE-GEL synergistically accelerated the healing of chronic wounds, exerting both rapid and prolonged effects.


Assuntos
Diabetes Mellitus/patologia , Hidrogéis/química , Fator de Crescimento Insulin-Like I/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Oxigênio/metabolismo , Quercetina/farmacologia , Absorção Cutânea , Cicatrização/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno/biossíntese , Modelos Animais de Doenças , Emulsões/química , Fator de Crescimento Epidérmico/farmacologia , Epiderme/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Peso Molecular , Nanopartículas/química , Nanopartículas/ultraestrutura , Octanos/química , Fator de Crescimento Derivado de Plaquetas/farmacologia , Protaminas/química , Absorção Cutânea/efeitos dos fármacos
10.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
11.
J Agric Food Chem ; 67(32): 8938-8949, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31361121

RESUMO

Polymeric pigments formed via ethyl linkages between grape tannins and anthocyanins are important to the development of stable red wine color. To determine the effect of tannin structure on the stability and color properties of ethyl-linked polymeric pigments, tannin fractions with average polymerization between 4 and 43 units were prepared from grape skins and seeds and combined with malvidin-3-glucoside (M3G) in model wine containing acetaldehyde. As tannin molecular mass increased, the reaction rate with M3G increased. Compared with skin tannins of comparable molecular mass, seed tannins reacted more rapidly with M3G but were prone to precipitation. This resulted in a loss of polymeric pigments formed from seed tannins, which was greater as tannin molecular mass increased. Aggregation occurred following the reaction of seed tannin with M3G, concomitant with precipitation. The aggregation-precipitation phenomenon was not observed for skin tannin-derived pigments, indicating a greater stability in solution than those formed from seed tannins.


Assuntos
Acetaldeído/química , Antocianinas/química , Pigmentos Biológicos/química , Extratos Vegetais/química , Sementes/química , Taninos/química , Vitis/química , Precipitação Química , Cor , Frutas/química , Cinética , Peso Molecular , Polimerização , Polímeros/química
12.
Bioresour Technol ; 291: 121799, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31351375

RESUMO

The aim of this work is to develop a novel green solvent based sustainable process to refine lignin into low molecular weight (LMW) and high molecular weight (HMW) fractions. Lignin dispersity reduction were experimentally determined using four solvent mixtures, and benchmarked against eight pure solvents. Data outputs were used for modelling the integrated fractionation process. Dispersity reduction of up to 73% was achieved for the high value LMW fraction. Also, a 90% reduction of energy requirement was achieved with an optimized process incorporating a mechanical vapor compression system. This study showed that solvent mixtures involving water can significantly reduce the cost, environment, health and safety impacts of lignin fractionation. Techno-economic evaluation confirmed the economic viability of a large-scale process processing 50 tonne/day of lignin.


Assuntos
Lignina/química , Fracionamento Químico , Peso Molecular , Solventes
13.
Plant Foods Hum Nutr ; 74(3): 414-420, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31278561

RESUMO

The amount of cold press oil manufacture is globally rising, which in turn leads to the accumulation of deoiled plant seeds at significant quantities and consequent manufacture of plant protein products. In this study, we made an attempt to analyze the protein profile of black cumin seed protein concentrates prepared by the alkali extraction-acid precipitation technique (AE-IP). The analytical strategy relied on gel-based proteome mapping which included two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). 14 different protein bands were identified, and in gel-trypsinolysis was carried out for the corresponding gel spots. Using the MASCOT database, current findings on 10 proteins were compared with the existing data. The highest similarity was 46 which was obtained between the highest pI black cumin protein observed here and the cyclin dependent kinase inhibitor of Arabidopsis thaliana. The molecular mass of the intact protein was determined by linear MALDI-TOF/TOF-MS as 23,711.2186 Da. The peptide constructs of this protein have been further studied in order to identify potential biological activity. Matching sequences generated bioactive peptides in silico such as IR, AL, and SL dipeptides during sequential enzymatic digestion with pepsin and trypsin. Since the majority of bioactivity investigations on black cumin seeds have been related to black cumin oil and its oil soluble components, the structure and bioactivities of black cumin proteins deserve further research.


Assuntos
Nigella sativa/metabolismo , Peptídeos/análise , Proteínas de Plantas/análise , Proteoma , Eletroforese em Gel Bidimensional , Peso Molecular , Proteômica , Sementes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismo
14.
J Microbiol Biotechnol ; 29(7): 1043-1052, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31353877

RESUMO

Active lipase-producing bacterium Burkholderia gladioli Bps-1 was rapidly isolated using a modified trypan blue and tetracycline, ampicillin (TB-TA) plate. The electro-phoretically pure enzyme was obtained by purification using ethanol precipitation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight was 34.6 kDa and the specific activity was determined to be 443.9 U/mg. The purified lipase showed the highest activity after hydrolysis with p-NPC16 at a pH of 8.5 and 50°C, and the Km, kcat, and kcat/Km values were 1.05, 292.95 s-1 and 279 s-1mM-1, respectively. The lipase was highly stable at 7.5 ≤ pH ≤ 10.0. K+ and Na+ exerted activation effects on the lipase which had favorable tolerance to short-chain alcohols with its residual enzyme activity being 110% after being maintained in 30% ethanol for 1 h. The results demonstrated that the lipase produced by the strain B. gladioli Bps-1 has high enzyme activity and is an alkaline lipase. The lipase has promising chemical properties for a range of applications in the food-processing and detergent industries, and has particularly high potential for use in the manufacture of biodiesel.


Assuntos
Burkholderia gladioli/enzimologia , Burkholderia gladioli/isolamento & purificação , Lipase/metabolismo , Biocatálise , Biocombustíveis , Burkholderia gladioli/crescimento & desenvolvimento , Burkholderia gladioli/metabolismo , Meios de Cultura , Detergentes , Estabilidade Enzimática , Etanol/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Lipase/química , Lipase/isolamento & purificação , Peso Molecular , Especificidade por Substrato , Temperatura Ambiente
15.
J Agric Food Chem ; 67(33): 9354-9361, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31339706

RESUMO

As a popular ingredient for western and traditional Chinese medicine, the root and rhizome of Chinese licorice (Glycyrrhiza uralensis Fisch.) is often administered in the form of a decoction. The protein nanoparticles (NPs) self-assembled during the process of decoction. A major constitutive protein (GLP) was purified and determined to have a molecular weight of 28 kDa with an N-terminal sequence of NPDGL IACYC GQYCW. Over 80% of the purified GLP self-assembled into spherical NPs with diameters of 74.1 ± 0.7 nm and ζ-potential of -24.3 ± 1.7 mV when boiled in Tris-HCl buffer (pH = 7.9, 20 mM) at 100 °C for 60 min. Each nanoparticle was estimated by the SEC-MALLS approach to be composed of approximately 23 protein molecules. The NPs and GLP showed low cellular toxicity upon four types of cells including MDCK, L-02, HepG2, and Caco2 cells, while the NPs promoted proliferation of normal hepatocytes by 67%. The NPs solubilized the insoluble astragaloside IV by encapsulation. The results suggest a great potential for GLP-NPs as a promising prototype of a type of drug vehicle, a novel source of bioactive nanomaterials from herbal proteins, as well as a new mode of function with herbal components.


Assuntos
Glycyrrhiza/química , Nanopartículas/química , Extratos Vegetais/química , Proteínas de Plantas/química , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Temperatura Alta , Humanos , Peso Molecular , Nanoestruturas/química , Extratos Vegetais/farmacologia , Proteínas de Plantas/farmacologia , Raízes de Plantas/química
16.
J Agric Food Chem ; 67(33): 9307-9313, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352784

RESUMO

Porphyra is one of the most consumed types of red algae. Porphyran is the major polysaccharide extracted from Porphyra, and it is composed of alternating 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) residues. ß-Porphyranases are promising tools for degrading porphyran; however, few enzymes have been reported, and the biochemical properties of porphyranases are still unclear. Here, a novel GH16 ß-porphyranase, designated as Por16A_Wf, was cloned from Wenyingzhuangia fucanilytica and expressed in Escherichia coli. Its biochemical properties and hydrolysis pattern were characterized. Por16A_Wf exhibited stable activity on a wide pH scale from 3.5 to 11.0. Glycomics analysis using LC-MS revealed that Por16A_Wf specifically hydrolyzed the glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-l-galactopyranose and methyl-d-galactose in -2 and +2 subsites, respectively. Por16A_Wf could be applied as a biotechnological tool for tailoring porphyran, which would serve in directional preparation of its disaccharide, producing products with various molecular weights and facilitating investigation of the structural heterogeneity of Porphyra polysaccharides.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sefarose/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Filogenia , Porphyra/química , Porphyra/metabolismo , Sefarose/química , Sefarose/metabolismo , Alinhamento de Sequência
17.
J Agric Food Chem ; 67(33): 9344-9353, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31361957

RESUMO

Anthocyanins determine the color and potential health-promoting properties of red fruit juices, but the juices contain remarkably less anthocyanins than the fruits, which is partly caused by the interactions of anthocyanins with the residues of cell wall polysaccharides like pectin. In this study, pectin was modified by ultrasound and enzyme treatments to residues of polysaccharides and oligosaccharides widely differing in their molecular weight. Modifications decreased viscosity and degrees of acetylation and methylation and released smooth and hairy region fragments. Native and modified pectin induced different effects on the concentrations of individual anthocyanins after short-term and long-term incubation caused by both hydrophobic and hydrophilic interactions. Results indicate that both pectin and anthocyanin structure influence these interactions. Linear polymers generated by ultrasound formed insoluble anthocyanin complexes, whereas oligosaccharides produced by enzymes formed soluble complexes with protective properties. The structure of the anthocyanin aglycone apparently influenced interactions more than the sugar moiety.


Assuntos
Antocianinas/química , Beta vulgaris/química , Pectinas/química , Acetilação , Cor , Frutas/química , Sucos de Frutas e Vegetais/análise , Interações Hidrofóbicas e Hidrofílicas , Metilação , Peso Molecular , Ultrassom , Viscosidade
18.
World J Microbiol Biotechnol ; 35(8): 114, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332537

RESUMO

N-acetyl-D-glucosamine (GlcNAc) is an important amino-monosaccharide with great potential for biotechnological applications. It has traditionally been produced by the chemical hydrolysis of chitin, despite certain industrial and environmental drawbacks, including acidic wastes, low yields and high costs. Therefore, enzymatic production has gained attention as a promising environmentally-friendly alternative to the chemical processes. In this study we demonstrate the GlcNAc bioproduction from colloidal α-chitin using an enzyme cocktail containing endochitinases and exochitinases (chitobiosidases and N-acetyl-glucosaminidases). The enzyme cocktail was extracted after fermentation in a bioreactor by Aeromonas caviae CHZ306, a chitinolytic marine bacterium with great potential for chitinase production. Hydrolysis parameters were studied in terms of temperature, pH, enzyme and substrate concentration, and reaction time, achieving over 90% GlcNAc yield within 6 h. The use of colloidal α-chitin as substrate showed a substantial improvement of GlcNAc yields, when compared with ß-chitin and α-chitin polymorphs. Such result is directly related to a significant decrease in crystallinity and viscosity from natural α-chitin, providing the chitinase with greater accessibility to the depolymerized chains. This study provides valuable information on the GlcNAc bioproduction from chitin using an enzymatic approach, addressing the key points for its production, including the enzyme cocktail composition and the substrate structures.


Assuntos
Acetilglucosamina/biossíntese , Aeromonas caviae/enzimologia , Quitina/metabolismo , Quitinases/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Hidrólise , Espectroscopia de Ressonância Magnética , Peso Molecular , Temperatura Ambiente , Viscosidade , Difração de Raios X
19.
Food Chem ; 298: 125090, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31272055

RESUMO

In this study, molecular properties of wheat starch from three different types of breads were analyzed using asymmetric flow field-flow (AF4) connected to multi-angle light scattering (MALS) and differential refractive index (dRI) detectors. This analysis allowed the determination of molecular properties, i.e. molar mass (M), root-mean-square radius (rrms), apparent density (ρapp) and conformation. Complementary analyses, such as resistant starch and amylose content, were also performed. The results show that wheat starch extracted from breads can have different properties reflected in changes in M, rrms and ρapp. In addition, the results suggest that some of the changes in molecular properties may be related to the presence of resistant starch.


Assuntos
Pão/análise , Fracionamento por Campo e Fluxo/métodos , Refratometria/métodos , Amido/química , Triticum/química , Amilose/análise , Bolívia , Análise de Alimentos/métodos , Peso Molecular , Espalhamento de Radiação
20.
Int J Nanomedicine ; 14: 4683-4695, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308653

RESUMO

Purpose: Clinical applications of curcumin (Cur) have been greatly restricted due to its low solubility and poor systemic bioavailability. Three-arm amphiphilic copolymer tricarballylic acid-poly (ε-caprolactone)-methoxypolyethylene glycol (Tri-CL-mPEG) nanoparticles (NPs) were designed to improve the solubility and bioavailability of Cur. The present study adopted a microchannel system to precisely control the preparation of self-assembly polymeric NPs via liquid flow-focusing and gas displacing method. Methods: The amphiphilic three-arm copolymer Tri-CL-mPEG was synthesized and self-assembled into nearly spherical NPs, yielding Cur encapsulated into NP cores (Cur-NPs). The obtained NPs were evaluated for physicochemical properties, morphology, toxicity, cellular uptake by A549 cells, release in vitro, biodistribution, and pharmacokinetics in vivo. Results: Rapidly fabricated and isodispersed Cur-NPs prepared by this method had an average diameter of 116±3 nm and a polydispersity index of 0.197±0.008. The drug loading capacity and entrapment efficiency of Cur-NPs were 5.58±0.23% and 91.42±0.39%, respectively. In vitro release experiments showed sustained release of Cur, with cumulative release values of 40.1% and 66.1% at pH 7.4 and pH 5.0, respectively, after 10 days post-incubation. The results of cellular uptake, biodistribution, and in vivo pharmacokinetics experiments demonstrated that Cur-NPs exhibited better biocompatibility and bioavailability, while additionally enabling greater cellular uptake and prolonged circulation with possible spleen, lung, and kidney targeting effects when compared to the properties of free Cur. Conclusion: These results indicate that Tri-CL-mPEG NPs are promising in clinical applications as a controllable delivery system for hydrophobic drugs.


Assuntos
Curcumina/farmacologia , Microfluídica/métodos , Nanopartículas/química , Polietilenoglicóis/química , Polímeros/química , Ácidos Tricarboxílicos/química , Células A549 , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcumina/química , Curcumina/farmacocinética , Liberação Controlada de Fármacos , Endocitose/efeitos dos fármacos , Humanos , Camundongos , Peso Molecular , Nanopartículas/ultraestrutura , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Ratos Sprague-Dawley , Distribuição Tecidual/efeitos dos fármacos
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