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1.
São Paulo; s.n; 2023. 78 p.
Tese em Português | LILACS | ID: biblio-1532219

RESUMO

Introdução: A diabetes mellitus (DM) é uma doença crônica não transmissível importante e crescente problema de saúde pública no mundo. Mudanças no estilo de vida, como um hábito alimentar saudável, contribuem para redução da glicemia e controle do diabetes. O café é um alimento amplamente consumido e rico em compostos fenólicos com propriedades antioxidantes, com estudos sugerindo que seu maior consumo pode estar associado a um menor risco de mortalidade no diabetes. Objetivos: Analisar os efeitos das bebidas à base de café em cápsula em enzimas do metabolismo glicídico e captação de glicose em modelo de células intestinais Caco-2. Métodos: As amostras de bebidas foram preparadas com cápsulas de café espresso regular e descafeinado com ou sem adição de leite. Essas bebidas à base de café foram submetidas à digestão in vitro e seus compostos fenólicos foram quantificados e identificados. Foram realizados ensaios de permeação e quantificação de glicose nas células Caco-2, ensaios da inibição da α-glicosidase, inibição α-amilase e inibição de dipeptidil peptidase-IV (DPP-IV). Análises da capacidade antioxidante foram realizadas por meio de ensaio da capacidade de absorbância de radical oxigênio (ORAC), inibição da peroxidação lipídica (TBARS) e, também foram analisados kits comerciais o ensaio da catalase e atividade antioxidante total (TAC). Os resultados foram expressos como média e desvio padrão. Resultados Não houve diferença estatística (p>0,05) na permeação de glicose entre as diferentes bebidas de café nas células Caco-2. Na análise da capacidade de inibição da enzima α-amilase o café regular apresentou melhor inibição na fase oral, e na fase intestinal o café descafeinado apresentou melhor resultado. A inibição da e α-glicosidase os cafés descafeinado e puro foram mais efetivos. Quanto à atividade da enzima catalase na porção apical, as menores concentrações de café regular e descafeinado foram mais efetivas. A melhor capacidade antioxidante foi observada no café descafeinado. Leite e cafeína foram efetivos em estimular a enzima DPPIV. Conclusão: Todas as bebidas apresentaram capacidade antioxidante, onde se destaca a superior capacidade antioxidante do café descafeinado. As bebidas puras foram mais efetivas para inibição das enzimas α-amilase e α-glicosidase após digestão e nas células Caco-2, leite e cafeína foram melhor ativadores de DPPIV.


Background: Diabetes mellitus (DM) is an important chronic non-communicable disease and a growing public health problem worldwide. Lifestyle changes, such as healthy eating habits, contribute to lowering blood glucose levels and controlling diabetes. Coffee is a widely consumed food rich in phenolic compounds with antioxidant properties, with studies suggesting that its higher consumption may be associated with a lower risk of mortality in diabetes. Aims: To analyze the effects of capsule coffee drinks on enzymes of glucose metabolism and glucose uptake in a Caco-2 intestinal cell model. Methods: The beverage samples were prepared with espresso and decaffeinated coffee capsules with or without added milk. These coffee drinks were subjected to in vitro digestion and their phenolic compounds were quantified and identified. Glucose permeation and quantification tests were carried out on Caco-2 cells, as well as α-glucosidase inhibition, α-amylase inhibition and dipeptidyl peptidase-IV (DPP-IV) inhibition tests. Antioxidant capacity analyses were carried out using the oxygen radical absorbance capacity (ORAC) assay, lipid peroxidation inhibition (TBARS), and the catalase assay and total antioxidant activity (TAC) were also analyzed using commercial kits. The results were expressed as mean and standard deviation. Results: There was no statistical difference (p>0.05) in glucose permeation between the different coffee drinks in Caco-2 cells. In the analysis of the ability to inhibit the α-amylase enzyme, regular coffee showed better inhibition in the oral phase, and decaffeinated coffee showed better results in the intestinal phase. Decaffeinated and pure coffees were more effective at inhibiting α-glucosidase. As for the activity of the enzyme catalase in the apical portion, the lower concentrations of normal and decaffeinated coffee were more effective. The best antioxidant capacity was observed in decaffeinated coffee. Milk and caffeine were effective in stimulating the DPPIV enzyme. Conclusion: All the beverages showed antioxidant capacity, with the superior antioxidant capacity of decaffeinated coffee standing out. The pure drinks were more effective in inhibiting the enzymes α-amylase and α-glucosidase after digestion and, in Caco-2 cells, milk and caffeine were better activators of DPPIV.


Assuntos
Café , Diabetes Mellitus , alfa-Amilases , Compostos Fenólicos , Inibidores da Dipeptidil Peptidase IV , Doenças não Transmissíveis , Glicosídeo Hidrolases , Antioxidantes
2.
Electron. j. biotechnol ; 50: 10-15, Mar. 2021. ilus, graf, tab
Artigo em Inglês | LILACS | ID: biblio-1292308

RESUMO

BACKGROUND: LXYL-P1-2 is the first reported glycoside hydrolase that can catalyze the transformation of 7-b-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT) by removing the D-xylosyl group at the C7 position. Successful synthesis of paclitaxel by one-pot method combining the LXYL-P1-2 and 10- deacetylbaccatin III-10-b-O-acetyltransferase (DBAT) using XDT as a precursor, making LXYL-P1-2 a highly promising enzyme for the industrial production of paclitaxel. The aim of this study was to investigate the catalytic potential of LXYL-P1-2 stabilized on magnetic nanoparticles, the surface of which was modified by Ni2+-immobilized cross-linked Fe3O4@Histidine. RESULTS: The diameter of matrix was 20­40 nm. The Km value of the immobilized LXYL-P1-2 catalyzing XDT (0.145 mM) was lower than that of the free enzyme (0.452 mM), and the kcat/Km value of immobilized enzyme (12.952 mM s 1 ) was higher than the free form (8.622 mM s 1 ). The immobilized form maintained 50% of its original activity after 15 cycles of reuse. In addition, the stability of immobilized LXYL-P1-2, maintained 84.67% of its initial activity, improved in comparison with free form after 30 d storage at 4 C. CONCLUSIONS: This investigation not only provides an effective procedure for biocatalytic production of DT, but also gives an insight into the application of magnetic material immobilization technology.


Assuntos
Paclitaxel/biossíntese , Glicosídeo Hidrolases/metabolismo , Cinética , Enzimas Imobilizadas , Nanopartículas , Imãs
3.
Arq. bras. med. vet. zootec. (Online) ; 72(4): 1504-1510, July-Aug. 2020. tab
Artigo em Português | LILACS, VETINDEX | ID: biblio-1131478

RESUMO

Foram determinados os valores energéticos e a composição bromatológica do resíduo seco de fecularia (RSF) para frangos de corte, na fase de crescimento, utilizando ou não enzimas carboidrases. Os tratamentos foram distribuídos em esquema fatorial 2x4 + ração referência, sendo uma RR sem adição de RSF e quatro tratamentos experimentais com 10%, 20%, 30% e 40% de inclusão do RSF e a suplementação ou não com carboidrases. A composição química encontrada para o RSF, na MN, foi de 89,86% de matéria seca, 0,98% de proteína bruta, 3519kcal kg-1 de energia bruta, 0,19% de extrato etéreo, 27% de fibra em detergente neutro, 19,5% de fibra em detergente ácido, 0,33% de cálcio, 0,43% de fósforo, 0,46% de potássio e 0,12% de magnésio. O uso de carboidrases proporcionou um aumento de 173 e 213kcal kg-1 nos valores de EMA e EMAn, respectivamente, resultando em 1828kcal kg-1 EMA e 1840kcal kg-1 EMAn. Concluiu-se que os maiores níveis de EMA e EMAn foram encontrados para o nível de inclusão médio do RSF de 35% e que a suplementação enzimática pode promover aumento desses parâmetros em até 12% em dietas para frangos de corte na fase de crescimento.(AU)


The energetic values and the bromatological composition of the dry residue of cassava (DRC) were determined for growing broilers with or without carbohydrase enzymes. The treatments were distributed in a 2x4 + reference diet factorial scheme, with one RD without addition of DRC and four experimental treatments with 10, 20, 30 and 40% inclusion levels of RSF and supplementation or not with carbohydrases. The chemical composition found for DRC in natural matter was 89.86% dry matter, 0.98% crude protein, 3519kcal kg-1 gross energy, 0.19% ether extract, 27% neutral detergent fiber, 19.5% of acid detergent fiber, 0.33% of calcium, 0.43% of phosphorus, 0.46% of potassium and 0.12% of magnesium. The use of carbohydrase resulted in an increase of 173 and 213kcal kg-1 in EMA and EMAn values, respectively, resulting in 1828kcal kg-1 EMA and 1840kcal kg-1 EMAn. It was concluded that the highest levels of AME and AMEn were found for the mean inclusion level of the DRC of 35% and that enzymatic supplementation may promote the increase of these parameters by up to 12% in broiler diets in the growth phase.(AU)


Assuntos
Animais , Fibras na Dieta/administração & dosagem , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , Amidos e Féculas , Ração Animal/análise , Metabolismo Energético , Glicosídeo Hidrolases/administração & dosagem
4.
Biosci. j. (Online) ; 36(1): 1-16, jan./feb. 2020. tab, ilus
Artigo em Inglês | LILACS | ID: biblio-1049184

RESUMO

The objective of this study was to evaluate the influence of agroforestry systems of different ages (AFS1: one-year old; AFS5: five-years old) on the biological attributes of soil; the following systems were used for comparison: a slash-and-burn (SBF) farming area, Caatinga which has been undergoing regeneration for 6 years (CaR6), and native Caatinga (NCa) in Brazil. Enzyme activity, abundance and composition of arbuscular mycorrhizal fungi (AMF), and production of glomalin-related soil proteins (GRSP) were evaluated at soil depths of 0­0.05 m. AMF species composition in the AFS was more similar to that in the NCa than in the SBF and CaR6 systems. In the rainy season, sporulation was most abundant in the AFS-1, CaR6, and SBF systems, whereas GRSP concentrations were highest in the AFS5 during the dry season. Acid phosphatase and arylsulfatase enzyme activity was lower in the AFS1 soils than in the NCa and SBF soils (rainy period), and levels of ß-glucosidase and fluorescein diacetate hydrolysis in the AFS were equal to or higher than those in the NCa in the dry season but lower in the rainy season. AFS thus appear to promote the maintenance of soil biological quality, and may be more sustainable than SBF farming systems in the Brazilian Caatinga over the long term.


O objetivo do estudo foi avaliar a influência de sistemas agroflorestais (AFS1: um ano de idade; AFS5: cinco anos de idade), nos atributos biológicos do solo usando como referência, uma área de agricultura de corte e queima (SBF), Caatinga em regeneração há 6 anos (CaR6), e Caatinga nativa (NCa), in Brasil. A atividade enzimática, a abundância e composição dos fungos micorrízicos arbusculares (AMF), e a produção de proteína do solo relacionada à glomalina (GRSP) foram avaliados, na profundidade de 0-5 cm do solo. A composição das espécies de AMF nos AFS foi mais semelhante a observada na NCa, do que os sistemas SBF e CaR6. Na estação chuvosa, a esporulação foi mais abundante em AFS-1, CaR6 and SBF quando comparada as outras áreas, enquanto a GRSP apresentou maiores teores no AFS5 no período seco. AFS1 apresentou atividade da fosfatase ácida e arilsulfatase inferiores tanto a NCa quanto a SBF, no período chuvoso. No período seco, a atividade de ß-glicosidase e a hidrólise do diacetato de fluoresceína (FDA) na AFS foram iguais ou superiores a Nca, mas menor no período chuvoso. Verifica-se que os AFS são potenciais para a manutenção da qualidade biológica do solo, podendo, em longo prazo, serem mais sustentáveis que a SBF, em ambiente de Caatinga.


Assuntos
Arilsulfatases , Solo , Fosfatase Ácida , Glicosídeo Hidrolases
5.
Electron. j. biotechnol ; 40: 71-77, July. 2019. tab, graf, ilus
Artigo em Inglês | LILACS | ID: biblio-1053491

RESUMO

Background: Burdock (Arctium lappa L.) is a fructan-rich plant with prebiotic potential. The aim of this study was to develop an efficient enzymatic route to prepare fructooligosaccharides (FOS)-rich and highly antioxidative syrup using burdock root as a raw material. Results: Endo-inulinase significantly improved the yield of FOS 2.4-fold while tannase pretreatment further increased the yield of FOS 2.8-fold. Other enzymes, including endo-polygalacturonase, endo-glucanase and endo-xylanase, were able to increase the yield of total soluble sugar by 11.1% (w/w). By this process, a new enzymatic process for burdock syrup was developed and the yield of burdock syrup increased by 25% (w/w), whereas with FOS, total soluble sugars, total soluble protein and total soluble polyphenols were enhanced to 28.8%, 53.3%, 8.9% and 3.3% (w/w), respectively. Additionally, the scavenging abilities of DPPH and hydroxyl radicals, and total antioxidant capacity of the syrup were increased by 23.7%, 51.8% and 35.4%, respectively. Conclusions: Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods.


Assuntos
Oligossacarídeos/química , Raízes de Plantas/química , Frutose/química , Glicosídeo Hidrolases/metabolismo , Antioxidantes/química , Oligossacarídeos/metabolismo , Poligalacturonase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Cromatografia Líquida de Alta Pressão , Radical Hidroxila , Arctium , Alimento Funcional , Polifenóis , Frutose/metabolismo , Antioxidantes/metabolismo
6.
Electron. j. biotechnol ; 36: 24-33, nov. 2018. graf, tab, ilus
Artigo em Inglês | LILACS | ID: biblio-1048179

RESUMO

Background: α-L-Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal α-L-1,2-, -1,3-, and -1,5- arabinofuranosyl residues in arabinose-containing polymers, and hence, it plays an important role in hemicellulose degradation. Herein, the bacterium Paenibacillus polymyxa, which secretes arabinofuranosidase with high activity, was selected for enzyme production, purification, and characterization. Results: Medium components and cultural conditions were optimized by the response surface method using shake flask cultures. Arabinofuranosidase production reached 25.2 U/mL under optimized conditions, which were pH 7.5, 28°C, and a basic medium supplemented with 1.5 g/L mannitol and 3.5 g/L soymeal. Furthermore, the arabinofuranosidase secreted by P. polymyxa, named as PpAFase-1, was partially purified from the supernatant using a DEAE Sepharose Fast Flow column and a hydroxyapatite column. The approximate molecular mass of the purified PpAFase-1 was determined as 56.8 kDa by SDS-PAGE. Protein identification by mass spectrometry analysis showed that the deduced amino acid sequence had significant similarity to the glycosyl hydrolase family 51. The deduced gene of 1515 bp was cloned and expressed in Escherichia coli BL21 (DE3) cells. Purified recombinant PpAFase-1 was active toward p-nitrophenyl-α-L-arabinofuranoside (pNPAraf). The Km and kcat values toward pNPAraf were 0.81 mM and 53.2 s−1 , respectively. When wheat arabinoxylan and oat spelt xylan were used as substrates, PpAFase-1 showed poor efficiency. However, a synergistic effect was observed when PpAFase-1 was combined with xylanase from Thermomyces lanuginosus. Conclusion: A novel GH51 enzyme PpAFase-1 was cloned from the genome of P. polymyxa and expressed in E. coli. This enzyme may be suitable for hemicellulose degradation on an industrial scale.


Assuntos
Paenibacillus polymyxa/enzimologia , Glicosídeo Hidrolases/metabolismo , Arabinose , Espectrometria de Massas , Celulose , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/biossíntese
7.
Biosci. j. (Online) ; 34(4): 830-847, july/aug. 2018. tab, ilus, graf
Artigo em Inglês | LILACS | ID: biblio-967017

RESUMO

Due to the toxicity and inefficiency of chemical fungicides to control infestation of Macrophomina phaseolina (Tassi) Goid which causes charcoal rot in plants, a biotechnological approach using - glucosidase (EC.3.2.1) as the alternative bioactive ingredient in fungicide is hereby, proposed. The extracellular enzyme was isolated from a highly efficient fungal antagonist, Trichoderma harzianum T12. The highly similar molecular masses obtained using SDS-PAGE (96 kDa) and MALDI-TOF mass spectrometry (98.3 kDa) affirmed that the -glucosidase was purified to homogeneity. Consequently, optimum catalytic parameters that rendered the highest enzyme activity were found to be: 45°C, pH 7, inoculum size of 10 % (w/v), supplementation with metal ions Zn2+ and Mn2+ ions, and Tween 80. Addition of wheat bran and (NH4)2SO4 as carbon and nitrogen sources also improved enzyme activity. BLASTn showed the sequence of -glucosidase T12 was highly identical to other -glucosidases viz. T. harzianum strain IOC-3844 (99%), T. gamsii and T. virens bgl1 (86 %) as well as T. reesei strain SJVTR and T. viride strain AS 3.3711 (84 %). Kinetic assessment showed that -glucosidase T12 catalyzes hydrolytic activity is characterized by a Km of 0.79 mM and Vmax of 8.45 mM min-1 mg-1 protein, with a corresponding kcat of 10.69 s-1.


Devido à toxicidade e ineficiência dos fungicidas químicos para controlar a infestação de Macrophomina phaseolina (Tassi) Goid que causa o apodrecimento das plantas, uma abordagem biotecnológica usando - glicosidase (EC.3.2.1) como o ingrediente bioativo alternativo do fungicida é por este meio, proposto. A enzima extracelular foi isolada de um antagonista fúngico altamente eficiente, o Trichoderma harzianum T12. As massas moleculares altamente similares obtidas usando SDS-PAGE (96 kDa) e espectrometria de massa MALDI-TOF (98,3 kDa) afirmaram que a -glicosidase foi purificada até a homogeneidade. Consequentemente, os parâmetros catalíticos ótimos que apresentaram a maior atividade enzimática foram: 45°C, pH 7, tamanho do inóculo de 10% (p / v), suplementação com íons de metais Zn2+ e Mn2+, e Tween 80. Adição de farelo de trigo e (NH4) 2SO4 como fontes de carbono e nitrogênio também melhoraram a atividade enzimática. O BLASTn mostrou que a sequência da -glicosidase T12 era altamente idêntica a outras -glicosidase viz. A estirpe T. harzianum IOC-3844 (99%), T. gamsii e T. virens bgl1 (86%) assim como a estirpe T. reesei SJVTR e a estirpe T. viride AS 3.3711 (84%). A avaliação cinética mostrou que -glicosidase T12 catalisa a actividade hidrolítica caracterizada por um Km de 0,79 mM e Vmax de 8,45 mM min-1 mg-1 de proteína, com um correspondente kcat de 10,69 s-1.


Assuntos
Trichoderma , Cinética , Fungos , Fungicidas Industriais , Glicosídeo Hidrolases , Biotecnologia
8.
Electron. j. biotechnol ; 32: 26-34, Mar. 2018. graf, tab
Artigo em Inglês | LILACS | ID: biblio-1022610

RESUMO

Background: A new ι-carrageenase-producing strain was screened from mangroves and authenticated as Pseudoalteromonas carrageenovora ASY5 in our laboratory. The potential application of this new strain was evaluated. Results: Medium compositions and culturing conditions in shaking flask fermentation were firstly optimized by single-factor experiment. ι-Carrageenase activity increased from 0.34 U/mL to 1.08 U/mL after test optimization. Optimal fermentation conditions were 20°C, pH 7.0, incubation time of 40 h, 15 g/L NaCl, 1.5% (w/v) yeast extract as nitrogen source, and 0.9% (w/v) ι-carrageenan as carbon source. Then, the crude ι-carrageenase was characterized. The optimum temperature and pH of the ι-carrageenase were 40°C and 8.0, respectively. The enzymatic activity at 35­40°C for 45 min retained more than 40% of the maximum activity. Meanwhile, The ι-carrageenase was inhibited by the addition of 1 mmol/L Cd2+ and Fe3+ but increased by the addition of 1 mmol/L Ag+, Ba2+, Ca2+, Co2+, Mn2+, Zn2+, Fe2+, and Al3+. The structure of oligosaccharides derived from ι-carrageenan was detected using electrospray ionization mass spectrometry (ESI-MS). The ι-carrageenase degraded ι-carrageenan, yielding disaccharides and tetrasaccharides as main products. Conclusions: The discovery and study of new ι-carrageenases are beneficial not only for the production of ι-carrageenan oligosaccharides but also for the further utilization in industrial production.


Assuntos
Proteínas de Bactérias/metabolismo , Pseudoalteromonas/enzimologia , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/biossíntese , Temperatura , Carbono/metabolismo , Carragenina/biossíntese , Espectrometria de Massas por Ionização por Electrospray , Fermentação , Concentração de Íons de Hidrogênio , Hidrólise , Nitrogênio/metabolismo
9.
Braz. j. microbiol ; 48(4): 612-614, Oct.-Dec. 2017. tab
Artigo em Inglês | LILACS | ID: biblio-889174

RESUMO

ABSTRACT Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296 bp and G + C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria.


Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Glicosídeo Hidrolases/genética , Microbiologia do Solo , Streptomyces/enzimologia , Streptomyces/isolamento & purificação , Proteínas de Bactérias/metabolismo , Composição de Bases , Brasil , Glicosídeo Hidrolases/metabolismo , Família Multigênica , Filogenia , Streptomyces/classificação , Streptomyces/genética
10.
Electron. j. biotechnol ; 29: 63-67, sept. 2017. ilus, tab, graf
Artigo em Inglês | LILACS | ID: biblio-1017249

RESUMO

Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Glicosídeo Hidrolases/metabolismo , Transformação Genética , Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estabilidade de RNA , Fermentação , Vetores Genéticos , Glicosídeo Hidrolases/genética
11.
Braz. j. microbiol ; 48(3): 427-441, July-Sept. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-889130

RESUMO

Abstract The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30 °C, 6% (v/v), inoculum size and 150 rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.


Assuntos
Aspergillus niger/metabolismo , beta-Frutofuranosidase/biossíntese , Glicosídeo Hidrolases/biossíntese , Microbiologia Industrial/métodos , Aspergillus niger/enzimologia , Aspergillus niger/genética , Aspergillus niger/crescimento & desenvolvimento , beta-Frutofuranosidase/genética , Reatores Biológicos/microbiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Glicosídeo Hidrolases/genética , Temperatura
12.
Electron. j. biotechnol ; 26: 46-51, Mar. 2017. graf, tab
Artigo em Inglês | LILACS | ID: biblio-1009650

RESUMO

Background: Current commercial production of isomalto-oligosaccharides (IMOs) commonly involves a lengthy multistage process with low yields. Results: To improve the process efficiency for production of IMOs, we developed a simple and efficient method by using enzyme cocktails composed of the recombinant Bacillus naganoensis pullulanase produced by Bacillus licheniformis, α-amylase from Bacillus amyloliquefaciens, barley bran ß-amylase, and α-transglucosidase from Aspergillus niger to perform simultaneous saccharification and transglycosylation to process the liquefied starch. After 13 h of reacting time, 49.09% IMOs (calculated from the total amount of isomaltose, isomaltotriose, and panose) were produced. Conclusions: Our method of using an enzyme cocktail for the efficient production of IMOs offers an attractive alternative to the process presently in use.


Assuntos
Oligossacarídeos/metabolismo , Amido/metabolismo , Enzimas/metabolismo , Isomaltose/metabolismo , Oligossacarídeos/biossíntese , Aspergillus niger/enzimologia , Temperatura , Bacillus/enzimologia , beta-Amilase/metabolismo , Glicosilação , Liquefação , alfa-Amilases/metabolismo , Fermentação , Glucosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio
13.
An. acad. bras. ciênc ; 89(1): 57-63, Jan,-Mar. 2017. tab
Artigo em Inglês | LILACS | ID: biblio-886625

RESUMO

ABSTRACT The present study evaluated the purification of inulinase by changing the ionic strength of the medium by addition of NaCl and CaCl2 followed by precipitation with n-propyl alcohol or iso-propyl alcohol. The effects of the concentration of alcohols and the rate of addition of alcohols in the crude extract on the purification yield and purification factor were evaluated. Precipitation caused an activation of enzyme and allowed purification factors up to 2.4-fold for both alcohols. The purification factor was affected positively by the modification of the ionic strength of the medium to 0.5 mol.L-1 NaCl before precipitation with the alcohol (n-propyl or iso-propyl). A purification factor of 4.8-fold and an enzyme yield of 78.1 % could be achieved by the addition of 0.5 mol.L-1 of NaCl to the crude extract, followed by the precipitation with 50 % (v/v) of n-propyl alcohol, added at a flow rate of 19.9 mL/min.


Assuntos
Concentração Osmolar , Precipitação Química , Álcoois/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/química , Valores de Referência , Sais/química , Solventes/química , Kluyveromyces/isolamento & purificação , Kluyveromyces/química , Cloreto de Cálcio/química , Cloreto de Sódio/química , Reprodutibilidade dos Testes , Meios de Cultura/química
14.
Electron. j. biotechnol ; 25: 13-20, ene. 2017. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1008291

RESUMO

Background: A simple and efficient strategy for agarase immobilization was developed with carboxyl-functionalized magnetic nanoparticles (CMNPs) as support. The CMNPs and immobilized agarase (agarase-CMNPs) were characterized by transmission electron microscopy, dynamic light scattering, vibrating sample magnetometry, scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, and zeta-potential analysis. The hydrolyzed products were separated and detected by ESI-TOF-MS. Results: The agarase-CMNPs exhibited a regular spherical shape with a mean diameter of 12 nm, whereas their average size in the aqueous solution was 43.7 nm as measured by dynamic light scattering. These results indicated that agarase-CMNPs had water swelling properties. Saturation magnetizations were 44 and 29 emu/g for the carriers and agarase-CMNPs, respectively. Thus, the particles had superparamagnetic characteristics, and agarase was successfully immobilized onto the supports. Agaro-oligosaccharides were prepared with agar as substrate using agarase-CMNPs as biocatalyst. The catalytic activity of agarase-CMNPs was unchanged after six reuses. The ESI-TOF mass spectrogram showed that the major products hydrolyzed by agarase-CMNPs after six recycle uses were neoagarotetraose, neoagarohexaose, and neoagarooctaose. Meanwhile, the end-products after 90 min of enzymatic treatment by agarase-CMNPs were neoagarobiose and neoagarotetraose. Conclusions: The enhanced agarase properties upon immobilization suggested that CMNPs can be effective carriers for agarase immobilization. Agarase-CMNPs can be remarkably used in developing systems for repeated batch production of agar-derived oligosaccharides.


Assuntos
Oligossacarídeos/metabolismo , Enzimas Imobilizadas , Nanopartículas de Magnetita/química , Glicosídeo Hidrolases/metabolismo , Termogravimetria , Difração de Raios X , Estabilidade Enzimática , Catálise , Microscopia Eletrônica de Transmissão , Magnetometria , Difusão Dinâmica da Luz , Glicosídeo Hidrolases/química
15.
J. venom. anim. toxins incl. trop. dis ; 22: 28, 2016. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-954789

RESUMO

Background: Wasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency. Methods: In the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay. Results: Two isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (42-97 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (α/β)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12. Conclusions: The wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.(AU)


Assuntos
Animais , Venenos de Vespas , Vespas , Clonagem de Organismos , Glicosídeo Hidrolases , Hialuronoglucosaminidase
16.
Braz. j. microbiol ; 46(4): 1053-1064, Oct.-Dec. 2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-769641

RESUMO

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.


Assuntos
Quitinases/análise , Quitinases/química , Quitinases/enzimologia , Quitinases/crescimento & desenvolvimento , Quitinases/metabolismo , /análise , /química , /enzimologia , /crescimento & desenvolvimento , /metabolismo , Proteínas Fúngicas/análise , Proteínas Fúngicas/química , Proteínas Fúngicas/enzimologia , Proteínas Fúngicas/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/enzimologia , Glicosídeo Hidrolases/crescimento & desenvolvimento , Glicosídeo Hidrolases/metabolismo , Micélio/análise , Micélio/química , Micélio/enzimologia , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Paquistão/análise , Paquistão/química , Paquistão/enzimologia , Paquistão/crescimento & desenvolvimento , Paquistão/metabolismo , Trichoderma/análise , Trichoderma/química , Trichoderma/enzimologia , Trichoderma/crescimento & desenvolvimento , Trichoderma/metabolismo
17.
Braz. j. pharm. sci ; 51(4): 931-947, Oct.-Dec. 2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-778412

RESUMO

abstract A series of N-substituted 2-{[5-(1H-indol-3-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl}acetamides (8a-w) was synthesized in three steps. The first step involved the sequential conversion of 2-(1H-indol-3-yl)acetic acid (1) to ester (2) followed by hydrazide (3) formation and finally cyclization in the presence of CS2 and alcoholic KOH yielded 5-(1H-indole-3-yl-methyl)-1,3,4-oxadiazole-2-thiol (4). In the second step, aryl/aralkyl amines (5a-w) were reacted with 2-bromoacetyl bromide (6) in basic medium to yield 2-bromo-N-substituted acetamides (7a-w). In the third step, these electrophiles (7a-w) were reacted with 4 to afford the target compounds (8a-w). Structural elucidation of all the synthesized derivatives was done by 1H-NMR, IR and EI-MS spectral techniques. Moreover, they were screened for antibacterial and hemolytic activity. Enzyme inhibition activity was well supported by molecular docking results, for example, compound 8q exhibited better inhibitory potential against α-glucosidase, while 8g and 8b exhibited comparatively better inhibition against butyrylcholinesterase and lipoxygenase, respectively. Similarly, compounds 8b and 8c showed very good antibacterial activity against Salmonella typhi, which was very close to that of ciprofloxacin, a standard antibiotic used in this study. 8c and 8l also showed very good antibacterial activity against Staphylococcus aureus as well. Almost all compounds showed very slight hemolytic activity, where 8p exhibited the least. Therefore, the molecules synthesized may have utility as suitable therapeutic agents.


resumo Uma série de acetamidas 2-{[5-(1H-indol-3-ilmetil)-1,3,4-oxadiazol-2-il]sulfanila} N-substituídas (8a-w) foi sintetizada em três fases. A primeira etapa envolveu a conversão sequencial de ácido 2-(1H-indol-3-il)acético (1) a éster (2), seguido por hidrazida (3) e, finalmente, a e ciclização na presença de CS2 e KOH alcoólico produziu 5-(1H-indol-3-il- metil)-1,3,4-oxadiazole-2-tiol (4). Na segunda etapa, aminas arílicas/aralquílicas(5a-w) reagiram com brometo de 2-bromoacetila (6​​), em meio básico, para se obter acetamidas 2-bromo-N-substituídas (7a-w). Na terceira etapa, estes eletrófilos (7a- w) reagiram com 4, para se obter os compostos alvo (8a-w). A elucidação estrutural de todos os derivados sintetizados foi realizada por 1H-NMR, IR e técnicas de espectrometria de EI-MS. Além disso, eles foram submetidos a triagem de atividade antibacteriana e hemolítica. Análise da inibição enzimática foi bem apoiada pelos resultados de docking molecular. Por exemplo, o composto 8q exibiu melhor potencial inibitório contra α-glicosidase, e os compostos 8g e 8b exibiram, comparativamente, melhor inibição contra butirilcolinesterase (BChE) elipoxigenase (LOX), respectivamente. Do mesmo modo os compostos 8b e 8c mostraram excelente potencial antibacteriano contra SalmonellaTyphi, semelhante ao do ciprofloxacino, antibiótico padrão usado neste estudo. Os compostos 8c e 8l também mostraram excelente potencial antibacteriano contra Staphylococcus aureus . Quase todos os compostos mostraram pequena atividade hemolítica, sendo que o composto 8p apresentou menor atividade. Assim, as moléculas sintetizadas podem ter a sua utilidade como agentes terapêuticos adequados.


Assuntos
Ácido Hidroxi-Indolacético/análise , Acetamidas/análise , Butirilcolinesterase/análise , Ensaio de Atividade Hemolítica de Complemento/classificação , Lipoxigenases/farmacocinética , Glicosídeo Hidrolases/farmacocinética
18.
Braz. j. microbiol ; 46(3): 911-920, July-Sept. 2015. tab, ilus
Artigo em Inglês | LILACS | ID: lil-755798

RESUMO

A new inulinase-producing strain was isolated from rhizosphere soils of Jerusalem artichoke collected from Shihezi (Xinjiang, China) using Jerusalem artichoke power (JAP) as sole carbon source. It was identified as an Aspergillus niger strain by analysis of 16S rRNA. To improve inulinase production, this fungus was subjected to mutagenesis induced by 60Co γ-irradiation. A genetically stable mutant (designated E12) was obtained and it showed 2.7-fold higher inulinase activity (128 U/mL) than the parental strain in the supernatant of a submerged culture. Sequential methodology was used to optimize the inulinase production of stain E12. A screening trial was first performed using Plackett-Burman design and variables with statistically significant effects on inulinase bio-production were identified. These significant factors were further optimized by central composite design experiments and response surface methodology. Finally, it was found that the maximum inulinase production (185 U/mL) could be achieved under the optimized conditions namely pH 7.0, yeast extract concentration of 5.0 g/L, JAP concentration of 66.5 g/L, peptone concentration of 29.1 g/L, solution volume of 49.4 mL in 250-mL shake flasks, agitation speed of 180 rpm, and fermentation time of 60 h. The yield of inulinase under optimized culture conditions was approximately 1.4-fold of that obtained by using basal culture medium. These findings are of significance for the potential industrial application of the mutant E12.

.


Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/genética , Reatores Biológicos/microbiologia , Glicosídeo Hidrolases/metabolismo , Helianthus/microbiologia , Aspergillus niger/metabolismo , China , Meios de Cultura , Etanol/metabolismo , Fermentação/fisiologia , Inulina/metabolismo , Tipagem Molecular , Mutação , Técnicas de Tipagem Micológica , Rizosfera , /genética , Microbiologia do Solo
19.
Braz. j. microbiol ; 46(3): 683-690, July-Sept. 2015. tab, ilus
Artigo em Inglês | LILACS | ID: lil-755831

RESUMO

An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg2+, Mn2+, K+; Ca2+, Na+, Ba2+, Zn2+, Cu2+, Co2+, Fe2+, Sr2+ and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.

.


Assuntos
Adaptação Fisiológica/fisiologia , Ágar/metabolismo , Glicosídeo Hidrolases/metabolismo , Pseudoalteromonas/enzimologia , Regiões Antárticas , Adaptação Fisiológica/genética , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Dissacarídeos/biossíntese , Sedimentos Geológicos/microbiologia , Glicosídeo Hidrolases/isolamento & purificação , Hidrólise , /genética
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