RESUMO
The present work aimed to characterize and compare the catalytic properties of amylases from Cunninghamella echinulata and Rhizopus microsporus. The highest production of amylase by C. echinulata, 234.94 U g-1 of dry substrate (or 23.49 U mL-1), was obtained using wheat bran as a substrate, with 50-55% initial moisture and kept at 28 °C for 48 h. The highest production of amylases by R. microsporus, 224.85 U g-1 of dry substrate (or 22.48 U mL-1), was obtained cultivating wheat bran with 65% initial moisture at 45 °C for 24 h. The optimal activity of the amylases was observed at pH 5.0 at 60 °C for C. echinulata enzymes and at pH 4.5 at 65 °C for R. microsporus. The amylases produced by C. echinulata were stable at pH 4.0-8.0, while the R. microsporus enzymes were stable at pH 4.0-10.0. The amylases produced by C. echinulata remained stable for 1 h at 50 °C and the R. microsporus amylases maintained catalytic activity for 1 h at 55 °C. The enzymatic extracts of both fungi hydrolyzed starches from different plant sources and showed potential for liquefaction of starch, however the amylolytic complex of C. echinulata exhibited greater saccharifying potential.
Assuntos
Amilases , Cunninghamella , Amilases/química , Fibras na Dieta , Amido , Concentração de Íons de HidrogênioRESUMO
The present study compared the production and the catalytic properties of amylolytic enzymes obtained from the fungi Lichtheimia ramosa (mesophilic) and Thermoascus aurantiacus (thermophilic). The highest amylase production in both fungi was observed in wheat bran supplemented with nutrient solution (pH 4.0) after 96 hours of cultivation, reaching 417.2 U/g of dry substrate (or 41.72 U/mL) and 144.5 U/g of dry substrate (or 14.45 U/mL) for L. ramosa and T. aurantiacus, respectively. The enzymes showed higher catalytic activity at pH 6.0 at 60°C. The amylases produced by L. ramosa and T. aurantiacus were stable between pH 3.5-10.5 and pH 4.5-9.5, respectively. The amylase of L. ramosa was stable at 55°C after 1 hour of incubation, whereas that of T. aurantiacus maintained 60% of its original activity under the same conditions. Both enzymes were active in the presence of ethanol. The enzymes hydrolyzed starch from different sources, with the best results obtained with corn starch. The enzymatic complex produced by L. ramosa showed dextrinizing and saccharifying potential. The enzymatic extract produced by the fungus T. aurantiacus presented only saccharifying potential, releasing glucose monomers as the main hydrolysis product.
Assuntos
Amilases/química , Fermentação , Mucorales/enzimologia , Thermoascus/enzimologia , Hidrólise , Microbiologia Industrial , Amido/metabolismoRESUMO
BACKGROUND: Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases. RESULT: All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi. CONCLUSIONS: Almost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting candidates for industrial processes that require lower temperatures.
Assuntos
Amilases/metabolismo , Celulases/metabolismo , Proteínas Fúngicas/metabolismo , Leveduras/enzimologia , Amilases/química , Amilases/genética , Regiões Antárticas , Celulases/química , Celulases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Temperatura , Leveduras/classificação , Leveduras/genética , Leveduras/isolamento & purificaçãoRESUMO
The haloalkaliphilic bacterium Kocuria sp. (HJ014) has the ability to produce extracellular amylase. The aim of this study was to purify and characterize this protein. The amylase enzyme with a specific activity of 753,502 U/mg was purified 5.7- fold using Sepharose 4B and Sephacryl S-300 gel filtration columns. The molecular weight of the enzyme was 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amylase showed maximum activity at pH 9 and 50°C in the presence of 3.5 M NaCl. The Km was 3.0 mg/ml and Vmax 90.09 U/ml. It was found that extracellular amylase from Kocuria sp. has a high industrial potential.
Assuntos
Amilases/isolamento & purificação , Micrococcaceae/enzimologia , Amilases/química , Eletroforese em Gel de Poliacrilamida , Peso MolecularRESUMO
Aim: To determine the relationship between the chemical composition of saliva, periodontal disease and dental calculus. Methods: An observational analytical cross-sectional study was conducted with patients over 55 years of age. Ethical principles of autonomy and risk protection were applied according to the international standards. Sociodemographic and diagnosis variables (presence of dental calculus and periodontal status) were considered to measure salivary concentrations of glucose (by the glucose oxidase/peroxidase method, amylase (by the colorimetric test), urea (by the amount of indophenol), total protein (by the Bradford method) and albumin (by the nephelometric method). Patients chewed a sterile rubber band and 3 mL of stimulated saliva were collected. The samples were stored at -5 °C, centrifuged at 2,800 rpm for 10 min, and the supernatant was removed and stored at -20 °C. Data were presented as frequencies and proportions for qualitative variables and measures of central tendency and dispersion for quantitative variables. Data were analyzed by either analysis of variance or Kruskal Wallis test . A p value <0.05 was considered statistically significant. Results: Significant relationships were observed between the concentration of salivary urea and periodontal status (p = 0.03) and the presence of dental calculus and urea (p = 0.04) was demonstrated. Conclusions: A relationship between the salivary urea concentration and the presence of periodontal disease and dental calculus is suggested.
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Estudos Transversais , Cálculos Dentários/química , Doenças Periodontais/diagnóstico , Doenças Periodontais/epidemiologia , Gengivite/diagnóstico , Gengivite/epidemiologia , Fatores Socioeconômicos , Saliva/química , Albuminas/análise , Albuminas/química , Amilases/análise , Amilases/química , Glucose/análise , Glucose/química , Proteínas/análise , Proteínas/química , Ureia/análise , Ureia/químicaRESUMO
A robust biocatalyst was produced by immobilization of alpha-amylase into mesoporous silica thin films with local order pore structure and 11 nm pore diameter, supported on glass stripes. The activity of this novel catalyst was evaluated for direct starch degradation. The catalyst films show excellent activity, and enhanced stability with respect to free enzyme at extreme conditions of pH and temperature. In addition, they can be easily separated from the reaction media and reused several times.
Assuntos
Amilases/química , Biopolímeros/química , Membranas Artificiais , Nanoestruturas/química , Dióxido de Silício/química , Amido/química , Catálise , Enzimas Imobilizadas/química , Teste de Materiais , Porosidade , Propriedades de SuperfícieRESUMO
A maca (Lepidium meyenii Walpers) é uma planta herbácea bienal da família Brassicae, cultivada principalmente na região dos Andes da América do Sul. A parte subterrânea vem sendo consumida por muito tempo devido a seu valor nutricional e energético, mas é mais conhecida no mercado peruano e internacional por alegadas propriedades terapêuticas. Esta raiz apresenta até 76% de carboidratos, dos quais 30% é amido. Este trabalho teve como objetivos estudar: as propriedades físico-químicas e funcionais do amido isolado; os parâmetros enzimáticos durante o armazenamento e a purificação parcial de enzimas amilolíticas. Em relação às propriedades do amido, este apresentou um teor de amilose de 20% valor semelhante aos encontrados em raízes e tubérculos similares. A turbidez das suspensões de amido apresentou estabilidade durante o armazenamento. A temperatura de gelatinização e a viscosidade da pasta foram a 45,7° e 46°C, respectivamente. Com base nos dados obtidos, o amido de maca seria indicado para alimentos que requeiram temperaturas moderadas no processamento, não sendo apropriado para o emprego em alimentos congelados. Os parâmetros enzimáticos medidos tais como teor de amido total, teor de açúcares solúveis, atividade amilolítica total, atividade de α e β amilases, não mostraram diferenças significativas entre as medidas durante um período de armazenamento de 16 dias. As microscopias eletrônicas de varredura (MEV) dos grânulos de amido mostraram grãos íntegros com superfícies lisas, com algumas depressões ao redor dos grânulos os quais poderiam indicar o inicio de ataque enzimático, ou fraturas na purificação. Em relação à purificação de enzimas amilolíticas, foi possível separar uma fração ativa com a carboximetilcelulose (CMC) seguida de cromatografia liquida de alta resolução (CLAE) que permitiu a separação de duas frações protéicas, analisadas por eletroforese SDS-PAGE e eletroforese bidimensional (2D). Os polipeptídeos identificados no gel 2D apresentaram...
Maca (Lepidium meyenii Walpers) is a biennial herbaceous plant from Brassicae family, grown mainly in the Andes of South America. The underground part has been consumed for a long time due to its nutritional value and energy, but is best known in the Peruvian and international market for alleged therapeutic properties. This root has up to 76% carbohydrates, of which 30% is starch. This work aimed to study: the physico-chemical properties of isolated starch, the enzymatic parameters during storage and partial purification of amylases. In relation to the properties of starch, the amylose content showed a 20% value similar to those found in roots and tubers alike. The turbidity of starch suspensions was stable during storage. The gelatinization temperature and viscosity of the paste were 45.7 ° and 46 ° C, respectively. Based on data obtained from the starch of litter would be given to foods that require moderate temperatures in processing and is not suitable for use in frozen foods. The enzymatic parameters measured such as total starch content, soluble sugars, total amylolytic activity, activity of α and β amylases, showed no significant differences between the measures over a storage period of 16 days. Electronic microscopy (SEM) of starch granules showed grains with smooth surfaces, with some depressions around the granules which could indicate the beginning of enzymatic attack, or fractures in the purification. Regarding the purification of amylases was possible to separate an active fraction with carboxymethylcellulose (CMC) followed by high-resolution liquid chromatography (HPLC) which allowed the separation of two protein fractions, analyzed by SDS-PAGE and two-dimensional electrophoresis (2D ). The polypeptides had a molecular mass between 22 and 27 kDa and isoelectric points ranging from 4.8 to 7.3.
Assuntos
Amido/isolamento & purificação , Enzimas/metabolismo , Lepidium/enzimologia , Fenômenos Químicos , Raízes de Plantas , Amilases/química , Amilose/química , Alegação de Propriedades Funcionais , Fenômenos de Química Orgânica , Fenômenos Fisiológicos VegetaisRESUMO
Rhizopus microsporus var. rhizopodiformis produced high levels of alpha-amylase and glucoamylase under solid state fermentation, with several agricultural residues, such as wheat bran, cassava flour, sugar cane bagasse, rice straw, corncob and crushed corncob as carbon sources. These materials were humidified with distilled water, tap water, or saline solutions--Segato Rizzatti (SR), Khanna or Vogel. The best substrate for amylase production was wheat bran with SR saline solution (1:2 v/v). Amylolytic activity was still improved (14.3%) with a mixture of wheat bran, corncob, starch and SR saline solution (1:1:0.3:4.6 w/w/w/v). The optimized culture conditions were initial pH 5, at 45 degrees C during 6 days and relative humidity around 76%. The crude extract exhibited temperature and pH optima around 65 degrees C and 4-5, respectively. Amylase activity was fully stable for 1 h at temperatures up to 75 degrees C, and at pH values between 2.5 and 7.5.
Assuntos
Amilases/química , Amilases/metabolismo , Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/métodos , Fibras na Dieta/microbiologia , Triticum/microbiologia , Zea mays/microbiologia , Ativação Enzimática , Estabilidade Enzimática , Especificidade da EspécieRESUMO
There is a wide range of biotechnological applications for amylases, including the textile, pharmaceutical, food and laundry industries. Hydrolytic enzymes are 100% biodegradable and enzymatic detergents can achieve effective cleaning with lukewarm water. Microorganisms and culture media were tested for amylase production and the best producer was Aspergillus niger L119 (3.9 U ml(-1) +/- 0.2) in submerged culture and its amylase demonstrated excellent activity at 50-55 degrees C and pH 4.0, remaining stable at 53 degrees C for up to 200 h. In order to establish the potential uses of this enzyme in detergents, different formulations were tested using the A. niger amylase extract. Enzyme activity was compared with three commercial formulations. The detergents are used in hospitals to clean surgical and endoscopy equipment. The presence of amylase in the formulation is because of its action within hospital drainage system, whether or not it has any function in cleaning the equipment.
Assuntos
Amilases/química , Aspergillus niger/enzimologia , Detergentes/química , Análise de Variância , Aspergillus niger/genética , Meios de Cultura , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Temperatura , Fatores de TempoRESUMO
An isolate of Trichoderma harzianum showing antagonistic activity against Crinipellis perniciosa, the causal agent of the witches' broom disease of cocoa, produces substantial amounts of hydrolytic enzymes. An amylase purified from isolate 1051 had a molecular mass of about 68.7 kDa. Maximal activity against soluble starch was determined at pH 4.0 and 60 degrees C. The K(m) and V(max) values were 3.5 mg ml(-1) and 1.67 mg min(-1) of reducing sugar. The end products were mostly malto-oligosaccharides. The enzyme also hydrolyzed glycogen, amylopectin, maltotriose, and maltotetraose, but not pullulan or cellobiose. Maltose was only barely hydrolyzed. The purified amylase exerted a discrete hydrolytic effect on the C. perniciosa cell wall in vitro as observed by scanning electron microscopic analysis. While Fe(3+), Al(3+), Zn(2+), and Cu(2+) were effective in inhibiting the purified amylase, Mn(2+) considerably enhanced the activity. Ca(2+), Mg(2+), and Co(2+) showed no substantial effect on enzyme activity.