RESUMO
In the present study, it was presented a strategy to maximize the cutinase production by solid-state fermentation from different microorganisms and substrates. The best results were observed using Fusarium verticillioides, rice bran being the main substrate. Maximum yield of cutinase obtained by the strain was 16.22 U/g. For concentration, ethanol precipitation was used, and the purification factor was 2.4. The optimum temperature and pH for enzyme activity were 35 °C and 6.5, respectively. The enzyme was stable at a wide range of temperature and at all pH values tested. The concentrated cutinase was used as an adjuvant in a formulation containing cutinase + bioherbicide. The use of enzyme increased the efficiency of bioherbicide, since cutinase was responsible to remove/degrade the cutin that recovery the weed leaves and difficult the bioherbicide absorption. Cutinase showed to be a promising product to be used in formulation of bioherbicides.
Assuntos
Hidrolases de Éster Carboxílico , Proteínas Fúngicas , Fusarium/enzimologia , Herbicidas/metabolismo , Controle Biológico de Vetores , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Herbicidas/química , Concentração de Íons de Hidrogênio , Oryza/químicaRESUMO
A novel tannase and gallic acid-producing Penicillium rolfsii (CCMB 714) was isolated from cocoa leaves from the South of Bahia. The influence of nutritional sources and the simultaneous effect of parameters involved in the fermentation process were available. Tannase (9.97 U mL-1) and gallic acid (9 mg mL-1) production were obtained in 48 h by submerged fermentation in non-optimized conditions. Among the carbon sources, tested gallic acid and tannic acid showed the highest tannase production (p<.05) when compared with methyl gallate and glucose. After optimization using the temperature and tannic acid concentration as variables with the Central Compound Rotational Design (CCRD), the maximal tannase production (25.6 U mL-1) was obtained at 29.8 °C and 12.7%, respectively, which represents an increase of 2.56 times in relation to the initial activity. The parameters optimized for the maximum production of gallic acid (21.51 mg mL-1) were 30 °C and 10% tannic acid. P. rolfsii CCMB 714 is a new strain with a high tannase and gallic acid production and the gallic acid produced is very important, mainly for its applications in the food and pharmaceutical industry.
Assuntos
Hidrolases de Éster Carboxílico/biossíntese , Proteínas Fúngicas/biossíntese , Ácido Gálico/metabolismo , Penicillium/metabolismo , Penicillium/isolamento & purificaçãoRESUMO
Background Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20-60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application.
Assuntos
Aspergillus/enzimologia , Hidrolases de Éster Carboxílico/biossíntese , Fermentação , Temperatura , Cinética , Concentração de Íons de HidrogênioRESUMO
Background A sequential statistical strategy was used to optimize tannase production from Aspergillus tubingensis using tea stalks by solid-state fermentation. Results First, using a Plackett-Burman design, inoculum size and incubation time (among seven tested variables) were identified as the most significant factors for tannase yield. The effects of significant variables were further evaluated through a single steepest ascent experiment and central composite design with response surface analysis. Under optimal conditions, the experimental value of 84.24 units per gram of dry substrate (U/gds) closely matched the predicted value of 87.26 U/gds. Conclusions The result of the statistical approach was 2.09 times higher than the basal medium (40.22 U/gds). The results were fitted onto a second-order polynomial model with a correlation coefficient (R²) of 0.9340, which implied an adequate credibility of the model.
Assuntos
Aspergillus/enzimologia , Chá , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/biossíntese , Análise de Variância , Modelos Estatísticos , Biomassa , FermentaçãoRESUMO
Gallic acid production in a batch bioreactor was evaluated using as catalytic material the mouldy polyurethane solids (MPS) obtained from a solid-state fermentation (SSF) bioprocess carried out for tannase production by Aspergillus niger GH1 on polyurethane foam powder (PUF) with 5 % (v/w) of tannic acid as inducer. Fungal biomass, tannic acid consumption and tannase production were kinetically monitored. SSF was stopped when tannase activity reached its maximum level. Effects of washing with distilled water and drying on the tannase activity of MPS were determined. Better results were obtained with dried and washed MPS retaining 84 % of the tannase activity. Maximum tannase activity produced through SSF after 24 h of incubation was equivalent to 130 U/gS with a specific activity of 36 U/mg. The methylgallate was hydrolysed (45 %) in an easy, cheap and fast bioprocess (30 min). Kinetic parameters of tannase self-immobilized on polyurethane particles were calculated to be 5 mM and 04.1 × 10(-2) mM/min for K M and V max, respectively. Results demonstrated that the MPS, with tannase activity, can be successfully used for the production of the antioxidant gallic acid from methyl-gallate substrate. Direct use of PMS to produce gallic acid can be advantageous as no previous extraction of enzyme is required, thus reducing production costs.
Assuntos
Aspergillus niger/metabolismo , Hidrolases de Éster Carboxílico/biossíntese , Proteínas Fúngicas/biossíntese , Ácido Gálico/análogos & derivados , Ácido Gálico/metabolismo , Taninos/metabolismo , Adsorção , Reatores Biológicos , Meios de Cultura/química , Dessecação , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Poliuretanos/químicaRESUMO
Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices. Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme. The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required. The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice. Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture. The enzyme showed the highest activity at pH 9.0 and 50°C. The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m.
Assuntos
Bebidas/microbiologia , Metabolismo dos Carboidratos , Hidrolases de Éster Carboxílico/biossíntese , Fermentação , Resíduos Industriais , Penicillium/enzimologia , Vitis/química , Agricultura , Aspergillus/efeitos dos fármacos , Aspergillus/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Fermentação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Penicillium/efeitos dos fármacos , Especificidade da Espécie , Taninos/farmacologia , TemperaturaRESUMO
Endophytic fungi, mostly belonging to the Ascomycota, are found in the intercellular spaces of the aerial plant parts, particularly in leaf sheaths, sometimes even within the bark and root system without inducing any visual symptoms of their presence. These fungi appear to have a capacity to produce a wide range of enzymes and secondary metabolites exhibiting a variety of biological activities. However, they have been only barely exploited as sources of enzymes of industrial interest. This review emphasizes the suitability and possible advantages of including the endophytic fungi in the screening of new enzyme producing organisms as well as in studies aiming to optimize the production of enzymes through well-known culture processes. Apparently endophytic fungi possess the two types of extracellular enzymatic systems necessary to degrade the vegetal biomass: (1) the hydrolytic system responsible for polysaccharide degradation consisting mainly in xylanases and cellulases; and (2) the unique oxidative ligninolytic system, which degrades lignin and opens phenyl rings, comprises mainly laccases, ligninases and peroxidases. The obvious ability of endophytic fungi to degrade the complex structure of lignocellulose makes them useful in the exploration of the lignocellulosic biomass for the production of fuel ethanol and other value-added commodity chemicals. In addition to this, endophytic fungi may become new sources of industrially useful enzymes such as lipases, amylases and proteases.
Assuntos
Ascomicetos/enzimologia , Endófitos/enzimologia , Proteínas Fúngicas/biossíntese , Amilases/biossíntese , Amilases/isolamento & purificação , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Reatores Biológicos , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/isolamento & purificação , Endófitos/genética , Fermentação , Proteínas Fúngicas/isolamento & purificação , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/isolamento & purificaçãoRESUMO
The optimization of tannase production by Lactobacillus plantarum CIR1 was carried out following the Taguchi methodology. The orthogonal array employed was L18 (2(1) × 3(5)) considering six important factors (pH and temperature, also phosphate, nitrogen, magnesium, and carbon sources) for tannase biosynthesis. The experimental results obtained from 18 trials were processed using the software Statistical version 7.1 using the character higher the better. Optimal culture conditions were pH, 6; temperature, 40 °C; tannic acid, 15.0 g/L; KH2PO4, 1.5 g/L; NH4Cl, 7.0 g/L; and MgSO4, 1.5 g/L which were obtained and further validated resulting in an enhance tannase yield of 2.52-fold compared with unoptimized conditions. Tannase production was further carried out in a 1-L gas-lift bioreactor where two nitrogen flows (0.5 and 1.0 vvm) were used to provide anaerobic conditions. Taguchi methodology allowed obtaining the optimal culture conditions for the production of tannase by L. plantarum CIR1. At the gas-lift bioreactor the tannase productivity yields increase 5.17 and 8.08-fold for the flow rates of 0.5 and 1.0 vvm, respectively. Lactobacillus plantarum CIR1 has the capability to produce tannase at laboratory-scale. This is the first report for bacterial tannase production using a gas-lift bioreactor.
Assuntos
Reatores Biológicos/microbiologia , Hidrolases de Éster Carboxílico/biossíntese , Lactobacillus plantarum/enzimologia , Bioengenharia , Biomassa , Desenho de Equipamento , Fermentação , Gases , Microbiologia Industrial , Cinética , Lactobacillus plantarum/crescimento & desenvolvimento , Taninos/metabolismoRESUMO
Pectin methylesterase removes the methyl groups from the main chain of pectin, the major component of the middle lamella of the plant cell wall. The enzyme is involved in plant cell-wall development, is part of the enzymatic arsenal used by microorganisms to attack plants and also has a wide range of applications in the industrial sector. Therefore, there is a considerable interest in studies of the structure and function of this enzyme. In this work, the pectin methylesterase from Sphenophorus levis was produced in Pichia pastoris and purified. Crystals belonging to the monoclinic space group C2, with unit-cell parameters a = 122.181, b = 82.213, c = 41.176â Å, ß = 97.48°, were obtained by the sitting-drop vapour-diffusion method and an X-ray diffraction data set was collected to 2.1â Å resolution. Structure refinement and model building are in progress.
Assuntos
Hidrolases de Éster Carboxílico/química , Proteínas de Insetos/química , Gorgulhos/enzimologia , Animais , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/isolamento & purificação , Cristalização , Cristalografia por Raios X , Escherichia coli , Proteínas de Insetos/biossíntese , Proteínas de Insetos/isolamento & purificaçãoRESUMO
Tannase from Aspergillus niger was partitioned in aqueous two-phase systems composed by polyethyleneglycol of molar mass 400, 600 and 1000 and potassium phosphate. Tannase was found to be partitioned toward the salt-rich phase in all systems, with partition coefficients lower than 0.5. Partition coefficients values and low entropic and enthalpic changes associated with tannase partition suggest that the entropic effect may be the driving force of the concentration of the enzyme in the bottom phase due to the high molar mass of the enzyme. The process was significantly influenced by the top phase/bottom phase volume ratio. When the fungal culture broth was partitioned in these systems, a good performance was found, since the enzyme recovery in the bottom phase of the system composed by polyethyleneglycol 1000 was around 96% with a 7.0-fold increase in purity.