Production and characterization of a thermostable ß-glucosidase from Myceliophthora heterothallica / Produção e caracterização de uma ß-glicosidase termoestável de Myceliophthora heterothallica

The conversion of biomass from agro-industrial residues into bioproducts is of great interest, especially to Brazil, where bioenergy has a huge potential for development. Enzymes involved in biodegradation of lignocellulosic biomass are those of the cellulase system, of which ß-glucosidase is a constituent. The production and characterization of ß-glucosidase by the thermophilic fungus Myceliophthora heterothallica by solid-state cultivation on different agro-industrial residues (sugarcane bagasse, sugarcane straw, wheat bran and a mixture of these three materials (1:1:1 w/w) was evaluated. Solid-state cultivation were conducted in 250 mL Erlenmeyer flasks, with 5 g of each substrate. Different culture parameters, such as supplementary nutrient solution to the substrate, supplementary nutrient solution pH, initial substrate moisture and fungus incubation temperature, were evaluated to establish conditions of higher enzyme production by the fungus The greatest production of enzymes occurred in a mixture of wheat bran, sugarcane bagasse and straw bagasse (1:1:1). The activity of ß-glucosidase was greater under the following conditions: nutrient solution composed of NH4NO3, MgSO4.7H2O and (NH4)2SO4 (0.1%), at pH 4.5 or 6.0, fungus incubation at 40°C or 45°C, initial moisture of substrate at 80%. Enzyme presented optimum pH at pH 5.0 and good pH stability. Best temperature was 65°C and enzyme showed 100% stability for 1h, up to 60°C. The use of agro-industrial residues provided good production of ß-glucosidase by fungus, with enzyme having the characteristics desirable from the industrial application.

A conversão da biomassa vegetal proveniente de resíduos agroindustriais em bioprodutos é de grande interesse, principalmente para o Brasil, onde a agroenergia possui grande potencial de desenvolvimento. Enzimas envolvidas na biodegradação da biomassa lignocelulósica fazem parte do grupo das celulases, no qual a 훽-glucosidase é um constituinte. O presente estudo avaliou a produção e caracterização de uma ß-glicosidase pelo fungo termofílico Myceliophthora heterothallica por cultivo em estado sólido de diferentes resíduos agroindustriais (bagaço de cana-de-açúcar, palha de cana-de-açúcar, farelo de trigo e em uma mistura dos três materiais (1:1: 1 p/p). O cultivo em estado sólido foi realizado em frascos Erlenmeyer de 250 mL, contendo 5 g de cada substrato. Diferentes parâmetros de cultivo, como solução nutriente suplementar ao substrato, pH da solução nutriente suplementar, umidade inicial do substrato e temperatura de incubação do fungo foram avaliados, visando estabelecer condições para maior produção da enzima pelo fungo. A maior produção da enzima ocorreu na mistura de farelo de trigo, e bagaço e palha de cana-de-açúcar (1:1:1). A atividade da ß-glicosidase foi maior nas seguintes condições: solução nutriente composta por NH4NO3, MgSO4.7H2O e (NH4)2SO4 (0,1%) com pH 4,5 e 6,0, temperatura de incubação do fungo a 40°C e 45°C, com umidade inicial do substrato em 80%. A enzima apresentou pH ótimo de 5,0, e boa estabilidade ao pH. A temperatura ótima foi de 65°C, e a enzima apresentou 100% de estabilidade por 1h, até 60°C. A utilização de resíduos agroindustriais proporcionou boa produção de ß-glicosidase pelo fungo, com a enzima apresentando características desejáveis para aplicação industrial.

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic ß-glucosidase.

The current study evaluated the production and characterization of ß-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of ß-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5-4.5 and at 70 °C. The enzyme exhibited high thermostability, for 1 h, up to 60 °C, and good tolerance to glucose (10 mM) and ethanol (10%). The optimization of fermentative parameters on the production of ß-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0 U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH4)2SO4 solution at pH 5.5-6.0 and fungus incubated at 40 °C. A more detailed study of ß-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.

Purification and Properties of Polygalacturonase Produced by Thermophilic Fungus Thermoascus aurantiacus CBMAI-756 on Solid-State Fermentation.

Polygalacturonases are enzymes involved in the degradation of pectic substances, being extensively used in food industries, textile processing, degumming of plant rough fibres, and treatment of pectic wastewaters. Polygalacturonase (PG) production by thermophilic fungus Thermoascus aurantiacus on solid-state fermentation was carried out in culture media containing sugar cane bagasse and orange bagasse in proportions of 30% and 70% (w/w) at 45°C for 4 days. PG obtained was purified by gel filtration and ion-exchange chromatography. The highest activity was found between pH 4.5 and 5.5, and the enzyme preserved more than 80% of its activity at pH values between 5.0 and 6.5. At pH values between 3.0 and 4.5, PG retained about 73% of the original activity, whereas at pH 10.0 it remained around 44%. The optimum temperature was 60-65°C. The enzyme was completely stable when incubated for 1 hour at 50°C. At 55°C and 60°C, the activity decreased 55% and 90%, respectively. The apparent molecular weight was 29.3 kDa, K m of 1.58 mg/mL and V max of 1553.1 µ mol/min/mg. The presence of Zn(+2), Mn(+2), and Hg(+2) inhibited 59%, 77%, and 100% of enzyme activity, respectively. The hydrolysis product suggests that polygalacturonase was shown to be an endo/exoenzyme.

Production of cellulolytic and hemicellulolytic enzymes from Aureobasidium pulluans on solid state fermentation.

This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL beta-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and beta-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75 degrees C, respectively. These enzymes remained stable between a wide range of pH. The beta-glucosidase was the most thermostable enzyme, remaining 100% active when incubated at 75 degrees C for 1 h.

Pectinase production by Penicillium viridicatum RFC3 by solid state fermentation using agricultural wastes and agro-industrial by-products

Pectin lyase and polygalacturonase production by newly isolated Penicillium viridicatum strain Rfc3 was carried out by means of solid fermentation using orange bagasse, corn tegument, wheat bran and mango and banana peels as carbon sources. The maximal activity value of polygalacturonase (Pg) (30 U.g-1) was obtained using wheat bran as carbon source while maximal pectin lyase (Pl) (2000 U.g-1) activity value was obtained in medium composed of orange bagasse. Mixtures of banana or mango peels with sugar cane bagasse resulted in increased Pg and Pl production compared to fermentations in which this residue was not used. The mixture of orange bagasse and wheat bran (50 percent) increased the production of Pg and Pl to 55 U.g-1 and 3540 U.g-1 respectively. Fractions of Pg and Pl, isolated by gel filtration in Sephadex G50, presented optimum activity at pH 5.0 and 10.5 respectively. Maximal activity of Pg and Pl fractions was determined at 55oC and 50oC respectively. Pg was stable in neutral pH range and at 40oC whereas Pl was stable in acidic pH and at 35oC, for 1h.