Purification and Biochemical Properties of a Glucose-Stimulated beta-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Production and purification of an Endo–1,4-beta-Xylanase from Humicola grisea var. thermoidea by electroelution

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated beta-glucosidase from the thermophilic fungus Humicola insolens

A mycelial beta-glucosidase from the thermophilic mold Humicola insolens was purified and biochemically characterized. The enzyme showed carbohydrate content of 21% and apparent molecular mass of 94 kDa, as estimated by gel filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a single polypeptide band of 55 kDa, suggesting that the native enzyme was a homodimer. Mass spectrometry analysis showed amino acid sequence similarity with a P-glucosidase from Humicola grisea var. thermoidea, with about 22% coverage. Optima of temperature and pH were 60 degrees C and 6.0-6.5, respectively. The enzyme was stable up to I h at 50 degrees C and showed a half-life of approximately 44 min at 55 degrees C. The beta-glucosidase hydrolyzed cellobiose, lactose, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-galactopyranoside, o-nitrophenyl-beta-D-galactopyranoside, and salicin. Kinetic studies showed that p-nitrophenyl-beta-D-fucopyranoside and cellobiose were the best enzyme substrates. Enzyme activity was stimulated by glucose or xylose at concentrations up to 400 mM, with maximal stimulatory effect (about 2-fold) around 40 mM. The high catalytic efficiency for the natural substrate, good thermal stability, strong stimulation by glucose or xylose, and tolerance to elevated concentrations of these monosaccharides qualify this enzyme for application in the hydrolysis of cellulosic materials. (C) 2009 Elsevier Ltd. All rights reserved.

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 A degrees C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 A degrees C and pH 6.5 for A. terricola, and 65 A degrees C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 A degrees C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t (50) of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4-3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and beta-xylosidase were detected which might act synergistically with xylanase.

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4-5 days in liquid medium at 40A degrees C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60-70A degrees C. The enzymes were stable for 30 min at 60A degrees C, maintaining 95-98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5-5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0-8.0, while the enzyme from A. niveus was more stable at pH 4.5-6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.

Produção de celulases fúngicas por fermentação em estado sólido e submersa utilizando biomassa lignocelulósica

Pós-graduação em Microbiologia - IBILCE

Isolamento de fungo produtor de enzimas xilanolíticas: produção e caracterização de xilanase

Pós-graduação em Alimentos e Nutrição - FCFAR

Produção e imobilização de celulases em matriz de agarose com diferentes ativações químicas

Pós-graduação em Biotecnologia - IQ

delta-Lactam derivative from thermophilic soil fungus exhibits in vitro anti-allergic activity

From cultures of thermophilic soil fungus Humicola grisea var thermoidea, a delta-lactam derivative (3-(2-(4-hydroxyphenyl)-2-oxoethyl)-5,6-dihydropyridin-2( 1H)-one) that displayed anti-allergic activity was isolated, which was predicted by in silico computational chemistry approaches. The in vitro anti-allergic activity was investigated by beta-hexosaminidase release assay in rat basophilic leukaemia RBL-2H3 cells. The delta-lactam derivative exhibited similar anti-allergic activity (IC50 = 18.7 +/- 6.7 mu M) in comparison with ketotifen fumarate (IC50 = 15.0 +/- 1.3 mu M) and stronger anti-allergic activity than azelastine (IC50 = 32.0 mu M). Also, the MTT cytotoxicity assay with RBL-2H3 cells showed that delta-lactam does not display cytotoxicity at concentrations lower than 50 mu M. This study suggests that the delta-lactam derivative has the potential to be used as a lead compound in the development of anti-allergic drugs for clinical use in humans.

Diversidade de Magnaporthe grisea em arroz de terras altas no sul do Estado do Tocantins, na safra 2008/09

A brusone do arroz, causada pelo fungo Magnaporthe grisea, é uma das mais importantes doenças orizícolas no Brasil e no mundo, principalmente em arroz de terras altas. Objetivou-se, neste trabalho, identificar as raças fisiológicas de M. grisea nas lavouras comerciais de arroz, cultivadas em sistema de terras altas, na região sul do Estado do Tocantins. Para isso, realizaram-se amostragens de plantas de arroz com sintomas de brusone, no campo experimental da Universidade Federal do Tocantins/Campus de Gurupi e em lavouras comerciais dos municípios de Aliança, Dueré, Figueirópolis e Peixe. Para identificação das raças de M. grisea, foram feitos isolamentos monospóricos em laboratório e plantio das linhagens diferenciadoras em casa de vegetação. Constataram-se 21 raças fisiológicas de M. grisea, distribuídas em seis grupos de raças da Série Internacional Diferenciadora, sendo IA e ID os grupos de maior prevalência, que apresentaram 52,38 e 14,28% dos isolados, respectivamente. As quatro raças mais prevalentes foram IA-1, IA-33, IC-1 e ID-9, sendo a IA-1 considerada muito agressiva. Maior número de raças foi encontrado no cultivar Primavera.

Eumycotic mycetoma due to Madurella grisea

The authors reported one case of eumycotic mycetoma due to Madurella grisea (black grains) occurred on the right foot of the patient studied. The structure, microscopic morphology and therapeutic evolution are also studied and reported.

Eumicetoma de grãos pretos por Madurella grisea: registro de dois casos

Os Autores registram dois casos de eumicetoma de grãos pretos, com localização podal, procedentes da Bahia, provocados por Madurella grisea Mackinnon et al., 1949. São estudadas a estrutura dos grãos, bem como as características micromorfológicas do fungo em vida saprofítica. Acreditam os Autores que estas observações correspondem ao sétimo e oitavo casos registrados na literatura do país, provocadas por este fungo. Os Autores consideram nomen dubium ou nomina confusa as seguintes espécies de Madurella: M. ramiroi, M. oswaldoi, M. bovoi, M. tozeuri, M. mansonii, M. brumpti, M. reynieri, M. americana, M. lackawanna e M. ikedae, o mesmo ocorrendo com a chamada Rubromadurella mycetomi. As únicas espécies válidas são Madurella mycetomatis McGinnis, 1980 (=Madurella mycetomi Brumpt, 1905) e Madurella grisea Mackinnon et al., 1949. Nos dois casos registrados o tratamento com itraconazol, por um período de 3 meses não fez regredir as lesões, havendo ligeira melhora clínica.

EUMYCETOMA BY Madurella grisea: REPORT OF THE FIRST CASE OBSERVED IN THE SOUTHERN BRAZILIAN REGION

The first case of eumycetoma by Madurella grisea occurred in Southern Brazilian Region is herein related. In addition, Brazilian literature on this subject was reviewed and, the geographic distribution of this eumycetoma is presented.

Eumicetoma por Madurella grisea: relato de caso

E relatado um caso de micetoma por Madurella grisea de localização podai em uma mulher do lar. A identificação da amostra foi feita através das características macro e microscópicas do agente em ágar Sabouraud e através da assimilação de açúcares. Este é o quinto caso de micetoma por Madurella grisea relatado no Brasil.