Prospecting Filamentous Fungi for Amylase Production: Standardization of Aspergillus japonicus Culture Conditions.

Aims: Prospecting different filamentous fungi for high production of amylases in standard conditions for future application in biotechnology industries. Methodology: Samples were collected in different field areas in the state of Bahia, Brazil, for isolating filamentous fungi. Fungi were grown in Petri dishes in a culture medium containing 4% Quaker® oatmeal and 2% bacteriological agar. Fungi screening was carried out in liquid medium containing 1% starch at 30°C and pH6.0 under static conditions for 4 days. Proteins and enzyme activities were determined by Bradford and DNS methods, respectively. A submerged fermentation was performed with different liquid media in order to obtain the best growth composition and enzyme production for the selected fungus. Several conditions such as time course of inactivation, pH, temperature, carbon and nitrogen sources were determined in the culture medium with the aim of improving amylase production and fungi growth. Various fibers and food residues were used as enzymatic inducers in a way to assess potential integration of this enzyme, producing microorganisms in the food industry. Results: A. japonicus proved to be the best degrader of fibers and food residues. After 4 days, A. japonicus enzyme production was maximal, with 44.65(±0.49) U/ml under static conditions. The maximal enzyme activity was obtained at pH 6.0, retaining its activity even at a higher pH. The optimum temperature was 25°C. The best carbon source was potato starch and the best food residues were orange bagasse and bark for the enzyme production by A. japonicus. Conclusion: The results suggest that A. japonicus is a good amylase producer for the degradation of fibers and food residues, indicating that it might become important for the food industry, bringing value to what is known as waste these days.

Screening of Filamentous Fungi for Xylanases and Cellulases Not Inhibited by Xylose and Glucose.

Aims: Screening different filamentous fungi for thermostable xylanases and cellulases that would not be inhibited by xylose and glucose, respectively. Methodology: Samples of fungi collected in the Atlantic forest region, Minas Gerais, Brazil, and some fungi from our Culture Collection were used in this screening. All fungi were grown in liquid media containing 1% sugar cane bagasse (SCB). After that, an aliquot of the crude broth was incubated at different temperatures (from 4 to 60 °C) in carboxymethyl cellulose (CMC) or xylan-media plates, for 12 hours. After this period, the plates were stained with Congo Red. Fungi that presented the best results (larger halos) were tested for the effect of adding xylose and glucose in the xylanase and cellulases activities, respectively. Crude extracts obtained from fungi grown in SCB were used for laccase and lichenase assay. Results: The screening on agar plates with CMC/xylan presented halos of different sizes. From all tested fungi, the best cellulase producer was Malbranchea pulchella, which also presented the most thermostable xylanase. Penicillium griseofulvum presented bigger halos at all temperatures tested, but the xylanase lost almost 14% of its stability in higher temperatures. The effect of xylose and glucose on the enzymatic activities recorded dosedependent. It was observed that 20% activation of the enzymes produced by M. pulchella with 30 mM glucose or 20 mM xylose to cellulase and xylanase, respectively. It was observed a loss of less than 20% for P. griseofulvum xylolytic activity using 50 mM xylose. Lichenase was detected in some fungi prospected but laccase was not detected. Conclusion: Malbranchea pulchella was a good producer of xylanase and cellulase tolerant to xylose and glucose, respectively. Other studies must be performed with this fungus so that it can be used in the future for biotechnological purposes.

Mycelial glucoamylases produced by the thermophilic fungus Scytalidium thermophilum strains 15.1 and 15.8: purification and biochemical characterization / Glucoamilases miceliais produzidas pelas linhagens 15.1 e 15.8 do fungo termofílico Scytalidium thermophilum: purificação e caracterização bioquímica

Two strains (15.1 and 15.8) of the thermophilic fungus Scytalidium thermophilum produced high levels of intracellular glucoamylases, with potential for industrial applications. The isoform I of the glucoamylase produced by 15.1 strain was sequentially submitted to DEAE-Cellulose and CM-Cellulose chromatography, and purified 141-fold, with 5.45 percent recovery. The glucoamylase of strain 15.8 was purified 71-fold by CM-Cellulose and Concanavalin A-Sepharose chromatography, with 7.38 percent recovery. Temperature and pH optima were in the range of 50-60ºC and 5.0-6.0, respectively, using starch and maltose as substrates. The glucoamylase of S. thermophilum 15.8 was more stable (t50 > 60 min) than that of S. thermophilum 15.1 (t50= 11-15 min), at 60ºC. The glucoamylase activities were enhanced by several ions (e.g. Mn2+ and Ca2+) and inhibited by β-mercaptoethanol. The glucoamylase from 15.1 strain showed a Km of 0.094 mg/ml and 0.029 mg/ml and Vmax of 202 U/mg prot and 109 U/mg prot, for starch and maltose, respectively. The hydrolysis products of starch and maltose, analyzed by TLC, demonstrated glucose as end product and confirming the character of the enzyme as glucoamylase. Differences were observed in relation to the products formed with maltose as substrate between the two strains studied. S. thermophilum 15.8 formed maltotriose in contrast with S. thermophilum 15.1.

Duas linhagens (15.1 e 15.8) do fungo termofílico Scytalidium thermophilum se mostraram produtoras de grandes quantidades de glucoamilases, com potencial aplicação industrial. A isoforma I de glucoamilase produzida pela linhagem 15.1 foi submetida seqüencialmente a cromatografia em colunas de DEAE-celulose e CM-celulose, sendo purificada 141 vezes com porcentagem de recuperação de 5,45 por cento. A glucoamilase da linhagem 15.8 foi purificada 71 vezes através do uso de colunas de cromatografia de CM-celulose e Concanavalina A-sepharose com porcentagem de recuperação de 7,38 por cento. Temperatura e pH ótimo foram de 50-60ºC e 5,0-6,0 respectivamente, utilizando-se amido e maltose como substratos. A glucoamilase de S. thermophilum 15.8 se mostrou mais estável (t50 > 60 min) que a de S. thermophilum 15.1 (t50 =11-15min) a 60ºC. As glucoamilases tiveram suas atividades enzimáticas aumentadas na presença de vários íons (ex: Mn2+, e Ca2+) e inibidas por β-mercaptoetanol. A glucoamilase da linhagem 15.1 apresentou um Km de 0,094 mg/ml e 0,029 mg/ml and Vmax de 202U/mg prot e 109U/mg prot, para amido e maltose respectivamente. A análise do produto da hidrólise de amido e maltose por TLC, demonstrou que o produto final era glucose, confirmando as características da enzima como glucoamilase. Diferenças entre as duas linhagens foram observadas com relação aos produtos formados tendo maltose como susbstrato, a linhagem 15.8 de S. thermophilum produziu maltotriose como produto final em contrate com a linhagem 15.1.

Cell wall deficiency in slime strains of Neurospora crassa: osmotic inhibition of cell wall synthesis and Beta-D-glucan synthase activity

1. The RCP-3 S/H mutant of Neurospora crassa was obtained by vegetative selection in medium of high osmolarity of a mycelial form an fz, sg, os-1 ("smile"-like) segregant. The mutant exhibits spheroplast-hyphal dimorphism conditioned by the osmolarity of the culture medium (pietro et al. (1990). Journal of General Microbiology, 136: 121-129). The carbohydrate composition of the cell wall of the mutant was different from that of the wild type in the absence of an alkalisoluble galactosaminoglycan polymer. Furthermore the mutant cell wall had a somewhat lower content of ß-glucan relative to that of chitin. 2. Increasing concentrations of sorbitol in the culture medium of the mutant inhibited by 10-fold the formation of cell wall relative to toal biomass. The cell wall of the mutant cultured in the presence of sorbitol lacked mannose-and galactose-containing polymers, and also showed progressively lower amounts of ß-glucan relative to chitin. 3. The activity of membrane-bound (1-3)-ß-D-glucan synthase from the mutant grown in the absence of sorbitol shared several properties with the wil type enzyme (i.e., Km app, Vmax, stability at 30ºC, activation by GTPyS, and dissociability by treatment with NaCl and Tergitol NP-40 into a membrane-bound catalytic center and GTP-binding activating protein). On the other hand, the enzyme from the mutant but not that from the wild type was inactivated by about 15 per cent by treatment with NaCl and detergent. 4. At high concentrations of sorbitol (1.0M) the RCP-3 S/H mutant exclusively produced spheroplasts devoid of (1-3)-ß-D-glucan synthase activity. The defect was at the level of the membrane-bound catalytic center. The activity of the GTP-binding activating factor was apparently normal in these cells. 5. These results suggest that the definitive loss of cell wall in the N. crassa "slime" RCP-3 S/H mutant was due to a defect in (1-3)-ß-D-glucan synthase activity which wass exaggerated in the presence of high osmolyte concentrations