A new extracellular glutaminase and urease-free L-asparaginase from Meyerozyma guilliermondii.

L-Asparaginase (L-ASNase) is a potent chemotherapeutic drug employed to treat leukemia and lymphoma. Currently, L-ASNases for therapeutic use are obtained from Escherichia coli and Dickeya chrysanthemi (Erwinia chrysanthemi). Despite their therapeutic potential, enzymes from bacteria are subject to inducing immune responses, resulting in a higher number of side effects. Eukaryote producers, such as fungi, may provide therapeutic alternatives through enzymes that induce relatively less toxicity and immune responses. Additional expected benefits from yeast-derived enzymes include higher activity and stability in physiological conditions. This work describes the new potential therapeutic candidate L-ASNase from the yeast Meyerozyma guilliermondii. A statistical approach (full factorial central composite design) was used to optimize L-ASNase production, considering L-asparagine and glucose concentration, pH of the medium, and cultivation time as independent factors. In addition, the crude enzymes were biochemically characterized, in terms of temperature and optimal pH, thermostability, pH stability, and associated glutaminase or urease activities. Our results showed that enzyme production increased after supplementing a pH 4.0 medium with 1.0% L-asparagine and 0.5% glucose during 75 h of cultivation. Under these optimized conditions, L-ASNase production reached 26.01 U mL-1, which is suitable for scale-up studies. The produced L-ASNase exhibits maximal activity at 37 °C and pH 7.0 and is highly stable under physiological conditions. In addition, M. guilliermondii L-ASNase has no associated glutaminase or urease activities, demonstrating its potential as a promising antineoplastic agent.

Thermophilic fungi in Araucaria Forest, Atlantic Forest Biome, Brazil.

Thermophilic fungi constitute an ecologically well-defined group, commonly found in environments wherever decomposition of organic matter takes place, making them self-heating. The importance of thermophilic fungus in ecosystems contrasts with the incompleteness of our understanding of the group's biogeography patterns, phylogenies and coevolution relationships. Actually, the lack of data about thermophilic fungi from the Brazil is a limiting factor that also contributes for this scenario. In order to reduce this gap of knowledge, we aimed to characterize thermophilic filamentous fungi in Araucaria Forest, Atlantic Forest biome. Species identification was achieved by using internal transcribed spacers (ITS) as molecular ribosomal markers. In total, 240 heat-tolerant fungal strains were isolated and identified as Thermothielavioides terrestris, Thielavia sp., Thermoascus crustaceus, Aspergillus fumigatus, Rhizomucor miehei, Rhizomucor pusillus, and Rhizopus microsporus. All thermophilic strains exhibited optimal growth at 45 °C. T. crustaceus, T. miehei e R. pusillus were the dominant species, with the frequencies of occurrence of 35.00%, 28.33% and 23.33%, respectively. Our data reveals the apparent diversity of the Neotropical realm and may serve as reference to future studies that will try to elucidate important aspects of group.

Defense responses of wheat plants (Triticum aestivum L) against brown spot as a result of possible elicitors application / Resposta de defesa das plantas de trigo (Triticum aestivum L) contra a mancha marrom diante da aplicação de possíveis elicitores

The objective of this research was to evaluate the response of wheat plants to the application of possible elicitor compounds against Bipolaris sorokiniana pathogen. This response was measured through the quantification of antioxidant enzymes, malondialdehyde and flavonoids, evaluation of the severity of brown spot disease and productivity in wheat, greenhouse and field crops. The treatments consisted of suspensions of endophytic fungi Aspergillus japonicus and Trichoderma tomentosum, salicylic acid, acibenzolar-S-methyl and fungicide. In the field trials, in 2015 and 2016, the development of the disease was lower and productivity was higher in all treatments, with emphasis on the fungicide. However, endophytic fungi suspensions demonstrated potential as growth promoters, disease severity reducers and protective antioxidant response activators, as they promoted significant increase in superoxide dismutase, catalase, glutathione and flavonoid enzymes.(AU)

O objetivo desta pesquisa foi avaliar a resposta de plantas de trigo diante da aplicação de possíveis compostos elicitores perante o patógeno Bipolaris sorokiniana. Tal resposta foi examinada por meio da quantificação de enzimas antioxidantes, malondialdeído e flavonoides, da análise da severidade da doença mancha marrom e da produtividade na cultura do trigo, em casa de vegetação e em campo. Os tratamentos consistiram em suspensões de fungos endofíticos Aspergillus japonicus e Trichoderma tomentosum, ácido salicílico, acibenzolar-S-metil e fungicida. Nos ensaios em campo, em 2015 e 2016, o desenvolvimento da doença foi menor e a produtividade foi superior em todos os tratamentos, com destaque para o fungicida. No entanto, as suspensões de fungos endofíticos demonstraram potencial como promotores de crescimento, redutores da severidade da doença e ativadores de resposta antioxidante protetora, pois promoveram o aumento significativo das enzimas superóxido dismutase e catalase, das glutationas e flavonoides.(AU)

Production of xylanases by an Aspergillus niger strain in wastes grain / Produção de xilanases por uma linhagem de Aspergillus niger em resíduos de grãos

Many fungi are used in order to extract products from their metabolism through bioprocesses capable of minimizing adverse effects caused by agro- industrial wastes in the environment. The aim of this study was to evaluate the xylanase production by an Aspergillus niger strain, using agro-industrial wastes as substrate. Brewer's spent grain was the best inducer of xylanase activity. Higher levels of xylanase were obtained when the fungus was grown in liquid Vogel medium, pH 5.0, at 30ºC, during 5 days. The temperature for optimum activity was 50ºC and optimum pH 5.0. The enzyme was stable at 50ºC, with a half-life of 240 min. High pH stability was verified from pH 4.5 to 7.0. These characteristics exhibited by A. niger xylanase turn this enzyme attractive for some industrial applications, such as in feed and food industries. Additionally, the use of brewer's spent grain, an abundantly available and low-cost residue, as substrate for xylanase production can not only add value and decrease the amount of this waste, but also reduce xylanase production cost.

Muitos fungos são utilizados com a finalidade de extrair produtos de seu metabolismo, por meio de bioprocessos capazes de minimizar efeitos nocivos que resíduos agroindustriais causam ao meio ambiente. O objetivo deste estudo foi avaliar a produção de xilanases por uma linhagem de Aspergillus niger, empregando resíduos agroindustriais como substrato. O bagaço de malte foi o melhor resíduo indutor da atividade xilanásica. Maiores níveis de xilanases foram obtidos quando o fungo foi cultivado em meio líquido de Vogel, pH 5,0, a 30ºC, durante cinco dias. A temperatura ótima estabelecida para a atividade xilanásica foi a de 50ºC e o pH ótimo 5,0. A enzima foi estável a 50ºC, apresentando uma meia vida de 240 min. Elevada estabilidade enzimática foi verificada entre os pH 4,5 e 7,0. As características bioquímicas exibidas pela xilanase produzida por A. niger tornam esta enzima atraente para determinadas aplicações industriais, como as indústrias de ração animal e alimentícia. Adicionalmente, a utilização do bagaço de malte, um resíduo disponível em abundância e de baixo custo como substrato para a produção de xilanases poderá não somente adicionar valor a este resíduo, como também reduzir os custos de produção destas enzimas.

Production, purification, and characterization of a major Penicillium glabrum xylanase using Brewer's spent grain as substrate.

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn(2+) and the reducing agents ß -mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg(2+), Zn(2+), and Cu(2+) as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.

Purification and characterization of two extracellular xylanases from Penicillium sclerotiorum: a novel acidophilic xylanase.

Two xylanases from the crude culture filtrate of Penicillium sclerotiorum were purified to homogeneity by a rapid and efficient procedure, using ion-exchange and molecular exclusion chromatography. Molecular masses estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 23.9 and 33.1 kDa for xylanase I and II, respectively. The native enzymes' molecular masses of 23.8 and 30.8 kDa were estimated for xylanase I and II, respectively, by molecular exclusion chromatography. Both enzymes are glycoproteins with optimum temperature and pH of 50 degrees C and pH 2.5 for xylanase I and 55 degrees C and pH 4.5 for xylanase II. The reducing agents beta-mercaptoethanol and dithio-treitol enhanced xylanase activities, while the ions Hg(2+) and Cu(2+) as well the detergent SDS were strong inhibitors of both enzymes, but xylanase II was stimulated when incubated with Mn(2+). The K (m) value of xylanase I for birchwood xylan and for oat spelt xylan were 6.5 and 2.6 mg mL(-1), respectively, whereas the K (m) values of xylanase II for these substrates were 26.61 and 23.45 mg mL(-1). The hydrolysis of oat spelt xylan by xylanase I released xylobiose and larger xylooligosaccharides while xylooligosaccharides with a decreasing polymerization degree up to xylotriose were observed by the action of xylanase II. The present study is among the first works to examine and describe an extracellular, highly acidophilic xylanase, with an unusual optimum pH at 2.5. Previously, only one work described a xylanase with optimum pH 2.0. This novel xylanase showed interesting characteristics for biotechnological process such as feed and food industries.

Cell-associated acid beta-xylosidase production by Penicillium sclerotiorum.

In recent decades, beta-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 degrees C, under stationary condition for six days. The optimum activity temperature was 60 degrees C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 degrees C, with half-life of 240 and 232min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The beta-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V(max) and K(m) values was 0.48micromol PNXPmin(-1)mg(-1) protein and 0.75mM, respectively. The enzyme was xylose tolerant with a K(i) of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.