Soil enzymatic activity under coffee cultivation with different water regimes associated to liming and intercropped brachiaria / Atividade enzimática de solo sob cultivo de café com diferentes regimes hídricos associados a calagem e braquiária nas entrelinhas

This research evaluated the effects of coffee cultivation with two different water regimes associated or not with liming and the presence/absence of brachiaria as intercrop on the activities of the soil enzymes ß-glucosidase, arylsulfatase and acid phosphatase. The study was carried out at the experimental farm of Embrapa Cerrados, using the cultivar IAC 144 (Coffea arabica L.), under a clayey dystrophic Cerrado Oxisol. Two water regimes (WR) were considered, WR1 with irrigation shifts throughout the year and WR3 with controlled water stress, for about 70 days, in the dry season. In each water regime, effects of lime application (with/without) and the presence/absence of brachiaria cultivated between the lines of coffee plants were evaluated. The activities of the enzymes ß-glucosidase, arylsulfatase and acid phosphatase were evaluated during the rainy and dry seasons. Liming and intercropped brachiaria positively affected the activities of the three enzymes assessed in this study at varying degrees, depending on season and/or the WR. Our findings evidenced that intercropped brachiaria in coffee rows was the factor that most positively impacted soil enzymes activities.

O objetivo desse trabalho foi avaliar os efeitos do cultivo do café sobre a atividade das enzimas do solo ß-glicosidase, arilsulfatase e fosfatase ácida em função de dois diferentes regimes hídricos associados ou não à calagem e ao cultivo de braquiária nas entrelinhas. Esse estudo foi realizado em um experimento conduzido no campo experimental do Centro de Pesquisa Agropecuária dos Cerrados, utilizando a cultivar IAC 144 (Coffea arabica L.), sob um Latossolo Vermelho distrófico argiloso. Foram considerados dois regimes hídricos (RH), RH1 com irrigação plena em turnos de rega ao longo do ano e RH3 com estresse hídrico controlado, por cerca de 70 dias, na época seca. Em cada regime hídrico foram avaliadas a aplicação ou não de calcário em cobertura e a presença ou ausência de braquiária cultivada nas entrelinhas das plantas do cafezal. As atividades das enzimas ß-glicosidase, arilsulfatase e fosfatase ácida foram avaliadas durante as estações chuvosa e seca. A calagem e a presença de braquiária nas entrelinhas tiveram efeito positivo sobre a atividade das três enzimas avaliadas nesse estudo, em graus variáveis, dependendo da época de coleta das amostras e/ou do RH. Nossos resultados evidenciam que a braquiária nas entrelinhas do café foi o fator de maior impacto positivo sobre a atividade enzimática do solo.

Analysis of polysaccharide hydrolases secreted by Aspergillus flavipes FP-500 on corn cobs and wheat bran as complex carbon sources.

Aspergillus flavipes FP-500 is a Mexican native strain that has been reported as a good producer of xylanases and pectinases; therefore, it promises a strong impact on biotechnology. To provide an overview of protein secretion by A. flavipes, we carried out a comparative proteome analysis of extracellular proteins in liquid cultures with two heterogeneous agro-industrial residues; corn cob (CC) and wheat bran (WB), as carbon sources. Extracellular proteins obtained from both cultures were identified using MS/MS spectrometry. We identified 134 proteins, which were classified into four groups: glycosyl hydrolases (GH), esterases/proteases, miscellaneous proteins, and unidentified proteins. Around 50% of the total proteins identified were GH such as xylanases, ß-xylosidases, ß-galactosidases, cellulolytic enzymes like ß-glucosidase, endoglucanases, and cellobiohydrolases. From this family, a core of 22 (16%) of the proteins identified were found in both substrates, CC and WB, whereas 30% and 54% were unique for CC and WB, respectively. In the esterases/proteases group, proteases, lipases and esterases like feruloylesterases, and acetyl-xylanesterase were identified. Proteins with diverse functions such as monophosphate dehydrogenase or N-acetylglucosaminidase were present. Here, we present strong evidences indicating that the composition and heterogeneity of the used carbon source determine the specific set of protein secreted by the fungus.

Diversity of Microbial Carbohydrate-Active enZYmes (CAZYmes) Associated with Freshwater and Soil Samples from Caatinga Biome.

Semi-arid and arid areas occupy about 33% of terrestrial ecosystems. However, little information is available about microbial diversity in the semi-arid Caatinga, which represents a unique biome that extends to about 11% of the Brazilian territory and is home to extraordinary diversity and high endemism level of species. In this study, we characterized the diversity of microbial genes associated with biomass conversion (carbohydrate-active enzymes, or so-called CAZYmes) in soil and freshwater of the Caatinga. Our results showed distinct CAZYme profiles in the soil and freshwater samples. Glycoside hydrolases and glycosyltransferases were the most abundant CAZYme families, with glycoside hydrolases more dominant in soil (∼44%) and glycosyltransferases more abundant in freshwater (∼50%). The abundances of individual glycoside hydrolase, glycosyltransferase, and carbohydrate-binding module subfamilies varied widely between soil and water samples. A predominance of glycoside hydrolases was observed in soil, and a higher contribution of enzymes involved in carbohydrate biosynthesis was observed in freshwater. The main taxa associated with the CAZYme sequences were Planctomycetia (relative abundance in soil, 29%) and Alphaproteobacteria (relative abundance in freshwater, 27%). Approximately 5-7% of CAZYme sequences showed low similarity with sequences deposited in non-redundant databases, suggesting putative homologues. Our findings represent a first attempt to describe specific microbial CAZYme profiles for environmental samples. Characterizing these enzyme groups associated with the conversion of carbohydrates in nature will improve our understanding of the significant roles of enzymes in the carbon cycle. We identified a CAZYme signature that can be used to discriminate between soil and freshwater samples, and this signature may be related to the microbial species adapted to the habitat. The data show the potential ecological roles of the CAZYme repertoire and associated biotechnological applications.

Comprehensive analysis of the cellulolytic system reveals its potential for deconstruction of lignocellulosic biomass in a novel Streptomyces sp.

The giant snail Achatina fulica is considered an invasive species in most territories in which it was introduced, due to its ability to process a large amount of lignocellulose as a consequence of the presence of a cellulolytic-associated microflora. Streptomyces are well known as crucial agents in the decomposition of complex polymers in soil environments and also as cellulolytic symbionts commonly associated with herbivore insects. Here, we employed a combination of genomic and biochemical tools for a detailed evaluation of the cellulolytic potential of Streptomyces sp. I1.2, an aerobic bacterium isolated from the intestinal lumen of A. fulica in a screening for cellulolytic bacteria. Genomic analysis revealed that the ratio and diversity of CAZy domains and GH families coded by Streptomyces sp. I1.2 are comparable to those present in other highly cellulolytic bacteria. After growth on crystalline cellulose or sugarcane bagasse as sole carbon sources, the functionality of several genes encoding endoglucanases, cellobiohydrolases, xylanases, CBMs, and one ß-glucosidase were confirmed by the combination of enzymatic activity measurements, zymography, TLC, and cellulose-binding assays. The endoglucanases secreted by this isolate were stable at 50 °C and exhibited activity over a broad pH range between 4.0 and 8.0. The endoglucanases and cellobiohydrolases secreted by Streptomyces sp. I1.2 exhibited specific activities that were similar to the levels present in a commercial cellulase preparation from Trichoderma reesei, while I1.2 xylanase levels were even 350 % higher. The results presented here show that Streptomyces sp. I1.2 is promising for future biotechnological applications, since it is able to produce endoglucanases, cellobiohydrolases, and xylanases in appreciable amounts when grown on a low-cost residue such as sugarcane bagasse.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan.

This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, ß-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. ß-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Screening of thermotolerant and thermophilic fungi aiming Beta-xylosidase and arabinanase production

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of β-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 ºC, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 ºC. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of β-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, β-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 ºC), while the correspondent extracellular activities were favorably secreted from cultures at 30 ºC. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.

Screening of thermotolerant and thermophilic fungi aiming β-xylosidase and arabinanase production

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of β-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 ºC, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 ºC. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of β-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, β-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 ºC), while the correspondent extracellular activities were favorably secreted from cultures at 30 ºC. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.

Screening of thermotolerant and thermophilic fungi aiming ß-xylosidase and arabinanase production.

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of ß-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 °C, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 °C. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of ß-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, ß-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 °C), while the correspondent extracellular activities were favorably secreted from cultures at 30 °C. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.

Crystallization and preliminary X-ray diffraction analysis of a new xyloglucanase from Xanthomonas campestris pv. campestris.

Xyloglucanases (Xghs) are important enzymes involved in xyloglucan modification and degradation. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacterium which produces a large number of glycosyl hydrolases (GH), but has only one family 74 GH (Xcc-Xgh). This enzyme was overexpressed in Escherichia coli, purified and crystallized. Diffraction data sets were collected for the native enzyme and its complex with glucose to maximum resolutions of 2.0 and 2.1 Å, respectively. The data were indexed in a hexagonal crystal system with unit-cell parameters a = b = 153.4, c = 84.9 Å. As indicated by molecular-replacement solution, the crystals belonged to space group P6(1).

Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements.

This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used.

Atividade das glicosidases na presença de chá verde e de chá preto / Activity of glycosidases in the presence of green tea and black tea

Várias plantas têm sido consideradas produtos terapêuticos, dentre elas destacam-se os chás verde e preto, popularmente utilizados para controle da hiperglicemia e obesidade. Objetivou-se neste trabalho avaliar o potencial inibitório sobre as enzimas α-amilase, α e β-glicosidases e o teor de compostos fenólicos do chá verde e do chá preto. O teor de compostos fenólicos encontrados foram de 80,8 ± 0,43 mg g-1 no chá preto e 32,0 ± 0,12 mg g-1 no chá verde. O chá verde e o chá preto, em condições de consumo, inibiram as enzimas em estudo, porém, após a simulação do fluido gástrico o inibidor presente no chá verde perdeu a ação. O chá preto deixou de inibir a α-amilase e apresentou inibição inalterada para α e β-glicosidases. Tais resultados sugerem que o chá preto pode ser auxiliar em dietas de restrição de carboidratos.

Several plants have been considered therapeutic products, including green and black tea, popularly used to control hyperglycemia and obesity. This study aimed to evaluate the inhibitory potential of the enzymes α-amylase, α and β-glycosidases, as well as the content of phenolic compounds in green tea and black tea. The concentrations of phenolic compounds found were 80.8 ± 0.43 mg g-1 in black tea and 32.0 ± 0.12 mg g-1 in green tea. Under the tested conditions of use, green and black tea inhibited the enzyme under study. However, after simulation of the gastric fluid, the inhibitor present in green tea lost its action. Black tea no longer inhibited a-amylase and showed unaltered inhibition for α and β-glycosidases. These results suggest that black tea can be helpful in diets restricting carbohydrates.

Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation.

AIM: To evaluate the solid-state fermentation (SSF) production of cellulase and hemicellulases (xylanases), by Penicillium echinulatum 9A02S1, in experiments carried out with different concentrations of the pretreated sugar cane bagasse (PSCB) and wheat bran (WB). METHODS AND RESULTS: This study reports the production of xylanolytic and cellulolytic enzymes by P. echinulatum 9A02S1 using a cheap medium containing PSCB and WB under SSF. The highest amounts of filter paper activity (FPA) could be measured on mixtures of PSCB and WB (32.89 +/- 1.90 U gdm(-1)). The highest beta-glucosidase activity was 58.95 +/- 2.58 U gdm(-1) on the fourth day. The highest activity for endoglucanases was 282.36 +/- 1.23 U gdm(-1) on the fourth day, and for xylanases the activity was around 10 U gdm(-1) from the second to the fourth day. CONCLUSIONS: The present work has established the potential of P. echinulatum for FPA, endoglucanase, beta-glucosidase and xylanase productions in SSF, indicating that WB may be partially substituted by PSCB. SIGNIFICANCE AND IMPACT OF THE STUDY: The incorporation of cheap sources, such as sugar cane bagasse, into media for the production of lignocellulose enzymes should help decrease the production costs of enzymatic complexes that can hydrolyse lignocellulose residues for the formation of fermented syrups, thus contributing to the economic production of bioethanol.

Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37

Entre 13 linhagens de fungos filamentosos isolados a partir de amostras de solo agrícola, tubérculos e de material em compostagem, duas foram selecionadas em função da capacidade de crescer em meio líquido contendo amido como única fonte de carbono, a 45ºC, e produzir consideráveis quantidades de glucoamilase. Essas linhagens, identificadas como Aspergillus flavus A1.1 e Thermomyces lanuginosus A13.37, foram utilizadas para desenvolvimento de experimentos para avaliar os efeitos do tipo de amido (milho e mandioca), do pH inicial do meio de cultura (4,0; 5,0 e 6,0) e da temperatura de incubação (40 e 45ºC), em um modelo fatorial (2x3x2), sobre a produção da glucoamilase. O tipo de amido usado como fonte de carbono para o cultivo dos fungos influenciou significativamente a produção de glucoamilase por A. flavus, sendo obtida uma maior quantidade da enzima em meio contendo amido de mandioca do que em meio com amido de milho. A produção da enzima por T. lanuginosus também foi maior em meio contendo amido de mandioca, porém, a diferença não foi estatisticamente significativa. As atividades enzimáticas sobre amido (0,3 per center), maltose (0,3 per center) ou sobre mistura de 0,3 per center de amido com 0,1 per center de maltose, indicaram que as enzimas de Aspergillus hidrolisaram, preferencialmente, o amido, embora tenham mostrado atividade considerável sobre a maltose. A maior liberação de glicose a partir da mistura de substratos sugeriu que o fungo em questão possa secretar dois tipos diferentes de enzimas. Enzimas produzidas por T. lanuginosus hidrolisaram o amido e a maltose e não liberaram maiores teores de glicose quando o substrato constou de mistura de amido e maltose. As enzimas de Aspergillus e Thermomyces apresentaram elevada temperatura ótima de atividade (65 e 70ºC, respectivamente) com boa termoestabilidade na ausência de substrato (manutenção de 50 per ceter da atividade por 5 e 8h respectivamente), além de estabilidade em ampla faixa de pH. Os resultados apresentados indicam uma importante fonte alternativa de glucoamilase para uso no processamento industrial de amido.

Activity of glycosidases from freshwater heterotrophic microorganisms on the degradation of extracellular polysaccharide produced by Anabaena spiroides (Cyanobacteria)

A atividade de glicosidases durante a degradação do polissacarídeo extracelular (EPS) produzido por Anabaena spiroides foi detectada e quantificada utilizando-se MUF-substratos (MUF-monossacarídeos). O consumo total do polissacarídeo efetuou-se em duas fases, uma primeira de alta atividade enzimática que rapidamente consumiu 41 per center do polissacarídeo e uma segunda, mais lenta, que consumiu o polissacarídeo restante (59 per center). A mudança de fase coincidiu com a sucessão de uma população de bactérias cocóides por outra de bacilos. A biomassa bacteriana, quantificada por contagens de células, aumentou com a degradação do EPS. As atividades registradas através dos substratos 4-MUF-a-D- e 4-MUF-b-D- glicosídeo foram mais altas quando comparadas aos demais substratos testados que foram: MUF-a-L-ramnopiranosídeo, MUF-b-D-galactosídeo, MUF-a-D-manopiranosídeo, MUF-b-D-fucosídeo, MUF-b-D-manopiranosídeo, MUF-a-L-arabinopiranosídeo, e MUF-b-L-fucosídeo. A fluorescência emitida a partir de cada um dos diferentes MUF-substratos foi, de modo geral, proporcional à concentração dos monossacarídeos correspondentes constituintes do polissacarídeo, um indício da susceptibilidade ao ataque enzimático microbiano do EPS produzido por A. spiroides.

A mixed mechanistic-electrostatic model to explain pH dependence of glycosyl hydrolase enzyme activity.

Glycosyl hydrolases are a vast group of enzymes that share a common topology at their active site with two acidic residues that are responsible for activity. In spite of their similarity, they exhibit a wide range of pH optima that must depend on other factors. Using structural and mechanistic knowledge about glycosyl hydrolases from families 7, 10, and 16, we have formulated a new mathematical model that can include not only the ionization behavior of the catalytic residues but also as many ionizable residues as desired in the active site. In addition, the model can incorporate electrostatic influences via acid dissociation equilibrium constants and chemical relationships such as hydrogen bonds. The results of the simulations indicate a clear shift in the pH dependence of activity for the enzymes only when a close interrelation (hydrogen bond) between the catalytic and auxiliary residues in the active site is taken into account. This explains the observations from mutagenesis studies that show this type of shift and cannot be explained by a purely electrostatic interaction theory. Moreover, the presence of the kind of chemical interaction proposed could provide stabilization of the activity in the presence of environmental, structural, pH and electrostatic variations. These findings and the implications for the design of new mutagenesis strategies are discussed. The results suggest a way to modify, via site-directed mutagenesis, the acid dissociation of one of the catalytic residues in the active site independently of the other, which could have clear advantages over the purely electrostatic modifications that usually affect both residues simultaneously.

Production and characterization of hemicellulase activities from Trichoderma harzianum strain T4.

Xylan and mannan are the major constituent groups of hemicellulose in the cell wall of higher plants. The mesophilic fungus Trichoderma harzianum strain T4 produces extracellular xylanase and mannanase activities when grown in the presence of oat (Avena sativa)-spelt xylan and wheat bran as the carbon sources respectively. After the growth procedure, the crude extracts were submitted to ultrafiltration in an Amicon system fitted with a 10 kDa-cut-off membrane. Mannanase activity was only detected in the concentrated sample, whereas xylanase was also found in the permeate after ultrafiltration. Xylanase from the concentrated sample showed highest activity at 40 degrees C and pH 5.0. Mannanase activity was optimal at 65 degrees C and pH 2.6. Xylanase was stable in the temperature range 40-70 degrees C, presenting full stability for at least 48 h. Xylanase retained 100% of its original activity after incubation for 48 h at 70 degrees C. Xylanase was also stable at pH 5.0 and 6.0 for 48 h. However, mannanase activity was markedly less stable. The enzyme lost 50% of its activity at 55 degrees C after 45 min, whereas at 60 degrees C its half-life was 20 min. The Michaelis-Menten constant K(m) and V(max) for mannanase and xylanase activities were also calculated. Xylanase had more affinity for soluble xylan, with K(m) and V(max) values of 1.61 mg/ml and 10.03 units/ml respectively. The K(m) and V(max) values for crude mannanase were 6.0 mg/ml and 20.1 units/ml respectively. Xylanase and mannanase were activated by dithiothreitol, L-cysteine and L-tryptophan. Xylanase was partially purified by gel-filtration (Sephadex G-50) and hydrophobic-interaction (Phenyl-Sepharose) chromatographies. The partially purified enzyme was stable over the pH range 5-7 and temperature range of 40-60 degrees C. It was more active on soluble oat-spelt xylan and was activated by dithiothreitol, L-cysteine and L-tryptophan.

Xylan-hydrolyzing enzyme system from Bacillus pumilus CBMAI 0008 and its effects on Eucalyptus grandis kraft pulp for pulp bleaching improvement.

The extracellular productions of beta-xylanase, beta-xylosidase, beta-glucosidase, beta-mannanase, arabinosidase, alpha-glucuronidase, alpha-galactosidase and Fpase from Bacillus pumilus CBMAI 0008 were investigated with three different xylan sources as substrate. The enzymatic profiles on birchwood, Eucalyptus grandis and oat were studied at alkaline and acidic pH conditions. B. pumilus CBMAI 0008 grown on the three carbon sources produced mainly beta-xylanase. At pH 10, the levels of xylanase were 328, 160 and 136 U/ml, for birch, oat and E. grandis, respectively. beta-Mannanase production was induced on E. grandis (5 U/ml) and arabinofuranosidase on oat (5 U/ml). Although small quantities of alpha-glucuronidase had been produced at pH 10, activity at pH 4.8 was 1.5 U/ml, higher than observed for Aspergillus sp. in literature reports. Preliminary assays carried out on E. grandis kraft pulp from an industrial paper mill (RIPASA S.A. Celulose e Papel, Limeira, SP, Brazil) showed a reduction of 0.3% of chlorine use in the pulp treated with the enzymes, resulting in increased brightness, compared to conventional bleaching. The enzymes were more efficient if applied before the initial bleaching sequence, in a non-pre-oxygenated pulp.

Lutzomyia longipalpis: pH in the gut, digestive glycosidases, and some speculations upon Leishmania development.

Screening for digestive glycosidases in different parts of the gut and associated organs of Lutzomyia longipalpis is reported. Searches for the enzymes were made in blood-fed and non-blood-fed females and the enzymes were characterized as soluble or membrane-bound molecules. A total of four different activities were detected, corresponding to the following specificities: an alpha-glucosidase, an N-acetyl-beta-d-glucosaminidase, an N-acetyl-beta-d-galactosaminidase, and an alpha-l-fucosidase. Their possible role and importance for Leishmania development are discussed and the alpha-glucosidase enzyme was partially characterized. The pH inside the gut of non-blood-fed phlebotomines was measured with pH indicator dyes. The pH ranges obtained for crop, midgut, and hindgut were, respectively, higher than pH 6, pH 6, and lower than pH 6. A hypothesis concerning these data and Leishmania development is proposed.

Assay for in vivo yeast invertase activity using NaF.

The methods used for invertase activity determination are based on the measurement of glucose or reducing sugars produced by the enzymatic hydrolysis of sucrose into glucose and fructose. When whole yeast cells are used in these assays, the monosaccharides formed by the action of the periplasmic enzyme can be taken up and metabolized, leading to errors on the enzyme activity determination. This study reports a method for a more accurate invertase activity measurement by blocking the glycolytic pathway. In this method the cells were preincubated with 50 mM sodium fluoride, and inhibitor of enolase. This in vivo measurement of the enzyme activity, under initial rate conditions, was performed using cell concentrations up to 64 mg cell/ml. The results obtained showed that this method is particularly useful for cells with low invertase activity.