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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.

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P32/gC1qR/HABP1 is a doughnut-shaped acidic protein, highly conserved in eukaryote evolution and ubiquitous in the organism. Although its canonical subcellular localization is the mitochondria, p32 can also be found in the cytosol, nucleus, cytoplasmic membrane, and it can be secreted. Therefore, it is considered a multicompartmental protein. P32 can interact with many physiologically divergent ligands in each subcellular location and modulate their functions. The main ligands are C1q, hyaluronic acid, calreticulin, CD44, integrins, PKC, splicing factor ASF/SF2, and several microbial proteins. Among the functions in which p32 participates are mitochondrial metabolism and dynamics, apoptosis, splicing, immune response, inflammation, and modulates several cell signaling pathways. Notably, p32 is overexpressed in a significant number of epithelial tumors, where its expression level negatively correlates with patient survival. Several studies of gain and/or loss of function in cancer cells have demonstrated that p32 is a promoter of malignant hallmarks such as proliferation, cell survival, chemoresistance, angiogenesis, immunoregulation, migration, invasion, and metastasis. All of this strongly suggests that p32 is a potential diagnostic molecule and therapeutic target in cancer. Indeed, preclinical advances have been made in developing therapeutic strategies using p32 as a target. They include tumor homing peptides, monoclonal antibodies, an intracellular inhibitor, a p32 peptide vaccine, and p32 CAR T cells. These advances are promising and will allow soon to include p32 as part of targeted cancer therapies.

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Copper (Cu) mining has to address a critical environmental issue related to the disposal of heavy metals and metalloids (HMs). Due to their deleterious effects on living organisms, Cu and arsenic (As) have gained global attention, and thus their monitoring in the environment is an important task. The aims of this study were: 1) to evaluate the alteration of soil enzyme activities (EAs) and soil microbial functional diversity with Cu/As contamination, and 2) to select the most reliable biochemical indicators of Cu/As contamination. A twelve-week soil experiment was performed with four increasing levels of Cu, As, and Cu/As from 150/15 to 1000/100 mg Cu/As kg-1. Soil enzyme activities and soil community-level physiological profile (CLPP) using MicroResp™ were measured during the experiment. Results showed reduced EAs over time with increasing Cu and Cu/As levels. The most Cu-sensitive EAs were dehydrogenase, acid phosphatase, and arylsulfatase, while arginine ammonification might be related to the resilience of soil microbial communities due to its increased activity in the last experimental times. There was no consistent response to As contamination with reduced individual EAs at specific sampling times, being urease the only EA negatively affected by As. MicroResp™ showed reduced carbon (C) substrate utilization with increasing Cu levels indicating a community shift in C acquisition. These results support the use of specific EAs to assess the environmental impact of specific HMs, being also the first assessment of EAs and the use of CLPP (MicroResp™) to study the environmental impact in Cu/As contaminated soils.

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The objective of this study was to investigate the impacts of high-intensity ultrasound treatments on the compositional, physicochemical, biochemical, functional and structural properties of canola protein isolates (CPI). Aqueous canola protein suspensions were sonicated at 40 kHz for 15 min and 30 min. The moisture content, water activity, bulk density and the L* and a* color parameters of the CPI decreased due to the ultrasound; however, the in vitro protein digestibility was not modified by the treatment. Glutelin (57.18%) was the main protein fraction in the canola protein isolate. SDS-PAGE demonstrated that there were no changes in the protein electrophoretic patterns, thus indicating that sonication did not break the covalent bonds. However, the ultrasound treatment improved the protein solubility, oil absorption capacity and the emulsifying, gelation and foaming properties, but these improvements depended on the pH and ultrasound exposure time. Scanning electron microscopy revealed that the ultrasound treatment disrupted the microstructure of the CPI by exhibiting larger aggregates as a lyophilized powder. In addition, there was an increase in the surface hydrophobicity and a decrease in the size of the particles of the canola protein due to the ultrasound effects, which indicates a destruction of the particles or a dissociation of the protein aggregates in the canola protein dispersions. These results suggest that ultrasound treatment is a valuable tool for improving the characteristics of canola proteins for use in foods.

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Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.

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This study evaluated the effect of the addition of the following ionic liquids (IL): choline chloride (CC), tetramethylammonium bromide (TB) and 1­ethyl­3­methylimidazolium bromide (EM), on some biochemical properties including enzymatic activity and different kinetic parameters of commercial proteases. The enzyme-IL combinations that showed the highest increases in enzyme activities were as follows: CC (0.5mM) and Neutrase® 0.8L; CC (5mM) and Flavourzyme® 500L; TB (2000mM) and Alcalase® 2.4L, with relative increases of 20, 15 and 150% in protease activities, respectively, compared to the control assays. The combination TB and Alcalase® 2.4L showed a reduction of 50% of the activation energy (Ea), an increase of the relation Vmax/Km of 35% and a 16-fold rise in the values of t1/2, and D. Neutrase® 0.8L combined with CC showed an increase of 20% in the relation Vmax/Km. The combination Flavourzyme® 500L and CC presented a 20% higher value of the relation Vmax/Km and a 2-fold increase in the values of t1/2 and D compared to the control assay. In summary, the most positive effects observed in this study included proteases with improved activity and stability properties and a greater affinity for the substrate.

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Background: α-Amylase is widely used in the starch processing, food and paper industries, hydrolyzing starch, glycogen and other polysaccharides into glucose, maltose and oligosaccharides. An α-amylase gene family from Aspergillus niger CBS513.88 encode eight putative α-amylases. The differences and similarities, biochemical properties and functional diversity among these eight α-amylases remain unknown. Results: The eight genes were cloned and expressed in Pichia pastoris GS115 by shaking-flask fermentation under the induction of methanol. The sequence alignment, biochemical characterizations and product analysis of starch hydrolysis by these α-amylases were investigated. It is found that the eight α-amylases belonged to three different groups with the typical structure of fungal α-amylase. They exhibited maximal activities at 30­40°C except AmyG and were all stable at acidic pH. Ca2+ and EDTA had no effects on the activities of α-amylases except AmyF and AmyH, indicating that the six amylases were Ca2+ independent. Two novel α-amylases of AmyE and AmyF were found. AmyE hydrolyzed starch into maltose, maltotriose and a small amount of glucose, while AmyF hydrolyzed starch into mainly glucose. The excellent physical and chemical properties including high acidic stability, Ca2+-independent and high maltotriose-forming capacity make AmyE suitable in food and sugar syrup industries. Conclusions: This study illustrates that a gene family can encode multiple enzymes members having remarkable differences in biochemical properties. It provides not only new insights into evolution and functional divergence among different members of an α-amylase family, but the development of new enzymes for industrial application.

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Background: ß-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of ß-galactosidase, but the properties of this enzyme are incompletely studied. Results: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative ß-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal ß-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4­5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All ß-galactosidases performed transgalactosylation activity optimally in an acidic environment. Conclusions: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three ß-galactosidase family members with remarkably different biochemical properties.

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This work evaluated a wild-type Streptomyces clavuligerus strain as a whole-cell lipase (Sc-WCL) producer by submerged fermentation. In an orbital shaker, lipase hydrolytic activity of 3000 U L-1, measured at pH 9.0 and 37 °C by using p-nitrophenyl palmitate as substrate, was achieved after 36 h fermentation using glycerol-free production medium in a baffled Erlenmeyer flask at 28 °C and pH 6.8. Maximum productivity of 52.5 U L-1 h-1 was achieved after 24 h in bioreactor using glycerol-free production medium at pH 6.8 and 28 °C, with agitation at 400 rpm and aeration at 1 vvm. Sc-WCL was shown to be more active at 60 °C and pH 10.7, while higher activity retention was observed at 30-40 °C after 1 h incubation at pH 10. Sc-WCL showed to have potential to be used as biocatalyst in hydrolysis and esterification reactions. In the hydrolysis of p-nitrophenyl palmitate, lyophilized Sc-WCL expressed a hydrolytic activity (330 units g-1 solid, measured at 37 °C and pH 9.0) around 100-fold higher than the ones declared by a supplier of lyophilized powders of mixtures of intracellular lipases from Thermus thermophiles and Thermus flavus (≥3.0 units g-1 solid, measured at 65 °C and pH 8.0). In the synthesis of butyl butyrate in anhydrous medium, 85% ester conversion was achieved at 37 °C after 8 h reaction. Thus, Sc-WCL showed to be a promising biocatalyst because it is cheaper than the isolated and purified lipases.

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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. Brewers 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 30؛C, during 5 days. The temperature for optimum activity was 50؛C and optimum pH 5.0. The enzyme was stable at 50؛C, 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 brewers 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.(AU)

Muitos fungos sمo utilizados com a finalidade de extrair produtos de seu metabolismo, por meio de bioprocessos capazes de minimizar efeitos nocivos que resيduos agroindustriais causam ao meio ambiente. O objetivo deste estudo foi avaliar a produçمo de xilanases por uma linhagem de Aspergillus niger, empregando resيduos agroindustriais como substrato. O bagaço de malte foi o melhor resيduo indutor da atividade xilanلsica. Maiores nيveis de xilanases foram obtidos quando o fungo foi cultivado em meio lيquido de Vogel, pH 5,0, a 30؛C, durante cinco dias. A temperatura َtima estabelecida para a atividade xilanلsica foi a de 50؛C e o pH َtimo 5,0. A enzima foi estلvel a 50؛C, apresentando uma meia vida de 240 min. Elevada estabilidade enzimلtica foi verificada entre os pH 4,5 e 7,0. As caracterيsticas bioquيmicas exibidas pela xilanase produzida por A. niger tornam esta enzima atraente para determinadas aplicaçُes industriais, como as indْstrias de raçمo animal e alimentيcia. Adicionalmente, a utilizaçمo do bagaço de malte, um resيduo disponيvel em abundância e de baixo custo como substrato para a produçمo de xilanases poderل nمo somente adicionar valor a este resيduo, como também reduzir os custos de produçمo destas enzimas.(AU)

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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.

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Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29 percent of the strains. Enterohaemolysin production was detected in 29 percent of the strains. Most of the bacterial strains (95 percent) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50 percent of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.

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