Cacti as low-cost substrates to produce L-asparaginase by endophytic fungi.

This study aimed to select endophytic fungi to produce L-asparaginase and partially optimising the production of the enzyme using cacti as substrate. Seventeen endophytes were assessed for intracellular enzymatic potential in modified Czapek Dox's medium using L-proline as an inducer. The best producer was evaluated for intracellular and extracellular enzymatic activity in modified Czapek Dox's medium using flours of Opuntia ficus-indica and Nopalea cochenillifera as substrate. The biomass and L-asparaginase production profile was analysed and the best conditions for enzyme production were verified using factorial design. Penicillium decaturense URM 7966, Diaporthe ueckerae URM 8321, and Colletotrichum annellatum URM 8538 produced 0.76 U g- 1, 0.87 U g- 1, and 0.74 U g- 1 L-asparaginase, respectively. Diaporthe ueckerae URM 8321 produced only intracellular L-asparaginase, using flours of N. cochenillifera (0.72 U g- 1) and O. ficus-indica (0.90 U g- 1) and the last was selected for the next steps. The ideal time for biomass and L-asparaginase production was 120 h. The best conditions for enzyme production (1.67 U g- 1) were initial pH 4.0, inoculum concentration 1% and cacti flour concentration 0.2%; where was observed an increase of 46.11% in compared to the initial production. Opuntia ficus-indica flour is indicated as an alternative low-cost substrate for the production of L-asparaginase by the endophytic fungus D. ueckerae URM 8321.

Application of aqueous biphasic systems as strategy to purify tannase from Aspergillus tamarii URM 7115.

The aims of the current study are to assess the influence of polyethylene glycol (PEG) concentration, molar mass, pH, and citrate concentrations on aqueous biphasic systems based on 24 factorial designs, as well as to check their capacity to purify tannase secreted by Aspergillus tamarii URM 7115. Tannase was produced through submerged fermentation at 26°C for 67 h in Czapeck-Dox modified broth and added with yeast extract and tannic acid. The factorial design was followed to assess the influence of PEG molar mass (MPEG 600; 4,000 and 8,000 g/ mol), and PEG (CPEG 20.0; 22.0 and 24.0% w/w) and citrate concentrations (CCIT 15.0, 17.5, and 20.0%, w/w), as well as of pH (6.0, 7.0, and 8.0) on the response variables; moreover, partition coefficient (K), yield (Y), and purification factor (PF) were analyzed. The most suitable parameters to purify tannase secreted by A. tamarii URM 7115 through a biphasic system were 600 (g/mol) MPEG, 24% (w/w) CPEG, 15% (w/w) CCIT at pH 6.0 and they resulted in 6.33 enzyme partition, 131.25% yield, 19.80 purification factor and 195.08 selectivity. Tannase secreted by A. tamarii URM 7115 purified through aqueous biphasic systems composed of PEG/citrate can be used for industrial purposes, since it presents suitable purification factor and yield.

Production, characterization of tannase from Penicillium montanense URM 6286 under SSF using agroindustrial wastes, and application in the clarification of grape juice (Vitis vinifera L.).

Tannase is an enzyme that hydrolyzes esters and lateral bonds of tannins, such as tannic acid, releasing glucose and gallic acid and stands out in the clarification of wines and juices. Fungi of the genera Aspergillus and Penicillium are excellent producers of this enzyme. The search for fungi that produce high levels of tannase as well as new substrates for the enzyme production by the SSF is required. The objectives of this study were to evaluate the production of tannase by Aspergillus and Penicillium species through SSF using leaves and agroindustrial waste barbados cherry and mangaba fruit as substrate, select the best producer, optimize production, characterize the crude enzyme extract, and apply it the clarification of grape juice. Selecting the best producer was performed by planning Placket-Burman and RSM. P. montanense showed highest activity with 41.64 U/mL after 72 h of fermentation residue using barbados cherry, with 3.5% tannic acid and 70% moisture. The enzyme showed the highest activity at pH 9.0 and 50°C. The tannase of P. montanense was stable over a wide pH range and temperature and, when applied to grape juice, showed higher efficiency by reducing 46% of the tannin content after incubation 120 m.

Production, Characterization and Application of a Thermostable Tannase from Pestalotiopsis guepinii URM 7114.

Tannase (EC 3.1.1.20) is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable effects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun (Syzygium cumini (L.) Skeels) leaves, and identified as Pestalotiopsis guepinii, in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL). Response surface methodology was employed to evaluate the effect of variables (pH and temperature), and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. Km was found to be 7.18·10-4 mol/L and vmax =250.00 U/mL. The tannase activity was the highest in the presence of Ca2+ at a concentration of 5·10-3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3% of its original activity after 6 h. The study contributes to the identification of microbial species that produce tannase, with potential application in biotechnology.

Molecular identification of Brazilian Fusarium strains: sources of proteases with milk-clotting properties.

Fusarium is a genus of ubiquitous fungi that comprises mycotoxigenic animal and plant pathogens. These fungi have the ability to exploit a wide range of substrates and hosts, indicating their great potential for enzyme production; however, this aspect is understudied. Therefore, the present study aimed for revaluating the identity of twenty-three Fusarium strains maintained in the University Recife Mycology (URM) culture collection, Brazil, and to evaluate their potential for proteases production and the milk-clotting activity of these proteases. According to phylogenetic analysis of translation elongation factor 1-alpha (TEF1) gene partial sequences, these strains belonged to 12 species representing four species complexes: Fusarium concolor, F. fujikuroi, F. incarnatum-equiseti, and F. oxysporum. Four of these species are putatively novel to science. Notably, novel associations of Fusarium spp. with certain hosts/substrates were documented. The proteolytic activity ranged from 1.67 U ml-1 to 22.03 U ml-1 among the evaluated fungal isolates, with specific proteolytic activity reaching 205.86 U mg-1. The values for coagulant activity and specific activity were up to 157.14 U ml-1 and 1,424.11 U mg-1, respectively. These results indicate the potential of URM Fusarium strains as a source for the production of enzymes of industrial interest. Additionally, they reinforce the importance of applying DNA-based methods for reviewing the identification of fungal strains preserved in biodiversity repositories.

Lichtheimia blakesleeana as a new potencial producer of phytase and xylanase.

Brazil is known for its great potential for production of renewable resources such as agro-industrial residues. These residues can be used as alternative sources of new products. Meanwhile, solid-state fermentation, with its advantages of energy conservation and pollution reduction, has been identified as a process of great potential for the production of bioactive compounds, especially enzymes. In the present work, a 2(3) factorial design was used to evaluate the effects of pH, temperature and moisture on the production of phytase and xylanase by Lichtheimia blakesleeana URM 5604 through the fermentation of citrus pulp. Statistical analyses of the results showed that the only the pH influenced the production of these enzymes, with the best phytase production (264.68 U/g) ocurring at pH 6.0, 34 °C, initial moisture 50%, after 48 hours of culture. The best conditions for xylanase production (397.82 U/g) were fermentation for 120 hours at pH 4.0, 26 °C and initial moisture of 70%. The best parameters for the simultaneous production of phytase (226.92 U/g) and xylanase (215.59 U/g) were determined to be initial moisture of 50%, pH 6.0, 26 °C, and 48 hours of fermentation.

Antarctic fungus proteases generate bioactive peptides from caseinate.

The extracellular serine protease produced by Acremonium sp. L1-4B isolated from the Antarctic continent, was purified and used for the proteolysis of bovine and caprine sodium caseinate. Protein hydrolysates were evaluated in vitro to determine their antioxidant and antihypertensive potential, and later characterized by mass spectrometry. Bovine and caprine hydrolysates produced over 24 h showed a higher content of copper chelation (25.8 and 31.2% respectively), also at this time the ABTS+• scavenging was 65.2% (bovine sample) and 67.5% (caprine sample), and bovine caseinate hydrolysate (8 h) exhibited higher iron chelation capacity (43.1%). Statistically (p < 0.05), caprine caseinate hydrolysates showed relatively higher antioxidant potential in this study. All hydrolysates showed antihypertensive potential; however peptides released from caprine caseinate after 8 h of hydrolysis were able to inhibit 75% of angiotensin-converting enzyme (ACE) activity. Nano-ESI-Q-TOF-MS/MS analysis prospected a total of 23 different peptide sequences in the bovine hydrolysate fraction, originated from the αS1- and ß-casein chain, whilst in caprine hydrolysate, 31 sequences were detected, all from ß-casein. The low molecular weight bovine and caprine hydrolysates obtained in this research have the potential to act in the prevention of disorders caused by oxidative reactions and in the regulation of blood pressure. These findings support the development of new functional food and nutraceutical formulations.

Biochemical and thermodynamic characteristics of a new serine protease from Mucor subtilissimus URM 4133.

A protease from the fungus Mucor subtilissimus URM 4133, capable of producing bioactive peptides from goat casein, was purified. SDS-PAGE and zymography showed a molecular mass of 30 kDa. The enzyme was active and stable in a wide pH range (6.0-10.5) and (5.0-10.5), respectively. Optimum temperature was at 45-50 °C and stability was above 80 % (40 °C/2 h). Activity was not influenced by ions or organic substances (Triton, Tween, SDS and DMSO), but was completely inhibited by PMSF, suggesting that it belongs to the serine protease family. The Km and Vmax were 2.35 mg azocasein.mL-1 and 333.33 U.mg protein-1, respectively. Thermodynamic parameters of irreversible denaturation (40-60 °C) were enthalpy 123.63 - 123.46 kJ.mol-1, entropy 120.24-122.28 kJ.mol-1 and Gibbs free energy 85.97 - 82.45 kJ.mol-1. Any peptide sequences compatible with this protease were found after analysis by MALDI-TOF, which suggests that it is a new serine protease.

Potencial antioxidante de hidrolisados proteicos obtidos a partir de caseinato de ruminantes pela ação de proteases comerciais / Potential antioxidant of protein hydrolysates obtained from ruminant caseinate by the action of commercial proteases

Introdução: a oxidação em sistemas biológicos está relacionada ao desenvolvimento de patologias em humanos. A ingestão de alimentos ricos em compostos químicos que exercem atividade antioxidante contribui para a prevenção e redução dos efeitos deletérios dos radicais livres formados no organismo. Peptídeos derivados das caseínas têm mostrado um elevado potencial como agentes antioxidantes. Objetivos: neste sentido, o presente estudo avaliou a atividade antioxidante de hidrolisados derivados de caseínas de leites das espécies bubalina, bovina e caprina, obtidos pela ação de diferentes proteases. Metodologia: inicialmente, as caseínas foram isoladas dos demais componentes do leite, depois foram submetidas ao processo de proteólise pelas enzimas bromelina, papaína, tripsina e neutrase, individualmente. A atividade antioxidante dos hidrolisados foi avaliada, através da capacidade de eliminação dos radicais: hidroxila (OH­Ë™), superóxido (O2­Ë™), 2,2 difenil-1-picrilhidrazil (DPPH˙), 2,2'azinobis-(3-ácido etilbenzotiazolino-6-sulfônico (ABTS˙), e quelante dos íons metálicos cobre (Cu2+) e ferro (Fe2+). Resultados: os resultados mostraram que a caseína bovina apresentou o menor (35,54%) grau de hidrólise e a caseína bubalina apresentou o maior (85,64%) grau de hidrólise pela ação da neutrase e bromelina após 480 minutos, respectivamente. O potencial para o sequestro dos radicais hidroxila variou entre 0 e 100%, superóxido superior a 80%, ABTS superior a 85%, DPPH entre 20 e 95% habilidade de quelar ferro entre 10 e 100% e cobre entre 14 e 80%. Conclusão: assim, a hidrólise das caseínas do leite bubalino, bovino e caprino foram capazes de produzir hidrolisados com elevado potencial antioxidante e que, mediante novos estudos, poderá vir ser incorporado em produtos alimentícios para o consumo humano.

Introduction: oxidation in biological systems is related to the development of pathologies in humans. The ingestion of foods rich in chemical compounds that exert antioxidant activity contributes to the prevention and reduction of the deleterious effects of free radicals formed in the body. Peptides derived from caseins have shown high potential as antioxidant agents. Objectives: the present study evaluated the antioxidant activity of casein hydrolysates derived from bubaline, bovine, and caprine milk obtained by the action of different proteases. Methodology: initially, the caseins were isolated from the other milk components, and then subjected to the proteolysis process by the enzymes bromelain, papain, trypsin and neutrase, individually. The antioxidant activity of the hydrolysates was evaluated, through the capacity of elimination of the radicals: hydroxyl (OH-˙), superoxide (O2-˙), 2,2 diphenyl-1-picrylhydrazyl (DPPH˙), 2,2'azinobis-(3-ethylbenzothiazolino-6-sulfonic acid (ABTS˙), and chelating of the metal ions copper (Cu2+) and iron (Fe2+). Results: the results showed that bovine casein showed the lowest (35.54%) degree of hydrolysis and bubaline casein showed the highest (85.64%) degree of hydrolysis by the action of neutrase and bromelin after 480 minutes, respectively. The potential for hydroxyl radical sequestration varied between 0 and 100%, superoxide higher than 80%, ABTS higher than 85%, DPPH between 20 and 95% and the ability to chelate iron between 10 and 100% and copper between 14 and 80%. Conclusion: thus, the hydrolysis of caseins from bubaline, bovine and goat milk were able to produce hydrolysates with high antioxidant potential and that, upon further studies, may be incorporated into food products for human consumption.

Potential application of waste from castor bean (Ricinus communis L.) for production for xylanase of interest in the industry.

Xylanases activity (XY) from Aspergillus japonicus URM5620 produced by Solid-State Fermentation (SSF) of castor press cake (Ricinus communis) on different conditions of production and extraction by PEG/citrate aqueous two-phase system (ATPS) were investigated. XY production was influenced by substrate amount (5-10 g), initial moisture (15-35 %), pH (4.0-6.0) and temperature (25-35 °C), obtaining the maximum activity of 29,085 ± 1808 U g ds-1 using 5.0 g of substrate with initial moisture of 15 % at 25 °C and pH 6.0, after 120 h of fermentation. The influence of PEG molar mass (1000-8000 g mol-1), phase concentrations (PEG 20.0-24.0 % w/w and sodium citrate 15-20 % w/w) and pH (6.0-8.0) on partition coefficient, purification factor, yield and selectivity of XY were determinate. Enzyme partitioning into the PEG rich phase was favored by M PEG 8000 (g mol-1), C PEG 24 % (w/w), C C 20 % (w/w) and pH 8.0, resulting in partition coefficient of 50.78, activity yield of 268 %, 7.20-fold purification factor and selectivity of 293. A. japonicus URM5620 has a potential role in the development of a bioprocess for the XY production using low-cost media. In addition, the present study proved it is feasible to extract xylanase from SSF by adopting the one step ATPS consisting of PEG/citrate.

Antioxidant Activities of Chicken Egg White Hydrolysates Obtained by New Purified Protease of Aspergillus avenaceus URM 6706

Abstract Protein hydrolysates originating from egg white have already been reported to be bioactive and, among their biological activities, possess the antioxidant property that protects the body from early ageing and diseases linked to oxidation. Therefore, the objective of this work was to evaluate the antioxidant activity of hydrolysates obtained by the hydrolysis of egg white from hen poultry. The protease produced by Aspergillus avenaceus URM 6706 was purified and subsequently applied to hydrolysate the egg white, and the degree of hydrolysis was verified during the protease exposure time (4-24 h). The hydrolysis was intensified over time of exposure to the protease. It was possible to detect the antioxidant activities of eliminating the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical (ABTS•+) from 97% to 99% and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) up to 27%, as well as the chelation of Cu2+ metal ions up to 62% and Fe2+ up to 54%. The elimination of ABTS•+ radical had a positive correlation with the degree of hydrolysis; however, all the other activities tested showed a negative correlation with the degree of hydrolysis. The results obtained suggest that the egg white of hen chicken represents a food source of animal origin with potential application in the functional food industry.

Extractive fermentation of xylanase from Aspergillus tamarii URM 4634 in a bioreactor.

Of the many reported applications for xylanase, its use as a food supplement has played an important role for monogastric animals, because it can improve the utilisation of nutrients. The aim of this work was to produce xylanase by extractive fermentation in an aqueous two-phase system using Aspergillus tamarii URM 4634, increasing the scale of production in a bioreactor, partially characterising the xylanase and evaluating its influence on monogastric digestion in vitro. Through extractive fermentation in a bioreactor, xylanase was obtained with an activity of 331.4 U mL(-1) and 72% yield. The xylanase was stable under variable pH and temperature conditions, and it was optimally active at pH 3.6 and 90 °C. Xylanase activity potentiated the simulation of complete monogastric digestion by 6%, and only Mg2+ inhibited its activity. This process provides a system for efficient xylanase production by A. tamarii URM 4634 that has great potential for industrial use.

Cellulase production by Aspergillus japonicus URM5620 using waste from castor bean (Ricinus communis L.) under solid-state fermentation.

The activity of ß-glucosidase (ßG), total cellulase (FPase) and endoglucanase (CMCase), produced by Aspergillus japonicus URM5620, was studied on solid-state fermentation using castor bean meal as substrate. The effect of the substrate amount, initial moisture, pH, and temperature on cellulase production was studied using a full factorial design (2(4)). The maximum ßG, FPase, and CMCase activity was 88.3, 953.4, and 191.6 U/g dry substrate, respectively. The best enzyme activities for all three enzymes were obtained at the same conditions with 5.0 g of substrate, initial moisture 15% at 25 °C and pH 6.0 with 120 h of fermentation. The optimum activity for FPase and CMCase was found at pH 3.0 at an optimum temperature of 50 °C for FPase and of 55 °C for CMCase. The cellulases were stable in the range of pH 3.0-10.0 at 50 °C temperature. The enzyme production optimization demonstrated clearly the impact of the process parameters on the yield of the cellulolytic enzymes.

Production and partial characterization of proteases from Mucor hiemalis URM3773 / Produção e caracterização parcial de proteases a partir de Mucor hiemalis URM3773

The current study evaluated the proteases production from 11 fungal species belonging to the genera Mucor, Rhizomucor and Absidia. The species were obtained from the Collection of Cultures URM at the Mycology Department-UFPE, Brazil. The best producing species was Mucor hiemalis URM 3773 (1.689 U mL-1). Plackett-Burman design methodology was employed to select the most effective parameter for protease production out of 11 medium components, including: concentration of filtrate soybean, glucose, incubation period, yeast extract, tryptone, pH, aeration, rotation, NH4Cl, MgSO4 and K2HPO4. Filtrated soybean concentration was the significant variable over the response variable, which was the specific protease activity. The crude enzyme extract showed optimal activity in pH 7.5 and at 50ºC. The enzyme was stable within a wide pH range from 5.8 to 8.0, in the phosphate buffer 0.1M and in stable temperature variation of 40-70ºC, for 180 minutes. The ions FeSO4, NaCl, MnCl2, MgCl2 and KCl stimulated the protease activity, whereas ZnCl2 ion inhibited the activity in 2.27%. Iodoacetic acid at 1mM was the proteases inhibitor that presented greater action.The results indicate that the studied enzyme have great potential for industrial application.

Foi avaliada a produção de proteases por 11 espécies fúngicas pertencentes aos gêneros Mucor, Rhizomucor e Absidia, obtidas da Coleção de Culturas URM do Departamento de Micologia- UFPE, Brasil. A melhor espécie produtora foi Mucor hiemalis URM3773 (1,689 U mL-1). A metodologia de planejamento Plackett-Burman foi empregada para selecionar o parâmetro mais efetivo para a produção de proteases através de 11 componentes do meio, incluindo: concentração do filtrado de soja, glicose, período de incubação, extrato de levedura, triptona, pH, aeração, rotação, NH4Cl, MgSO4 e K2HPO4. A variável significante sobre a variável- resposta, atividade proteásica específica, foi a concentração do filtrado de soja. O extrato enzimático bruto apresentou atividade ótima ao pH 7,5 a 50ºC. A enzima foi estável em uma ampla variação de pH de 5,8­8,0 em tampão fosfato 0,1M e termicamente estável a uma variação de 40-70°C, durante 180 minutos. Os íons FeSO4, NaCl, MnCl2, MgCl2 e KCl estimularam a atividade proteásica, enquanto que o íon ZnCl 2 inibiu 2,27% da atividade. O inibidor de proteases que teve maior ação foi o ácido iodoacético a 1mM. Os resultados obtidos indicam que a enzima estudada tem grande potencial de aplicação industrial.

Potencial do latex da fruta pão (Artocarpus altilis) como agente coagulante do leite

Breadfruit is an exotic tree in Brazil, very well acclimatized. Despite of being a laticifer plant, the knowledge about its latex is scarce. However, it is known that proteolytic enzymes represents over 50% of the latex composition in laticifers plants. The aim of this study was to evaluate the potential of breadfruit (Artocarpus altilis var. Apyrena) latex as a source of milk clotting proteases and partially characterize it. The proteolytic activity of the fraction of crude extract was assessed using azocasein and quantitation of total protein, the bicinchoninic acid (BCA). Milk-clotting activity was analyzed using skim milk at 12%. The enzyme activity was analyzed under different temperature conditions (35 to 80°C) and pH (5.8 to 10.7) presenting optimum activity in alkaline pH (8.5) and 50°C; being stable to the two variables at 120 minutes during the test. The clotting activity was directly proportional to the temperature at the better concentration of CaCl2 (10mmol L-1). The results indicate that enzyme is a possible replacement for calf rennet.

A fruta-pão é uma árvore exótica no Brasil, onde se aclimatou muito bem. Embora seja uma planta lactífera, o conhecimento sobre seu látex é escasso. No entanto, sabe-se que enzimas proteolíticas correspondem a mais de 50% da composição do látex em plantas lactíferas. O objetivo deste estudo foi avaliar o potencial do látex da fruta-pão (Artocarpus altilis var. Apyrena) como fonte de protease coagulante do leite e caracterizar parcialmente a enzima. A atividade proteolítica da fração de extrato bruto foi avaliada utilizando azocaseína e, para a quantificação das proteínas totais, o ácido bicinconínico (BCA). A atividade de coagulação do leite foi testada utilizando leite desnatado a 12%. A protease foi testada em diferentes condições de temperatura (35 a 80°C) e pH (5,8 a 10,7) e apresentou atividade ótima a 50°C e pH alcalino (8,5) sendo estável a estas variáveis durante 120 minutos. A atividade coagulante no leite foi diretamente proporcional à temperatura na melhor concentração de CaCl2 a 10µmol L-1. Os resultados indicam que a enzima analisada é uma possível alternativa à quimosina.

Production and stability of protease from Candida buinensis.

Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations carried out at variable temperature, pH, and soybean flour concentration, according to a 2(3) full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL) when cultivated at 35 degrees C, pH 6.5, and using soybean flour concentrations in the range 0.1-0.5% (w/v). The cell-free supernatants showed the highest activity at 25 degrees C and pH 7.0, and satisfactory stability in the ranges 25-30 degrees C and pH 7-9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; DeltaH* = 37.3 kJ/mol; DeltaS* = -197.5 J/mol K; DeltaG* = 101 kJ/mol).

Partial characterization of an inulinase produced by Aspergillus japonicus URM5633

Enzymes obtained by fermentation processes offer a number of advantages and have been widely researched and used throughout the world. This study aimed to partially characterise an inulinase produced from palm and cassava peel. The enzyme was produced via the solid-state fermentation of Aspergillus japonicus URM5633. The optimal temperatures were 50ºC and 55ºC, and the optimal pH values were 5.2 and 3.4 for inulinase fermentatively produced from palm and cassava peel, respectively. The thermostability measurements for inulinase produced in palm showed that the relative activity remained below 100% until 30 minutes of stability for all temperatures, but reached 106.8% at a temperature of 50ºC after 60 minutes. Inulinase from the crude extract of cassava peel was pH stable and only decreased to 55% of the maximal activity over the course of the assay, suggesting that this enzyme can be used in inulinase production and can be utilized in food industries.

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

The partial characterization and purification of milk clotting enzyme obtained from the (root latex) of Jacaratia corumbensis O. kuntze was studied, by fractional precipitation with ammonium sulphate and ion exchange chromatography. The ammonium sulphate precipitate showed five fractions (AS1- 0-20 percent; AS2 - 20-40 percent; AS3 - 40-60 percent; AS4 - 60-80 percent; AS5 - 80-100 percent) and among the fractions obtained, the 40-60 percent fraction (AS3) showed the highest milk clotting activity with a purification factor of 1.2 fold in relation to the crude extract. This fraction when applied on Mono Q column yielded two protein peaks (p1 and p2), but p1 pool showed the best milk-clotting activity. The optimal pH for the crude and partially purified extract was 6.5 and 7.0, respectively. The maximum milk-clotting activity was at 55ºC for the both crude and partially purified extracts. The enzyme was inhibited by iodoacetic acid which suggested that this enzyme was a cysteine protease, with molecular weight of 33 kDa.

A enzima coagulante de leite obtida de látex de raiz de Jacaratia corumbensis O. kuntze foi caracterizada parcialmente e purificada, por precipitação fracionária com sulfato de amônio e cromatografia de troca de íon. Foram utilizadas cinco frações de sulfato de amônio (AS1 - 0-20 por cento; AS2 - 20-40 por cento; AS3 - 40-60 por cento; AS4 - 60-80 por cento; AS5 - 80-100 por cento), a fração 40-60 por cento (AS3) mostrou alta atividade coagulante com um fator de purificação de 1,2 vezes em relação ao extrato bruto. Esta fração foi aplicada em coluna Mono Q obtendo dois picos de proteína (p1 e p2), o p1 mostrou melhor atividade coagulante. O pH ótimo para o extrato bruto e parcialmente purificado foi 6,5 e 7,0, respectivamente. A atividade coagulante foi atingida a 55ºC para ambos os extratos, bruto e parcialmente purificado. A enzima foi inibida por ácido iodoacético que sugere que esta enzima é uma cisteína protease, com peso molecular de 33 kDa.

Extraction of amylase from fermentation broth in poly (Ethylene Glycol) salt aqueous two-phase system

Studies were carried out on the partition of amylase from Bacillus subtilis in a minimal medium at 37 °C and 110 rpm. Enzyme recovery was carried out in aqueous two-phase system PEG-Phosphate salt were carried out. The best purification factor (5.4) was obtained in system PEG 1000 (16.7 percent w/w) with potassium phosphate (14.8 percent w/w), at pH 6.0, resulting in a recovery of 45.2 percent activity enzymatic in the salt-rich phase.

Enzimas amilolíticas têm sido amplamente investigadas com a finalidade de melhorar os processos industriais para a degradação do amido. Foi determinado que a extração da enzima em sistema bifásico aquosos é um método aplicável para separação e purificação de biomoléculas em misturas. Vários sistemas compostos de soluções aquosas de polietilenoglicol e fosfato foram avaliados. Estudos de produção em meio mínimo suplementado, à 37°C, com uma velocidade de agitação de 110rpm e recuperação da amilase a partir do Bacillus subtilis em sistema bifásico aquoso PEG-fosfato foram avaliados. O melhor fator de purificação (5.4) foi obtido no sistema PEG 1000 (16.7 por cento w/w) com fosfato de potássio (14.8 por cento w/w), a pH 6.0, resultando na recuperação da atividade enzimática de 45.2 por cento na fase rica em sal.

Purification of plasmid (pVaxLacZ) by hydrophobic interaction chromatography

O DNA plasmidial tem sua aplicação na terapia gênica, tendo que apresentar elevado grau de pureza. Um método de purificação foi descrito neste trabalho, que inclui precipitação com sulfato de amônio seguida da cromatografia de interação hidrofóbica, usando a resina phenyl sepharose 6 fast flow. Ocorreu aumento da pureza das amostras analisadas, em torno de 30% após a precipitação com sulfato de amônio. Houve significativa redução dos contaminantes após a eluição das amostras na cromatografia de interação. O maior rendimento obtido foi 53% e fator de purificação 3,1 vezes após a eluição na cromatografia de interação hidrofóbica. A vantagem deste método é a possibilidade de explorar o caráter hidrofóbico, constituído de material genético de fita simples, tais como RNA e DNA genômico e proteínas, que podem interferir na liberação deste material para possíveis aplicações em terapia gênica.