Production and biochemical and biophysical characterization of fibrinolytic protease of a Mucor subtilissimus strain isolated from the caatinga biome.

Cardiovascular diseases, resulting from the deposition of clots in blood vessels, are the leading cause of death worldwide. Fibrinolytic enzymatic activity can catalyze blood clot degradation. Findings show that 36 fungal isolates recovered from Caatinga soils have the potential to produce fibrinolytic protease under submerged conditions. About 58 % of the isolates displayed fibrinolytic activity above 100 U/mL, with Mucor subtilissimus UCP 1262 being the most active. The protease was biochemically and biophysically characterized, showing that the enzyme had a high affinity for SAApNA substrate and was significantly inhibited by fluoride methyl phenyl sulfonyl-C7H7FO2S, suggesting that it is a chymotrypsin-like serine protease. The highest enzyme activity was detected at pH 5.0 and 28 °C. This fibrinolytic protease's far-UV circular dichroism (CD) showed that its secondary structure was primarily α-helical. The purified fibrinolytic enzyme may represent a novel therapeutic agent for treating thrombosis. At temperatures above 65 °C, the enzyme lost all its secondary structure. Its melting temperature was 58.1 °C, the denaturation enthalpy 85.1 kcal/mol, and the denaturation entropy 0.26 kcal/K∙mol.

Seafood Waste as Attractive Source of Chitin and Chitosan Production and Their Applications.

Chitosan is a cationic polymer obtained by deacetylation of chitin, found abundantly in crustacean, insect, arthropod exoskeletons, and molluscs. The process of obtaining chitin by the chemical extraction method comprises the steps of deproteinization, demineralization, and discoloration. To obtain chitosan, the deacetylation of chitin is necessary. These polymers can also be extracted through the biological extraction method involving the use of microorganisms. Chitosan has biodegradable and biocompatible properties, being applied in the pharmaceutical, cosmetic, food, biomedical, chemical, and textile industries. Chitosan and its derivatives may be used in the form of gels, beads, membranes, films, and sponges, depending on their application. Polymer blending can also be performed to improve the mechanical properties of the bioproduct. This review aims to provide the latest information on existing methods for chitin and chitosan recovery from marine waste as well as their applications.

Economic microbiological conversion of agroindustrial wastes to fungi chitosan.

To investigate the simultaneous influence of different non-nutritional factors in production and physical-chemical characteristics of chitosan obtained by Syncephalastrum racemosum we used individually agroindustrial wastes as the only nutritional sources for fungus growth. The growth conditions were evaluated according to Factorial Design, 24 with three central points in order to determine the mainly factors for maximum production of microbiological chitosan in submerged culture. Syncephalastrum racemosum grown in corn steep liquor and yield up to 7.8 g chitosan/kg of substrate in the best condition by factorial design. The microbiological chitosan obtained has deacetilation degree 88.14%, crystallinity rate of 55.96%, mass decomposition process at 304.43 ºC, and low molecular weight. To fast production we performed a kinetic study and confirmed that at 36 h the chitosan production is higher and the physical-chemical characteristics were maintained. This research describes, for the first time, the factorial study of chitosan production by Syncephalastrum racemosum in agroindustrial wastes and its economic potential for commercialization.

Growth of Cunninghamella elegans UCP 542 and production of chitin and chitosan using yam bean medium

Microbiological processes were used for chitin and chitosan productions by Cunninghamella elegans (UCP 542) grown in a new economic culture medium. The assay was carried out to evaluate the growth of C. elegans using yam bean (Pachyrhizus erosus L. Urban) medium, in different times of growth (24, 48, 72 and 96 hrs), incubated at 28°C in an orbital shaker at 150 rpm. The lyophilized biomass was determined by gravimetry. The polysaccharides were extracted by alkali-acid treatment, and characterized by infrared spectroscopy, titration and viscosity. C. elegans grown in the yam bean medium and produced higher yields of biomass (24.3 g/ mL) in 96 hrs. The high level was chitosan (66 mg/g), and chitin (440 mg/g) were produced at 48 and 72 hrs of growth, respectively. The polysaccharides showed degree of deacetilation and viscosimetric molecular weight as: 6.2 percent and 3.25 x10(4) g/mol for chitin, and 85 percent and 2.72 x 10(4) g/mol for chitosan, respectively. The results obtained suggest high biotechnological potential of yam bean as an economic source to produce chitin and chitosan by C. elegans. In addition, the new medium using yam bean for production of the chitin and chitosan may be used for many purposes to reduce the cost price of fermentation processes.

Production and stability studies of the bioemulsifier obtained from a new strain of Candida glabrata UCP 1002

Evaluation of both tenso-active and emulsifying activities indicated that a biosurfactant was produced by the newly isolated and promising strain Candida glabrata isolated from mangrove sediments. The extracellular water-soluble emulsifying agent was isolated and identified as a heteropolymer. The maximum of bioemulsifier production was observed when the strain was grown on soluble and insoluble substrates cotton seed oil plus glucose, reaching values of 10.0 g/l after 144 hrs at 200 rpm. The cell-free culture broth containing the examined agent lowered the surface tension of the medium to 31 mN/m. Stable and compact emulsions with emulsifying activity of 75 percent of cotton seed oil were detected. The emulsification capacity remained practically unaltered within a wide pH (2-12), temperature (4-80°C) ranges and under NaCl concentrations up to 10 percent.

Two new meroterpenoids, penisimplicin A and B, isolated from Penicillium simplicissimum.

In the course of our research for new antifungal agents, two new meroterpenoids, penisimplicin A (1) and B (2), were isolated from Penicillium simplicissimum. The absolute structures of 1 and 2 were established by spectroscopic and chemical investigation. Penisimplicin A (1) and B (2) are rare examples of D-ring seco-meroterpenoids. Since compounds 1 and 2 showed no antifungal activities, the isolation of the compounds with antifungal activity will be attempted.

Partition of proteins in aqueous two-phase systems based on Cashew-nut tree gum and poly(ethylene glycol)

A partição de duas proteínas, albumina de soro bovino (BSA) e tripsina foi estudada no sistema bifásico aquoso Polietileno glicol(PEG) - Goma do cajueiro. O diagrama de fases foi estabelecido para a Goma do Cajueiro e para PEG de peso molecular 1500 em duas diferentes temperaturas. A influência de vários parâmetros na partição destas proteínas, incluindo concentração dos polímeros, pH, adição de sal e temperatura foi investigada. Os resultados desta pesquisa demonstraram a importância das características da proteína na partição em sistemas bifásicos aquosos.

Heavy metal biosorption by chitin and chitosan isolated from Cunninghamella elegans (IFM 46109)

Chitin and chitosan were extracted from mycelial biomass of Cunninghamella elegans and the performance for copper, lead and iron biosorption in aqueous solution was evaluated. The growth curve of C. elegans was accomplished by determination of biomass, pH, glucose and nitrogen consumption. Chitin and chitosan were extracted by alkali-acid treatment and the yields were 23.8 and 7.8 percent, respectively. For the adsorption analysis, the process of heavy uptake metal sorption was evaluated using polysaccharides solutions (1 percent w/v). The rate of metallic biosorption was dependent upon the concentration and pH of metal solutions, and the best results were observed with pH 4.0. Chitosan showed the highest affinity for copper and chitin for iron adsorption. The results suggest that C. elegans (IFM 46109) is an attractive source of production of chitin and chitosan, with a great potential of heavy metals bioremediation in polluted environments.

Effects of phosphorus on polyphosphate accumulation by Cunninghamella elegans

O crescimento, consumo de fosfato e glicose, bem como o conteúdo de fósforo, a distribuicão, estrutura e localizacão de polifosfato foram avaliados no micélio de Cunninghamella elegans cultivado em meios contendo diferentes concentracões de fosfato. Os resultados permitiram verificar a influência dessas concentracões de fosfato sobre o crescimento do fungo estudado. A maior concentracão de fosfato proporcionou maior rendimento da biomassa ao longo do crescimento. Uma relacão entre consumo de fosfato e glicose do meio foi observada em relacão ao crescimento e a quantidade de polifosfato total nos micélios cultivados nos diferentes meios de cultivo. Distintos métodos de extracão permitiram identificar e quantificar as diferentes fracões do polifosfato celular de Cunninghamella elegans. A citoquímica ultrastrutural foi utilizada com sucesso para identificar a localizacão e a distribuicão de polifosfato em Cunninghamella elegans. Os resultados revelaram diferencas no padrão de marcacão citoquímica nas diferentes fases do crescimento e meios de cultivo. Uma marcacão uniforme do polifosfato foi observada sobre a superfície celular, em especial, na parede celular e na membrana citoplasmática. Produtos de reacão resultantes da marcacão citoquímica foram também visualizados em estruturas trabeculares, vacuolares, vesiculares, sob a forma de corpos eletrondensos e grânulos dispersos no citoplasma. Os resultados demonstraram o potencial de Cunninghamella elegans na acumulacão de polifosfato, sugerindo uma possível aplicacão em processos biotecnológicos.

New bioemulsifiers produced by Candida lipolytica using D-glucose and babassu oil as carbon sources

Candida lipolytica IA 1055 produced extracellular biosurfactants with emulsification activity by fermentation using babassu oil and D-glucose as carbon sources. Natural seawater diluted at 50 percent supplemented with urea, ammonium sulfate, and phosphate was used as economic basal medium. The best results were achieved with the YSW-B2 medium, which contained urea, ammonium sulfate, and babassu oil and with YSW-B3 medium, which contained urea, ammonium sulfate, phosphate, and babassu oil, kept under fed batch fermentation for 60 hours with 5 percent of babassu oil. For the two media, the maximum specific growth rates were 0.02 h-1 and 0.04 h-1; the generation times were 34.6 h-1 and 17.3 h-1, and the emulsification activities were 0.666 and 0.158 units, respectively. The molecules of these new bioemulsifiers were contituted of carbohydrates, proteins and lipids.

Effect of medium components and time of cultivation on chitin production by Mucor circinelloides (Mucor javanicus IFO 4570) -- a factorial study.

Chitin production of mycelia from Mucor circinelloides (Mucor javanicus) was studied with a two-level factorial design in all combinations of the following factors in the culture medium: time of cultivation, concentration of D-glucose, L-asparagine and thiamine. Chitin was characterised by infrared and nuclear magnetic resonance spectroscopy. The four factors showed statistically significant (95%) positive main effects on chitin production, without interactions between them. The highest chitin yield (23.9%) was obtained with the medium containing 60 g/l of glucose, 3 g/l of asparagine and 0.008 mg/l of thiamine and is comparable to the highest in the literature.

Protoplast formation and regeneration from Streptomyces clavuligerus NRRL 3585 and clavulanic acid production

Protoplasts of the wild type Streptomyces clavuligerus NRRL 3585 (ATCC 27064) were formed from spores cultures obtained in the lag, exponential and stationary growth phases by using 0.5 percent glycine in the culture medium. The protoplasts were obtained by treatment of the cells with lysozyme (Ec û3.2.1.17) 40,000 U (1mg/ml), in an osmotic solution for 90 min at 28oC. The frequency of regenerated protoplasts in the lag phase was 1.7x103 CFU/ml (28,97 percent), in the beginning of the exponential phase 0.4x102 CFU/ml (31.67 percent), in the exponential growth phase 2.5x103 CFU/ml (46.30 percent) and 1.0x105 CFU/ml in stationary phase (48.45 percent). Antibiotic production and activity of regenerated protoplasts were observed in all phases, except in the lag phase. The protoplast formation and regeneration techniques resulted in a new isolate strain of Streptomyces clavuligerus that produced approximately 2.5 fold more clavulanic acid.

Production of extracellular proteases by Mucor circinelloides using d-glucose as carbon source / substrate

Recently, some Mucorales species have been reported as protease producers. The production of extracellular proteases by Mucor circinelloides using glucose as substrate was studied. Experiments were carried out with different D-glucose concentrations (40, 60 and 80 g/L). Biomass, pH and protease activity were determined. Although biomass production had reached best yields for the medium containing D-glucose in a concentration of 80 g/L, the enzymatic production was higher when the substrate concentration was reduced to 40 g/L. The yield factor for product on cell growth and the yield factor for product on carbon substrate were higher when the microorganism grew in medium containing 40 g/L glucose. The kinetics parameters suggest that this strain seems to be promising as an alternative microorganism for protease production.

Polyphospate in zygomycetes: a cytochemical study

The inorganic phosphorus content, and distribution, structure and localization of polyphosphate in mycelia of zygomycetous fungi was evaluated. Ultrastructural cytochemistry was successfully used to identifying the localization and distribution of polyphosphate in Absidia cylindrospora, Gongronella butleri and Mucor javanicus. The results revealed differences in the cytochemical staining pattern in all species studied and a uniform labeling on the cellular surface (cell wall and citoplasmic membrane). Reaction products were observed in intracellular structures and cellular membrane. Intracellular staining was observed in trabecullar, vacuolar and vesicular structures, in dense bodies and in the cytoplasm. However, the cytochemical staining intensity varied during the cellular growth. Analytical procedure revealed the phosphorus content during cell growth. The results demonstrated that phosphorus content varied during cultivation time and could be related to the polyphosphate cytochemical staining. Polyhosphate functions such as storage and utilization are discussed.

Qualidade microbiológica do mapará (Hypophtalmus edentatus) salgado, comercializado em Fortaleza, CE / Microbiological quality of saltet-fish samples of mapará marked in Fortaleza, CE

Foram analisadas 48 amostras de peixe mapará (Hypophtalmus edentatus) salgado, coletadas em dois pontos de comercializaçäo (A e B) estabelecidos na rua Conde d'Eu, centro de Fortaleza, no período de abril a setembro de 1999. As amostras apresentaram Micrococcus, bolores e leveduras em números significantes. Os valores de Microccus se apresentaram no intervalo entre 1,0 x 1000 a 1,8 x 10000 UFC/g para as amostras do ponto de comercializaçäo A e 1,15 x 1000 a 1,43 x 10000 UFC/g para amostras do ponto B, enquanto os de leveduras se mostraram nos intervalos <10 a 8,5 x 1000 e <10 a 9,2 x 1000 UFC/g para A e B, respectivamente. Doze colônias de Penicillium spp. foram identificadas nas 48 amostras. Leveduras do gênero Candida spp. foram identificadas nas amostras provenientes dos pontos A e B, com predominância da espécie Candida albicans. Näo foi observada a presença de Staphylococcus aureus nas amostras.

Faster Chitosan productions by mucoralean strains in submerged culture

Production of chitosan was conducted using two Mucoralean strains, Mucor racemosus and Cunninghamella elegans. Chitosan was extracted from mycelia of M. racemosus and C. elegans at different growth phases on YPD medium. In both fungi, chitosan was rapidly produced, while highest yield of extractable chitosan was found in 24h of cultivation in submerged culture. The yield of chitosan isolated from dry mycelia of M. racemosus was about 40 per cente higher than from C. elegans. The degree of N-acetylation of chitosan was 49 per cente in M. racemosus and 20 per cente in C. elegans, and the D-glucosamine contents were about 48 per cente and 90 per cente, respectively.

Extraction and characterization of proteins from cell walls of two strains of paracoccidioides brasiliensis

En el presente trabajo se analizaron las proteínas de la pared celular de 2 cepas de paracoccidioides brasiliensis en fase levaduriforme (PbHC-PE y Pb 18). Las proteínas fueron extraidas por tres difrentes métodos y estudiadas por electroféresis SDS-PAGE. Los resultados de los perfiles de las dos cepas fueron diferentes, permitiendo la posibilidad de su uso como marcadores quimiotaxonómicos. Se observó una secreción transitoria de la proteína gp 43 a través de la pared celular de las cepas de p. brasiliensis.

Atividade antimicrobiana e citotóxica de extratos brutos de Fuligo septica (L.) Wigg. E Tubifera microsperma (Berk. & Curt.) Martin (Myxomycetes) / Antibiotic and cytotoxic activities of crude extracts from Fuligo septica (L.) Wigg. and Tubifera microsperma (Berk. & Curt.) Martin (Myxomycetes)

Este trabalho apresenta as atividades citotóxica e antimicrobiana de extratos clorofórmico, acetônico e metanólico de Fuligo septica (L.) Wigg. e extrato metanólico de Tubifera microsperma (Berk. & Curt.) Martin contra bactérias Gram negativas, Gram positivas, álcool-ácido resistentes, fungos filamentosos e leveduras. Os extratos de F. septica foram ativos contra Bacillus subtilis e Candida albicans, enquanto o extrato de T. microsperma inibiu S. aureus, Mycobacterium smegmatis e C. albicans. A atividade citotóxica em células da linha contínua KB foi mais eficiente com o extrato acetônico de F. septica (DI50÷22µg/ml)