Seleção de fungos produtores de β-D-frutosiltransferase por fermentação em estado sólido

A enzima β-D-frutosiltransferase é responsável pela síntese de FOS (frutooligossacarídeos) a partir de sacarose por reação de transfrutosilação é produzida por diferentes micro-organismos, principalmente por fungos filamentosos. O objetivo deste trabalho foi selecionar a melhor linhagem fúngica produtora da -D-frutosiltransferase por fermentação em estado sólido, bem como o método de extração. A fermentação em estado sólido utilizando o substrato farelo de trigo umedecido com solução de sacarose atingindo 70% de umidade na concentração de esporos de 107 no tempo de 96 horas de crescimento. Todas as linhagens manipuladas apresentaram atividade hidrolítica, no entanto apenas uma linhagem não demonstrou atividade transfrutosilação. O isolado SIS 14 que pertence ao gênero Aspergillus sp. destacou-se pelos maiores valores em atividade no método de extração utilizando água destilada, apresentando 300,90 U/mL na atividade de transfrutosilação e na atividade hidrolítica de 155,74 U/mL. Contudo, pode-se perceber que dos solventes estudados a água destilada foi melhor obtendo o valor em atividade de transfrutosilação, como também a linhagem SIS 14 é promissora para a produção da β-D-frutosiltransferase

Effect of pH and temperature on phytase and biomass production by submerged fermentation with Aspergillus niger var. phoenicis URM 4924

Phytase production and biomass was evaluated in present work by submerged fermentation with Aspergillus niger var. phoenicis URM 4924. Experimental assays were done under different conditions of pH (4.0 to 8.0) and temperature (25 to 35 ºC), and the influence of these variables on the responses was studied through a 22 central composite design and response surface methodology. Phytase and biomass production were affected by the pH and temperature used during submerged fermentation. Phytase activity was increased in up to 7.8-fold (from 1.04 to 8.09 U/mL) and the ergosterol content was increased in up to 38-fold (from 9.3 to 354.09 μg/mL). The maximum values of both responses were achieved when using pH 4.0 and 30 ºC

Integrated process production and extraction of the fibrinolytic protease from Bacillus sp. UFPEDA 485

Fibrinolytic proteases are enzymes that degrade fibrin; these enzymes are a promising alternative for thrombolytic therapy, and microorganisms produce them. The aim of this study was to evaluate the optimum conditions for the integrated production and purification of fibrinolytic protease from Bacillus sp. UFPEDA 485. Extractive fermentation was carried out in a culture medium containing soybean flour and by adding polyethylene glycol (PEG) and Na2SO4 according to a 23 experimental design. In all assays, the enzyme preferentially partitioned to the bottom phase (K < 1), with an optimum activity of 835 U ml−1 in the bottom phase (salt-rich phase). The best conditions for extractive fermentation were obtained with 18 % PEG 8000 and 13 % Na2SO4. Characterization showed that it is a metalloprotease, as a strong inhibition—residual activity of 3.13 %—occurred in the presence of ethylenediaminetetraacetic acid. It was also observed that enzymatic activity was stimulated in the presence of ions: CaCl2 (440 %), MgCl2 (440 %), FeSO4 (268 %), and KCl (268 %). The obtained results indicate that the use of a low-cost substrate and the integration of fermentation with an aqueous two-phase system extraction may be an interesting alternative for the production of fibrinolytic protease.The authors thank CAPES (National Council for the Improvement of Higher Education) for the scholarship and CNPq (National Counsel of Technological and Scientific Development) and RENORBIO for the financial support

Production and characterization of new fibrinolytic protease from Mucor subtillissimus UCP 1262 in solid-state fermentation

Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25˚C for 72 hours. The optimum temperature for the produced enzyme was 45˚C and most of its original activity was retained after being subjected to 80˚C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+; but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy

Recovery and partitioning of fibrinolytic protease from Bacillus sp. UFPEDA 485 by aqueous two-phase systems

Fibrinolytic proteases produced by Bacillus sp. has attracted interest in the pharmaceutical industry as a promising alternative in thrombolytic therapy due to their effectiveness in degrading fibrin, its production requiring the development of an efficient recovery process. Aqueous two-phase systems (ATPS) have been recognized as an efficient and economical process for recovering enzymes. To optimize the recovery of fibrinolytic protease from the fermentation broth of Bacillus sp. UFPEDA 485, a 23 full factorial design was used to evaluate the influence of the three independent variables PEG molar mass (MPEG), PEG concentration (CPEG) and sodium sulfate concentration (CNa2SO4) on the partition coefficient (K), purification factor (PF) and yield recovery (Y) of fibrinolytic protease in PEG/Na2SO4 aqueous two-phase system. For all ATPS studied, enzymes partitioned to the top phase and the highest extraction was obtained for MPEG 6000 g.mol-1, CPEG 24 % (w/w) and CNa2SO4 11.6 % (w/w) with K = 5.03; PF = 3.30; Y = 91.40% and Fibrinolytic activity in the top phase 821 U.mL-1. Findings reported here show that ATPS composed of PEG/Na2SO4 is a valuable strategy for the extraction of fibrinolytic protease and can be considered a promising method for the extraction of enzymes in industrial scale

Partitioning and purification of polygalacturonases produced by Aspergillus niger URM 5162 using PEG-phosphate in an aqueous two-phase system

Pectinases, or pectinolytic enzymes, are naturally produced by plants, filamentous fungi, bacteria and yeasts. The pectinases are of great importance to clarify and reduce viscosity in fruit juices, improving and increasing tbe filtration efficiency. When used in the crushing of grapes or wine must improve juice extraction, reduce the time to clarify and enhance tbe content ofterpenes in wine. The filamentous fungi most frequently used fur industrial purposes because as much as 90% ofthe enzyme can be excreted into the culture medium. The partitioning and purification of polygalacturonases (PG) produced by Aspergillus niger URM 5162 were investigated in aqueous two-phase systems (ATPS), furmed by polyetbylene glycol and phosphate salts (PE(ijlhosphate). To evaluate the effect oftbe 4 independent variables- molar mass ofpolyetbylene glycol (PEG) (400-8000 g1nol MPEG), PEG concentration (12.5-17.5%, w/w- CPEG), phosphate concentration (15-25%, ...W, CPHOS) and pH (6.0, 8.0) - on the 3 response variables: partition coefficient (K), activity yield (Y) and purification fàctor (PF), a fuctorial design (24) was used. The endo-polygalacturonases (endo-PG) were prefurentially partitioned in tbe top phase. For endo-PG, the highest values for the response variables K, Y and PF of 1.23, 74.04% and 8.18, respectively, were obtained for a CPEG of 12.5% (...W), MPEG of8000 g1nol, and CPHOS of25% (w/w) at pH 6.0. Also, exo-polygalacturonases (exo-PG) were preferentially partitioned in the top phase. ln tbis case, the highest values ofK (2.40), Y (33.33%), and PF (1.98) were obtained with a MPEG of 8000 g1nol, CPEG of 12.5% (...W), and CPHOS of25% (...W) at pH 6.0. ln both cases, MPEG had a positive influence on K, Y and PF. The conditions ofMPEG 8000 g1nol, CPEG of 12.5% (...W), and CPHOS of25% (...W) at pH 6.0 were considered the most suitable for tbe purification of PG produced by A. niger URM 5162. Furtbermore, MPEG and CPHOS were the most important independent variables. The PEG/phosphate system is a useful cost-effective altemative for PG purification

Production and characterization of protease from Penicillium aurantiogriseum URM 4622

Proteases with new properties are required due to their increasing industrial importance. In this work, the optimal fermentation conditions for the production of a protease from Penicillium aurantiogriseum dierchx (URM-4622) are presented together with partial characterization of the protease catalytic properties. The batch fermentation conditions that allow for the highest specific proteolytic activity are 26 ºC, pH 7.0, and 25 % saturation dissolved O2 concentration. The obtained protease is stable over a wide range of pH (5.8 to 9.5) and temperature (25 to 40 ºC) values. In the presence of Zn2+ a 26 % reduction in the enzyme proteolytic activity occurs and, in contrast, Mn2+ enhances its activity by 28.9 %. 96.2 % and 70.8 % of the protease activity are maintained after 90 min incubation in 5 and 10 % (v/v) H2O2 aqueous solutions, respectively. PMSF inhibition reveals that this enzyme is a serine protease. Protease is able to hydrolyze different proteins

Fibrinolytic protease production by new Streptomyces sp. DPUA 1576 from Amazon lichens

Background: Streptomyces sp. DPUA 1576 from Amazon lichens was studied to protease and fibrinolytic production. A 22 factorial experimental design was applied to optimize its protease enzyme production using two independent variables, namely soybean flour and glucose concentrations. Results: The optimal conditions to obtain high protease production (83.42 U/mL) were 1.26% soybean flour and 1.23% glucose concentration. A polynomial model was fitted to correlate the relationship between the two variables and protease activity. In relation to fibrinolytic activity, the highest activity of 706.5 mm2 was obtained at 1.7% soybean flour and 1.0% glucose concentration, which was 33% higher than plasmin. Fibrinolytic production was not optimized in the studied conditions. Conclusions: These results show that the optimization of the culture medium can enhance protease production, thus becoming a good process for further research. In addition, Streptomyces sp. DPUA 1576, isolated from Amazon lichens, might be a potential strain for fibrinolytic protease production

Fibrinolytic protease production by new Streptomyces sp. DPUA 1576 from amazon lichens

Background Streptomyces sp. DPUA 1576 from Amazon lichens was studied to protease and fibrinolytic production. A 22 factorial experimental design was applied to optimize its protease enzyme production using two independent variables, namely soybean flour and glucose concentrations. Results The optimal conditions to obtain high protease production (83.42 U/mL) were 1.26% soybean flour and 1.23% glucose concentration. A polynomial model was fitted to correlate the relationship between the two variables and protease activity. In relation to fibrinolytic activity, the highest activity of 706.5 mm2 was obtained at 1.7% soybean flour and 1.0% glucose concentration, which was 33% higher than plasmin. Fibrinolytic production was not optimized in the studied conditions. Conclusions These results show that the optimization of the culture medium can enhance protease production, thus becoming a good process for further research. In addition, Streptomyces sp. DPUA 1576, isolated from Amazon lichens, might be a potential strain for fibrinolytic protease production.The authors thank CAPES (National Council for the Improvement of Higher Education) for the scholarship and CNPq/RENORBIO (National Counsel of Technological and Scientific Development, N. 55146/2010-3) and FACEPE (Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco, 0158-2.12/11) for the financial support