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

Marcos, João Carlos

Marques, D. A. V.

Nascimento, J. C. S.

Porto, Ana L. F.

Porto, Tatiana Souza

Souza, F. A. S. D.

Teixeira, J. A.

Universidade do Minho: RepositoriUM

 XIX SIMPÓSIO NACIONAL DE BIOPROCESSOS X SIMPÓSIO DE HIDRÓLISE ENZIMÁTICA DE BIOMASSAS  30 de julho-02 de agosto de 2013 Foz de Iguaçu, PR, Brasil       RECOVERY AND PARTITIONING OF FIBRINOLYTIC PROTEASE FROM Bacillus sp. UFPEDA 485 BY AQUEOUS TWO-PHASE SYSTEMS    F. A. S. D. SOUZA1, J. C. S. NASCIMENTO1, , D. A. V. MARQUES1,  J. C. MARCOS4, T. S. PORTO2, J. A. TEIXEIRA3, and A. L. F. PORTO 1  1 Federal Rural University of Pernambuco, Department of Morphology and Animal Physiology, Recife, Brazil 2 Federal Rural University of Pernambuco, Academic Unit of Garanhuns, Brazil 3 University of Minho, Centre of Biological Engineering, Braga, Portugal 4 University of Minho, Department of Chemistry, Braga, Portugal. E-mail: fabiana.americasouza@yahoo.com.br  ABSTRACT – 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.  1. INTRODUCTION  Fibrinolytic proteases are hydrolytic enzymes that dissolve blood clots. The formation of blood clots is a natural phenomenon of protection of the human body to prevent excessive bleeding from injuries and wounds, but may sometimes block blood flow causing cardiovascular disorders. Thrombolytic therapy using fibrinolytic proteases has shown to be a potential solution to many vascular disorders. The fibrinolytic proteases from Bacillus sp. have attracted the interest of researchers and the pharmaceutical industry because of its efficiency as thrombolytic agents in fibrinolysis Raafat et al. (2012). Currently, there is an industrial need for techniques for the recovery of biomolecules enabling rapid, efficient and low cost processes. Conventional techniques used nowadays for the extraction of products such as ultrafiltration, precipitation and chromatography, are not considered viable for the industry because of the high cost and time consuming process.  Therefore, the search for alternative methods has been constant. The application of ATPS in  XIX SIMPÓSIO NACIONAL DE BIOPROCESSOS X SIMPÓSIO DE HIDRÓLISE ENZIMÁTICA DE BIOMASSAS  30 de julho-02 de agosto de 2013 Foz de Iguaçu, PR, Brasil       the recovery of products meets the criteria required by industry. An ATPS is formed when combinations of hydrophilic solutes (two polymers, or a polymer and a salt) in aqueous solution above critical concentrations occur. (Yavari et al. 2013)  The purpose of this study was to analyze the optimal conditions for the extraction of fibrinolytic protease from Bacillus sp. UFPEDA 485, using aqueous two-phase system (PEG/Na2SO4)  2. MATERIALS AND METHODS  2.1. Microorganism and culture condition  The Bacillus sp. UFPEDA 485 strain was obtained from Culture Collection of Department of Antibiotics, at the Federal University of Pernambuco, Brazil. The stock culture was maintained in nutrient broth in cryotubes (10% v/v glycerol) at - 80 ◦C. The soybean medium (MS-2) described by Porto et al. (1996), was used for inoculum growth and for the fibrinolytic proteases production process. The medium composition was: filtered soy flour (2% w/v), K2HPO4 (0.435% w/v) and 1% of mineral solution containing: FeSO4.7H2O (100 mg); MnCl2.4H2O (100 mg), and ZnSO4.H2O (100 mg) of distilled water q.s.p 100 mL, NH4Cl (0.1% w/v); MgSO4.7H2O (0.06% w/v), glucose (1% w/v). The process for enzymatic production occurred in shake flasks of 250 mL capacity with 100 mL of production medium at pH 7.8, 150 rpm, at 37 °C for 48 hours.   2.2. Analytical determinations and aqueous two phase systems (ATPS)  Total protein concentration was determined by the Bradford method using as standard bovine serum albumin (BSA) Bradford (1976) Fibrinolytic activity was determined according to Wang et al. (2011).  One unit of fibrinolytic protease activity was defined as the amount enzyme required to produce an increase in absorbance equal to 0.01 per minute at 275 nm. The aqueous two-phase system and the methodology of analysis of the results were in according Medeiros e Silva et al., 2013. The concentrations of Na2SO4 and PEG were adjusted according to the planned factorial design (Table 1).  2.4. Statistical analysis and experimental design   A 23 full factorial design was used to analyze the influence of the three independent variables: MPEG; CPEG and CNa2SO4. The effects of these three independent variables on enzyme recovery were determined based on response variables enzyme K, PF and Y. The experimental design was composed of 8 runs and 4 repetitions at the central point, needed to calculate the pure error. Statistical significance of the variables was determined at 5% probability level (p<0.05).  The analysis of the results was carried with the program Statistic version 8.0  3. RESULTS AND DISCUSSION  Effect of independents variables on the partition coefficient (K), Purification factor (PF) and yield recovery (Y) of fibrinolytic protease  XIX SIMPÓSIO NACIONAL DE BIOPROCESSOS X SIMPÓSIO DE HIDRÓLISE ENZIMÁTICA DE BIOMASSAS  30 de julho-02 de agosto de 2013 Foz de Iguaçu, PR, Brasil        For all the runs, the fibrinolytic protease partition occurred on the top phase rich in PEG, indicating an affinity between the enzyme and the PEG. The best result was obtained at the central point, using MPEG 6000 g.mol-1, CPEG 24% and CNa2SO4 11.6%. For this assay, the obtained values at the mean center point were K = 5.03; PF = 3.30; Y = 91.40% and FA = 821 U. mL-1.   Table 1. Matrix of the full factorial design 23 with the results of the fibrinolytic protease extraction by ATPS. Run Independent variables Responses 1MPEG (g.mol-1) 2 CPEG (%) 3 CNa2SO (%) 4 K 5 Y (%) 6 PF 1 4000 18 10 4.28 87.30 2.71 2 8000 18 10 3.00 66.88 2.05 3 4000 30 10 3.84 80.20 2.97 4 8000 30 10 2.84 68.16 2.58 5 4000 18 13.2 4.05 85.35 2.60 6 8000 18 13.2 2.82 67.23 2.37 7 4000 30 13.2 3.66 72.69 2.99 8 8000 30 13.2 3.16 57.48 2.07 9 6000 24 11.6 4.78 92.56 3.36 10 6000 24 11.6 5.16 90.96 3.30 11 6000 24 11.6 5.30 91.61 3.28 12 6000 24 11.6 4.87 90.47 3.26 (1) PEG molar mass (g mol-1); (2) PEG concentration (%); (3) Na2SO4 concentration (%); (4) Partition coefficient (K); (5) yield recovery (Y) and (6) Purification factor (PF).The pure error estimate for K, Y, and PF was 0.06, 0.81 and 0.00, respectively.  The influence of the independent variables, on the responses is described in Table 2.  Table 2. Influence of independent variable on response parameters  Factor Effect estimate K Y PF (1)MPEG - 5.79* - 25.70* - 17.80* (2)CPEG - 0.93 - 11.02*    7.05* (3)CNa2SO4             - 0.38  - 7.73*               - 2.20 1*2               1.47    4.41*   - 3.31* 1*3               0.79               - 0.34               - 0.83 2*3               0.80   - 6.48*               - 5.66* 1*2*3               0.64   - 2.13               - 7.83* (1) PEG molar mass (g mol-1); (2) PEG concentration (%); (3) Na2SO4 concentration (%); Partition coefficient (K); Purification factor (PF) and yield recovery (Y).  * Statistically significant values at the 95% confidence level. The pure error estimate for K, Y, and PF was 0.06, 0.81 and 0.00, respectively  The presented results are corroborated by Kirsch et al, 2012, since in all ATPSs tested, the protease from Lentinus citrinus partitioned to the top phase with the best result obtained using MPEG 6000 g.mol-1 The increase of the MPEG, increased the hydrophobicity of the top phase, reducing the free space available for biomolecules and generating an exclusion effect. A similar result was observed by Medeiros e Silva et al, 2013, in the extraction of fibrinolytic protease from  XIX SIMPÓSIO NACIONAL DE BIOPROCESSOS X SIMPÓSIO DE HIDRÓLISE ENZIMÁTICA DE BIOMASSAS  30 de julho-02 de agosto de 2013 Foz de Iguaçu, PR, Brasil       Streptomyces sp. DPUA1576. These authors verified that the decrease on MPEG from 8000 to 1500 (g.mol-1) causes an improvement on fibrinolytic protease partition to the top PEG rich phase.   The interaction between MPEG and CPEG favored the increase of the Y and a 91.40% recovery was achieved. These results indicate that none of these independent variable acts alone in the response variables and a synergism occurs. A similar result was observed by Neves et al, 2012, in the extraction of a phytase from Absidia blakesleeana URM5604. The reported positive interaction of MPEG with CPEG indicates that an increase in PEG molar mass and PEG concentration enhanced the recovery. The best result for the recovery was 89.67%. The increase of the CPEG exerted a positive effect and favored the increase in the PF. The best result was 3.30 for the purification factor. Results corroborating this work were obtained by Kirsch et al, 2012.  4. CONCLUSION  According to the presented results, the ATPS composed of PEG/sodium sulfate proved to be a promising method for fibrinolytic protease extraction from the fermentation broth of Bacillus sp. UFPEDA 485.  5. REFERENCES  KIRSCH, L.S.; PINTO, A.C.S.;  TEIXEIRA, M.F.S.; PORTO, T.S.; PORTO, A.L.F.P. Partition of proteases from Lentinus citrinus DPUA 1535 by the peg/phosphate aqueous two-phase system. Quim. Nova, v. 35, n. 10, p. 1912-1915, 2012. MEDEIROS E SILVA, G.M.; MARQUES, D.A.V.; PORTO, T.S.; LIMA FILHO, J.L.; TEIXEIRA, J.A.C.; PESSOA-JÚNIOR, A.; PORTO, A.L.F. Extraction of fibrinolytic proteases from Streptomyces sp. DPUA1576 using PEG-phosphate aqueous two-phase systems. Fluid Phase Equilib., v. 339, p. 52-57, 2013. NEVES, M.L.C.; PORTO, T.S.; SOUZA-MOTTA, C.M.; SPIERF, M.R.; SOCCOLF, C.R.; Moreira, K.A.; PORTO, A.L.F. Partition and recovery of phytase from Absidia blakesleeana URM5604 using PEG–citrate aqueous two-phase systems. Fluid Phase Equilib., v. 318, p. 34-39, 2012. PORTO, A.L.F.; CAMPOS-TAKAKI, G.M.; LIMA-FILHO, J.L. Effects of culture conditions on protease production by Streptomyces clavuligerus growing soy bean flour medium. Appl. Biochem. Biotechnol., v. 60, p. 115-122, 1996. RAAFAT, A.I.; ARABY, E.; LOTFY, S. Enhancement of fibrinolytic enzyme production from Bacillus subtilis via immobilization process onto radiation synthesized starch/dimethylaminoethyl methacrylate hydrogel. Carbohydr. Polym., v. 87, n. 2, p.1369-1374, 2012. WANG, S-L.; WU, Y-Y. and LIANG, T-W. Characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. New Biotechnol. v. 28, n. 2, p. 196-202, 2011. YAVARI, M.; PAZUKIB, G.R.; VOSSOUGHIA, M.; MIRKHANIA, S.A.; SEIFKORDI, A.A. Partitioning of alkaline protease from Bacillus licheniformis (ATCC 21424) using PEG-K2HPO4 aqueous two-phase system. Fluid Phase Equilib., v. 337, p. 1- 5, 2013.   

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

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