Modeling Of Ion Exchange Expanded-bed Chromatography For The Purification Of C-phycocyanin

This work is focused on the experimental evaluation and mathematical modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin from crude fermentative broth containing Spirulina platensis cells. Experiments were carried out in different expansion degree to evaluate the process performance. The experimental breakthrough curves were used to estimate the mass transfer and kinetics parameters of the proposed model, using the Particle Swarm Optimization algorithm (PSO). The proposed model satisfactorily fitted the experimental data. The results from the model application pointed out that the increase in the initial bed height does not influence the process efficiency, however enables the operation of expanded-bed column at high volumetric flow rates, improving the productivity. It was also shown that the use of mathematical modeling was a good and promising tool for the optimization of chromatographic processes. © 2013 Elsevier B.V.12817378Patil, G., Raghavarao, K.S.M.S., (2007) Biochem. Eng. J., 34, p. 156Soni, B., Kalavadia, B., Trivedi, U., Madamwar, D., (2006) Process Biochem., 41, p. 2017Estrada, J.E.P., Bescós, P.B., Fresno, A.M.V., (2001) IL Farmaco, 56, p. 497Romay, C., Remírez, D., González, R., (2001) Rev. Cubana Invest. Bioméd., 20, p. 38Reddy, C.M., Subhashini, J., Mahipal, S.V.K., Bhat, V.B., Reddy, P.S., Kiranmai, G., Madyastha, K.M., Reddanna, P., (2003) Biochem. Biophys. Res. Commun., 304, p. 385Eriksen, N.T., (2008) Appl. Microbiol. Biotechnol., 80, p. 1Ling, T.C., Loong, C.K., Tan, W.S., Tey, B.T., Abdullanh, W.M.W., Ariff, A., (2004) J. Microbiol., 42 (3), p. 228Moraes, C.C., Kalil, S.J., (2009) Bioresour. Technol., 100, p. 5312Wang, G., (2002) Chromatographia, 56, p. 509Niu, J.-F., Wang, G.-C., Lin, X.-Z., Zhou, B.-C., (2007) J. Chromatogr. B, 850, p. 267Kaczmarski, K., Bellot, J.-C., (2005) J. Chromatogr. A, 1069, p. 91Xia, H.-F., Lin, D.-Q., Yao, S.-J., (2007) J. Chromatogr. A, 1145, p. 58Pai, A., Gondkar, S., Sundaram, S., Lali, A., (1999) Bioseparation, 8, p. 131. , NetherlandsBermejo, R., Felipe, M.A., Talavera, E.M., Alvarez-Pez, J.M., (2006) Chromatographia, 63, p. 59Moares, C.C., Ores, J.V., Costa, J.A.V., Kalil, S.J., (2011) Chromatographia, 74, p. 307Bermejo, R., Ramos, A., (2012) Chromatographia, 75, p. 195Nayak, D.P., (2001) Studies of Protein Purification on Expanded Bed. Doctor of Philosophy in Biotechnology, , University of Pune, índiaJungbauer, A., (2005) J. Chromatogr. A, 1065, p. 3Moraes, C.C., Mazutti, M.A., Rodrigues, M.I., Maugeri, F., Kalil, S.J., (2009) J. Chromatogr. A, 1216, p. 4395Burkert, C.A.V., Mazutti, M., Barbosa, G., Maugeri Filho, F., (2011) Process Biochem., 46, p. 1270Costa, J.A.V., Linde, G.A., Atala, D.I.P., Mibileli, G.M., Krüger, R.T., (2000) World J. Microbiol. Biotechnol., 16, p. 15Moraes, C.C., Burkert, J.F.M., Kalil, S.J., (2010) J. Food Biochem., 34, p. 133(2002), Amersham Biosciences, InstructionsRichardson, J.F., Zaki, W.N., (1954) Trans. Inst. Chem. Eng., 32 (1), p. 35Li, P., Xiu, G., Rodrigues, A.E., (2005) AIChE J., 51, p. 2965Chang, Y.K., Chase, H.A., (1996) Biotechnol. Bioeng., 49 (2), p. 512Vennapusa, R., Hunegnaw, S.M., Cabrera, R.B., Fernandez-Lahore, M., (2008) J. Chromatogr. A, 1181, p. 9Deuflhard, E., Hairer, E., Zugck, J., (1987) Numer. Math., 51, p. 501Schwaab, M., Biscaia, E.C., Monteiro, J.L., Pinto, J.C., (2008) Chem. Eng. Sci., 63, p. 1542Kalil, S.J., Maugeri-Filho, F., Rodrigues, M.I., (2005) Process Biochem., 40, p. 581Tong, X.-D., Xue, B., Sun, Y., (2003) Biochem. Eng. J., 16, p. 265Yun, J., Lin, D.Q., Yao, S.J., (2005) J. Chromatogr. A, 1095, p. 16Anspach, F.B., Curbelo, D., Hartmann, R., Garke, G., Deckwer, W.-D., (1999) J. Chromatogr. A, 865, p. 12

Purification of Peroxidase from Rice Bran Using Expanded-Bed Ion-Exchange Chromatography

Peroxidase catalyzes the oxidation of various substrates at the expense of hydrogen peroxide. Among the various techniques used for the purification of enzymes, expanded-bed adsorption chromatography is particularly popular because it offers several advantages, such as greater interactions between adsorbents and target molecules, increased overall yield, potential for a scale-up and shorter process times. It relies on the interaction between charged molecules in the mobile phase (i.e. buffer and sample) and oppositely charged groups coupled to the resin in the expanded-bed form. Other chromatographic techniques are also commonly used for peroxidase purification and characterization; however, there are no reports in the literature about the use of expanded-bed adsorption chromatography for this purpose. In this paper, the purification of peroxidase from rice bran using ion-exchange chromatography (expanded-bed column) was investigated. Chromatographic assays were carried out using STREAMLINE SP cationic resin with different buffer solutions and pH values in the equilibrium, washing and elution steps. The use of 0.025 mol/l sodium acetate buffer at pH 4.5 (during equilibration and washing) and 4.7 (during elution) allowed for the purification of peroxidase from rice bran, resulting in a purification factor and enzyme recovery of 2.4-fold and 41%, respectively

Kinetics And Thermal Properties Of Crude And Purified β-galactosidase With Potential For The Production Of Galactooligosaccharides

β-Galactosidase is an enzyme that catalyzes the hydrolysis of lactose. It has potential importance due to various applications in the food and dairy industries, involving lactose-reduced ingredients. The properties of two b-galactosidase enzymes, crude and purified, from different sources, Kluyveromyces marxianus CCT 7082 and Kluyveromyces marxianus ATCC 16045, were analyzed. The pH and temperature optima, deactivation energy, thermal stability and kinetic and thermodynamic parameters were determined, as well as the ability to hydrolyze lactose and produce galactooligosaccharides. Purification process improved the properties of the enzymes, and the results showed that purified enzymes from both strains had a higher optimum temperature, and lower values of Km, thus showing greater affinity for o-nitrophenyl-b-D-galactopiranoside than the crude enzymes. The production of galactooligosaccharides was also greater when using purified enzymes, increasing the synthesis by more than 30 % by both strains.5114552Gekas, V., López-Leiva, M., Hydrolysis of lactose: A literature review (1985) Process Biochem., 20, pp. 1-12Braga, A.R.C., Gomes, P.A., Kalil, S.J., Formulation of culture medium with agroindustrial waste for b-galactosidase production from Kluyveromyces marxianus ATCC 16045 (2012) Food Bioprocess Technol., 5, pp. 1653-1663Mahoney, R.R., Galactosyl-oligosaccharide formation during lactose hydrolysis: A review (1998) Food Chem., 63, pp. 147-154Sako, T., Matsumoto, K., Tanaka, R., Recent progress on research and applications of non-digestible galacto-oligosaccharides (1999) Int. Dairy J., 9, pp. 69-80Sears, P., Wong, C.H., Toward automated synthesis of oligosaccharides and glycoproteins (2001) Science,, 291, pp. 2344-2350Ladero, M., Santos, A., García-Ochoa, F., Kinetic modeling of lactose hydrolysis with an immobilized b-galactosidase from Kluyveromyces fragilis (2000) Enzyme Microbial Technol., 27, pp. 583-592Szczodrak, J., Hydrolysis of lactose in whey permeate by immobilized b-galactosidase from Kluyveromyces fragilis (2000) J. Mol. Catal. B: Enzym., 10, pp. 631-637Jurado, E., Camacho, F., Luzon, G., Vicaria, J.M., Kinetic models of activity for b-galactosidases: Influence of pH, ionic concentration and temperature (2004) Enzyme Microbial Technol., 34, pp. 33-40Ustok, F.I., Tari, C., Harsa, S., Biochemical and thermal properties of b-galactosidase enzymes produced by artisanal yoghurt cultures (2010) Food Chem., 119, pp. 1114-1120Manera, A.P., Ores, J.C., Ribeiro, V.A., Burkert, C.A.V., Kalil, S.J., Optimization of the culture medium for the production of b-galactosidase from Kluyveromyces marxianus CCT 7082 (2008) Food Technol. Biotechnol., 46, pp. 66-72Pinheiro, R., Belo, I., Mota, M., Growth and b-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure (2003) Lett. Appl. Microbiol., 37, pp. 438-442Lukondeh, T., Ashbolt, N.J., Rogers, P.L., Fed-batch fermentation for production of Kluyveromyces marxianus FII 510700 cultivated on a lactose-based medium (2005) J. Ind. Microbiol. Biotechnol., 32, pp. 284-288de Medeiros, F.O., Burkert, C.A.V., Kalil, S.J., Purification of b-galactosidase by ion exchange chromatography: A study of the elution using an experimental design (2012) Chem. Eng. Technol., 35, pp. 911-918de Medeiros, F.O., Alves, F.G., Lisboa, C.R., de Souza Martins, D., Burkert, C.A.V., Kalil, S.J., Ultrasonic waves and glass pearls: A new method of extraction of b-galactosidase for use in laboratory (2008) Quimica Nova,, 31, pp. 336-339. , in PortugueseWhitaker, J.R., (1994) Principles of Enzymology for the Food Sciences, , Marcel Dekker, New York, NY, USAInchaurrondo, V.A., Yantorno, O.M., Voget, C.E., Yeast growth and b-galactosidase production during aerobic batch cultures in lactose-limited synthetic medium (1994) Process Biochem., 29, pp. 47-54Manera, A.P., Costa, F.A.A., Rodrigues, M.I., Kalil, S.J., Maugeri Filho, F., Galacto-oligosaccharides production using permeabilized cells of Kluyveromyces marxianus (2010) Int. J. Food Eng., 6, pp. 1-15Brady, D., Marchant, R., McHale, L., McHale, A.P., Isolation and partial characterization of b-galactosidase activity produced by a thermotolerant strain of Kluyveromyces marxianus during growth on lactose-containing media (1995) Enzyme Microbial Technol., 17, pp. 696-699Lai, L.S., Chang, P.C., Chang, C.T., Isolation and characterization of superoxide dismutase from wheat seedlings (2008) J. Agric. Food Chem., 56, pp. 8121-8129Alcántara, A.R., Borreguero, I., López-Belmonte, M.T., Sinisterra, J.V., Covalent Immobilization of Crude and Partially-Purified Lipases onto Inorganic Supports: Stability and Hyperactivation (1998) Progress in Biotechnology, pp. 571-576. , Elsevier Science B.V., Amsterdam, The Netherlands , A. Ballesteros, F.J. Plou, J.L. Iborra, P.J. Hailing (Eds.)Naidu, G.S.N., Panda, T., Studies on pH and thermal inactivation of pectolytic enzymes from Aspergillus niger (2003) Biochem. Eng. 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New Creatinine- and Cystatin C-Based Equations to Estimate GFR without Race.

Current equations for estimated glomerular filtration rate (eGFR) that use serum creatinine or cystatin C incorporate age, sex, and race to estimate measured GFR. However, race in eGFR equations is a social and not a biologic construct. We developed new eGFR equations without race using data from two development data sets: 10 studies (8254 participants, 31.5% Black) for serum creatinine and 13 studies (5352 participants, 39.7% Black) for both serum creatinine and cystatin C. In a validation data set of 12 studies (4050 participants, 14.3% Black), we compared the accuracy of new eGFR equations to measured GFR. We projected the prevalence of chronic kidney disease (CKD) and GFR stages in a sample of U.S. adults, using current and new equations. In the validation data set, the current creatinine equation that uses age, sex, and race overestimated measured GFR in Blacks (median, 3.7 ml per minute per 1.73 m <sup>2</sup> of body-surface area; 95% confidence interval [CI], 1.8 to 5.4) and to a lesser degree in non-Blacks (median, 0.5 ml per minute per 1.73 m <sup>2</sup> ; 95% CI, 0.0 to 0.9). When the adjustment for Black race was omitted from the current eGFR equation, measured GFR in Blacks was underestimated (median, 7.1 ml per minute per 1.73 m <sup>2</sup> ; 95% CI, 5.9 to 8.8). A new equation using age and sex and omitting race underestimated measured GFR in Blacks (median, 3.6 ml per minute per 1.73 m <sup>2</sup> ; 95% CI, 1.8 to 5.5) and overestimated measured GFR in non-Blacks (median, 3.9 ml per minute per 1.73 m <sup>2</sup> ; 95% CI, 3.4 to 4.4). For all equations, 85% or more of the eGFRs for Blacks and non-Blacks were within 30% of measured GFR. New creatinine-cystatin C equations without race were more accurate than new creatinine equations, with smaller differences between race groups. As compared with the current creatinine equation, the new creatinine equations, but not the new creatinine-cystatin C equations, increased population estimates of CKD prevalence among Blacks and yielded similar or lower prevalence among non-Blacks. New eGFR equations that incorporate creatinine and cystatin C but omit race are more accurate and led to smaller differences between Black participants and non-Black participants than new equations without race with either creatinine or cystatin C alone. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)

Optimization of fermentation conditions for P450 BM-3 monooxygenase production by hybrid design methodology

Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E. coli. Operational conditions for maximum production were determined with twelve parameters under consideration: the concentration of FeCl(3), induction at OD(578) (optical density measured at 578 nm), induction time and inoculum concentration. Initially, Plackett-Burman (PB) design was used to evaluate the process variables relevant in relation to P450 BM-3 production. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM-3 production was improved to 57.90×10(−3) U/ml by the best combinations of the physicochemical parameters at optimum levels of 0.12 mg/L FeCl(3), inoculum concentration of 2.10%, induction at OD(578) equal to 1.07, and with 6.05 h of induction

Enhanced production of elastase by Bacillus licheniformis ZJUEL31410: optimization of cultivation conditions using response surface methodology

Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cultures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410