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Selective Extraction and Purification of Papain Using Polyethylene Glycol (PEG 4000)/Potassium Citrate Aqueous Two Phase
Corresponding Author(s) : Yanmin Lu
Asian Journal of Chemistry,
Vol. 26 No. 12 (2014): Vol 26 Issue 12
Abstract
Mathematical models concerning the purification of papain from the crude papain in an aqueous two-phase system which are composed of polyethylene glycol (PEG 4000)/potassium citrate are established with response surface methodology. The concentration of PEG 4000 and potassium citrate, the concentration of additional salt (KCl), the temperature and pH on the papain purification have been investigated. Results showed that papain tends to partitions to the more hydrophilic salt-rich phase. The steepest ascent method was used to locate the optimal domain. Papain was successfully extracted to the bottom phase of a system composed of 15 % (w/w) PEG 4000, 17 % (w/w) citrate and 5.0 % (w/w) KCl at pH 5.0 and 30 °C. Under those conditions, the specific activity, purification factor and activity yield for papain were 1703.02 U/mg, 1.89 and 94 %, respectively. The purity of extracted papain was confirmed by SDS-PAGE analysis.
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- C. Qihe, H. Guoqing, J. Yingchun and N. Hui, Food Chem., 98, 624 (2006); doi:10.1016/j.foodchem.2005.06.043.
- S.S. Khaparde and R.S. Singhal, Bioresour. Technol., 78, 1 (2001); doi:10.1016/S0960-8524(00)00178-4.
- I.F. Starley, P. Mohammed, G. Schneider and S.W. Bickle, Burns, 25, 636 (1999); doi:10.1016/S0305-4179(99)00056-X.
- S. Nitsawang, R. Hatti-Kaul and P. Kanasawud, Enzyme Microb. Technol., 39, 1103 (2006); doi:10.1016/j.enzmictec.2006.02.013.
- S.N. Su, H.L. Nie, L.M. Zhu and T.X. Chen, Bioresour. Technol., 100, 2336 (2009); doi:10.1016/j.biortech.2008.11.048.
- Y.-Q. Ling, H.-L. Nie, S.-N. Su, C. Branford-White and L.-M. Zhu, Sep. Purif. Technol., 73, 343 (2010); doi:10.1016/j.seppur.2010.04.020.
- T.J. Ridgway and G.A. Tucker, Enzyme Microb. Technol., 24, 225 (1999); doi:10.1016/S0141-0229(98)00109-4.
- K.S.M.S. Raghavarao, N.K. Rastogi, M.K. Gowthaman and N.G. Karanth, Adv. Appl. Microbiol., 41, 97 (1995); doi:10.1016/S0065-2164(08)70309-5.
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- L. Fukal, J. Kas and E. Paluska, J. Chromatogr. A, 285, 365 (1984); doi:10.1016/S0021-9673(01)87776-1.
- F. D’Souza and A. Lali, Biotechnol. Tech., 13, 59 (1999); doi:10.1023/A:1008843731758.
- M. Li, J.W. Kim and T.L. Peeples, J. Biotechnol., 93, 15 (2002); doi:10.1016/S0168-1656(01)00382-0.
- P.A. Albertsson, Partitioning of Cell Particles and Macromolecules, Wiley, New York, edn 3 (1986).
- T.M. Przybycien, N.S. Pujar and L.M. Steele, Curr. Opin. Biotechnol., 15, 469 (2004); doi:10.1016/j.copbio.2004.08.008.
- J. Thommes and M. Etzel, Biotechnol. Prog., 23, 42 (2007); doi:10.1021/bp0603661.
- A.M. Azevedo, A.G. Gomes, P.A.J. Rosa, I.F. Ferreira, A.M.M.O. Pisco and M.R. Aires-Barros, Sep. Purif. Technol., 65, 14 (2009); doi:10.1016/j.seppur.2007.12.010.
- R. Kuboi, W.H. Wang and I. Komasawa, Kagaku Kogaku Ronbunshu, 16, 772 (1990); doi:10.1252/kakoronbunshu.16.772.
- J. Vernau and M.R. Kula, Biotechnol. Appl. Biochem., 12, 397 (1990).
- X.Q. Xie, Y. Wang, J. Han and Y.S. Yan, Anal. Chim. Acta, 687, 61 (2011); doi:10.1016/j.aca.2010.12.012.
- M.M. Bradford, Anal. Biochem., 72, 248 (1976); doi:10.1016/0003-2697(76)90527-3.
- R. Arnon, Methods Enzymol., 19, 226 (1970); doi:10.1016/0076-6879(70)19017-3.
- U.K. Laemmli, Nature, 227, 680 (1970); doi:10.1038/227680a0.
- T.S. Meyer and B. Lamberts, Biochim. Biophys. Acta, 107, 144 (1965); doi:10.1016/0304-4165(65)90403-4.
- C.K. Su and B.H. Chiang, Process Biochem., 41, 257 (2006); doi:10.1016/j.procbio.2005.06.026.
- A.L. Ahmad, C.J.C. Derek and M.M.D. Zulkali, Sep. Purif. Technol., 62, 702 (2008); doi:10.1016/j.seppur.2008.03.028.
- I.F. Ferreira, A.M. Azevedo, P.A.J. Rosa and M.R. Aires-Barros, J. Chromatogr. A, 1195, 94 (2008); doi:10.1016/j.chroma.2008.04.077.
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- Y.M. Lu, Y.Z. Yang, X.D. Zhao and C.B. Xia, Food Bioprod. Process., 88, 40 (2010); doi:10.1016/j.fbp.2009.12.002.
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- M. Antov and R. Omorjan, Bioprocess Biosyst. Eng., 32, 235 (2009); doi:10.1007/s00449-008-0243-3.
References
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S.S. Khaparde and R.S. Singhal, Bioresour. Technol., 78, 1 (2001); doi:10.1016/S0960-8524(00)00178-4.
I.F. Starley, P. Mohammed, G. Schneider and S.W. Bickle, Burns, 25, 636 (1999); doi:10.1016/S0305-4179(99)00056-X.
S. Nitsawang, R. Hatti-Kaul and P. Kanasawud, Enzyme Microb. Technol., 39, 1103 (2006); doi:10.1016/j.enzmictec.2006.02.013.
S.N. Su, H.L. Nie, L.M. Zhu and T.X. Chen, Bioresour. Technol., 100, 2336 (2009); doi:10.1016/j.biortech.2008.11.048.
Y.-Q. Ling, H.-L. Nie, S.-N. Su, C. Branford-White and L.-M. Zhu, Sep. Purif. Technol., 73, 343 (2010); doi:10.1016/j.seppur.2010.04.020.
T.J. Ridgway and G.A. Tucker, Enzyme Microb. Technol., 24, 225 (1999); doi:10.1016/S0141-0229(98)00109-4.
K.S.M.S. Raghavarao, N.K. Rastogi, M.K. Gowthaman and N.G. Karanth, Adv. Appl. Microbiol., 41, 97 (1995); doi:10.1016/S0065-2164(08)70309-5.
K. Brocklehurst, J. Carlsson, M.P.J. Kierstan and E.M. Crook, Biochem. J., 133, 573 (1973).
D.E. Burke, S.D. Lewis and J.A. Shafer, Arch. Biochem. Biophys., 164, 30 (1974); doi:10.1016/0003-9861(74)90004-6.
L. Fukal, J. Kas and E. Paluska, J. Chromatogr. A, 285, 365 (1984); doi:10.1016/S0021-9673(01)87776-1.
F. D’Souza and A. Lali, Biotechnol. Tech., 13, 59 (1999); doi:10.1023/A:1008843731758.
M. Li, J.W. Kim and T.L. Peeples, J. Biotechnol., 93, 15 (2002); doi:10.1016/S0168-1656(01)00382-0.
P.A. Albertsson, Partitioning of Cell Particles and Macromolecules, Wiley, New York, edn 3 (1986).
T.M. Przybycien, N.S. Pujar and L.M. Steele, Curr. Opin. Biotechnol., 15, 469 (2004); doi:10.1016/j.copbio.2004.08.008.
J. Thommes and M. Etzel, Biotechnol. Prog., 23, 42 (2007); doi:10.1021/bp0603661.
A.M. Azevedo, A.G. Gomes, P.A.J. Rosa, I.F. Ferreira, A.M.M.O. Pisco and M.R. Aires-Barros, Sep. Purif. Technol., 65, 14 (2009); doi:10.1016/j.seppur.2007.12.010.
R. Kuboi, W.H. Wang and I. Komasawa, Kagaku Kogaku Ronbunshu, 16, 772 (1990); doi:10.1252/kakoronbunshu.16.772.
J. Vernau and M.R. Kula, Biotechnol. Appl. Biochem., 12, 397 (1990).
X.Q. Xie, Y. Wang, J. Han and Y.S. Yan, Anal. Chim. Acta, 687, 61 (2011); doi:10.1016/j.aca.2010.12.012.
M.M. Bradford, Anal. Biochem., 72, 248 (1976); doi:10.1016/0003-2697(76)90527-3.
R. Arnon, Methods Enzymol., 19, 226 (1970); doi:10.1016/0076-6879(70)19017-3.
U.K. Laemmli, Nature, 227, 680 (1970); doi:10.1038/227680a0.
T.S. Meyer and B. Lamberts, Biochim. Biophys. Acta, 107, 144 (1965); doi:10.1016/0304-4165(65)90403-4.
C.K. Su and B.H. Chiang, Process Biochem., 41, 257 (2006); doi:10.1016/j.procbio.2005.06.026.
A.L. Ahmad, C.J.C. Derek and M.M.D. Zulkali, Sep. Purif. Technol., 62, 702 (2008); doi:10.1016/j.seppur.2008.03.028.
I.F. Ferreira, A.M. Azevedo, P.A.J. Rosa and M.R. Aires-Barros, J. Chromatogr. A, 1195, 94 (2008); doi:10.1016/j.chroma.2008.04.077.
W.B. Zhi, J.N. Song, F. Ouyang and J.X. Bi, J. Biotechnol., 118, 157 (2005); doi:10.1016/j.jbiotec.2005.03.017.
D.L. Qiao, B. Hu, D. Gan, Y. Sun, H. Ye and X.X. Zeng, Carbohydr. Polym., 76, 422 (2009); doi:10.1016/j.carbpol.2008.11.004.
Y.M. Lu, Y.Z. Yang, X.D. Zhao and C.B. Xia, Food Bioprod. Process., 88, 40 (2010); doi:10.1016/j.fbp.2009.12.002.
J.A. Asenjo, A.S. Schmidt, F. Hachem and B.A. Andrews, J. Chromatogr. A, 668, 47 (1994); doi:10.1016/0021-9673(94)80090-1.
A.S. Schmidt, A.M. Ventom and J.A. Asenjo, Enzyme Microb. Technol., 16, 131 (1994); doi:10.1016/0141-0229(94)90076-0.
M. Antov and R. Omorjan, Bioprocess Biosyst. Eng., 32, 235 (2009); doi:10.1007/s00449-008-0243-3.