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Study on the Cellulase Components β-Endoglucanase Purification by Chromatography
Corresponding Author(s) : Hong Wu
Asian Journal of Chemistry,
Vol. 25 No. 3 (2013): Vol 25 Issue 3
Abstract
Two active components, EgI and EgII, of b-endoglucanase were purified by utilizing salting out method, as well as twice Sephadex G-75 and DEAE-Sephadex A-50 chromatography from the fermentation broth of Trichoderma reesei QM9414. The relative molecular weights of EgI and EgII were 65.47 and 57.04 kDa, respectively, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimal temperatures of EgI and EgII were 50 ºC and 55 ºC, respectively and the optimal pH levels were 4.8 and 5.0. The experiment on the effect of different metal ions showed that: K+ could activate EgI and EgII activities and Fe3+, Cu2+, Zn2+, Ca2+ and Mn2+ were the inhibitors of EgI and EgII. EgI and EgII with good thermal stability, in temperature range of 30-60 ºC with 0.5 h, EgI and EgII can maintain more than 78 % enzyme activity, EgI and EgII are heat-resistant. The Km of EgI and EgII was detected using the Lineweaver-Burk plot method, which showed that their Michaelis constants values were 0.238 g/L and 0.266 g/L, yEgI = 0.6078x + 2.5558, yEgII = 0.6665x + 2.5046, respectively. EgI and EgII are new discoveries according to their characterization. Apparently, the present research built a foundation for the further study of b-endoglucanase in T. reesei QM9414.
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References
P. Tomme, A.J. Warren and N.R. Gilkes, Microb. Physiol., 37, 1 (1995).
D.J.T. Hill and T.T. Le, Polym. Degrad. Stab., 51, 211 (1996).
C.Y. Wang, Y.R. Hsieh, C.C. Ng, H. Chan, H.T. Lin, W.S. Tzeng and Y.T. Shyu, Enzyme Microb. Technol., 44, 373 (2009).
A.M. de Castro, M.C. Ferreira, J.C. da Cruz, K.C.N.R. Pedro, D.F. Carvalho, S.G.F. Leite and N. Pereira Jr., Enzyme Res., Article ID 854526 (2010).
J. Woodward, H.J. Marquess and C.S. Picker, Prep. Biochem., 16, 337 (1986).
L. Bao, Q. Huang, L. Chang, J.G. Zhou and H. Lu, J. Mol. Catal. B, 73, 104 (2011).
T. Watanbe, T. Sato, S. Yoshioka, T. Koshijima and M. Kuwahara, Eur. J. Biochem., 209, 651 (1992).
J. Zhou, Y.-H. Wang, J. Chu, Y.-P. Zhuang, S.-L. Zhang and P. Yin, Bioresour. Technol., 99, 6826 (2008).
L.J. Yin, H.H. Lin and Z.R. Xiao, J. Marine Sci. Technol. Taiwan, 18, 466 (2010).
F. Colussi, L.C. Textor, V. Serpa, R.N. Maeda, N. Pereira Jr and I. Polikarpov, Acta Crystallogr., F66, 1041 (2010).
X.Y. Ye and K.J. Cheng, Int. J. Biochem. Cell Biol., 33, 87 (2001).
M.M. Bradford, Anal. Biochem., 72, 248 (1976).
A.M. Scrivener and M. Slaytor, Molec. Biol., 24, 223 (1994).
H. Chanzy, B. Henrissat, R. Vuong and M. Schuelein, FEBS Lett., 153, 113 (1983).
D.C. Irwin, M. Spezio, L.P. Walker and D.B. Wilson, Biotech. Bioeng., 42, 1002 (1993).
M. Linder and T.T. Teeri, Proc. Nat. Acad. Sci. USA, 93, 251 (1996).
Y.-J. Lee, B.-K. Kim, B.-H. Lee, K.-I. Jo, N.-K. Lee, C.-H. Chung, Y.-C. Lee and J.-W. Lee, Bioresour. Technol., 99, 378 (2008).
V.T.O. Santos, P.J. Esteves, A.M.F. Milagres and W. Carvalho, J. Ind. Microbiol. Biotechnol., 38, 1089 (2011).
Z.A. Olama, M.A. Hamza, M.M. El-Sayed and M. Abdel-Fattah, Food Chem., 47, 221 (1993).
C. Chararas, R. Eberhard, J.E. Courtois and F. Petek, Insect. Biochem., 13, 213 (1983).