Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/68797
標題: Recovery of active N-acetyl-D-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers
作者: Lu, S.C.
Lin, S.C.
關鍵字: Epimerase
Inclusion bodies
Refolding
Solubilization
d-neuraminic acid
acetylneuraminic acid
sialic-acid
protein
aggregation
circular-dichroism
egg-yolk
renaturation
membrane
bacteria
aldolase
期刊/報告no:: Enzyme and Microbial Technology, Volume 50, Issue 1, Page(s) 65-70.
摘要: Overexpression of recombinant N-acetyl-D-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coil led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08 +/- 0.02 U/mg, was similar to that of the native protein, 2.13 +/- 0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Iris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41 +/- 10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32 +/- 5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins. (C) 2011 Elsevier Inc. All rights reserved.
URI: http://hdl.handle.net/11455/68797
ISSN: 0141-0229
文章連結: http://dx.doi.org/10.1016/j.enzmictec.2011.09.010
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