文献类型：期刊文献

英文题名：Expression, purification and characterization of a recombinant Lipomyces starkey dextranase in Pichia pastoris

作者：Chen, Lin[1];Zhou, Xiangshan[1];Fan, Weimin[1];Zhang, Yuanxing[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2008

卷号：58

期号：1

起止页码：87

外文期刊名：PROTEIN EXPRESSION AND PURIFICATION

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000253568700010)】；

语种：英文

外文关键词：dextranase; Lipomyces starkeyi; Pichia pastoris; expression; purification

摘要：The DEX gene encoding an extracellular dextranase from Lipomyces starkeyi was cloned into vector pPIC9k-His6 and was expressed in Pichia pastoris GS115 strain under the control of AOX1 promoter. After 107 h of the 5 L-scaled fermentation, wet cells weight of the recombinant P. pastoris Mut(+) strain reached to 588.6 g/L, and the concentration of dextranase and enzyme activity in the supernatant were 0.46 g/L and 83900 U/L, respectively. The activity of dextranase was improved 17.56-fold by cation-exchange chromatography only with a final yield of 71.61% and the specific activity of the purified enzyme was 181.96 U/mg. The purified dextranase, analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. Then the factors affecting the dextranase activity were evaluated. The optimal temperature and pH was 30 degrees C and pH 4.5, respectively. Metal ions Al3+, Cu2+, Fe3+, and SDS could completely inhibit the enzyme activity, whereas Mg2+ enhanced 145% of the enzyme activity. These characters are much different from what was previously reported for the L. starkeyi dextranase that was either expressed in S. cerevisiae or purified from natural L. starkeyi. (c) 2007 Elsevier Inc. All rights reserved.