文献类型： 外文期刊

作者： Liu, Z. W. ¹ ; Yin, H. X. ¹ ; Yi, X. P. ¹ ; Zhang, A. L. ¹ ; Luo, J. X. ² ; Zhang, T. Y. ² ; Fu, C. Y.; Zhang, Z. H. ¹ ;

作者机构： 1.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Haikou 571101, Hainan, Peoples R China

2.Sun Yat Sen Univ, Key Lab Gene Engn, Minist Educ, Guangzhou 510275, Guangdong, Peoples R China

3.Sun Yat Sen Univ, Dept Biochem, Guangzhou 510275, Guangdong, Peoples R China

4.Sun Yat Sen Univ, Dept Biochem, Guangzhou 510275, Guangdong, Peoples

关键词： alpha-Amylase;Constitutive expression;Pichia pastoris;High-density cell culture;Bioactivity

期刊名称：MOLECULAR BIOLOGY REPORTS （ 影响因子：2.316； 五年影响因子：2.357 ）

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收录情况： SCI

摘要： alpha-amy gene amplified from barley genome was cloned into MCS of pGAP9K to generate pGAP9K-alpha-amy which was then transformed into Pichia pastoris GS115 by electroporation. Transformants with multi-copies and high expression for the foreign gene were selected on G418 containing plate and expression analysis. The fermentation was carried out in a 50 l bioreactor with 20 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 54 h at 30A degrees C. Under the control of GAP promoter (pGAP), alpha-amy gene was constitutively expressed. At the end of the fermentation, the alpha-AMY expression reached 125 mg/l, while the biomass growth was 186 as measured by absorption of 600 nm. The secreted alpha-AMY was purified to 97.5% by SP-Sepharose FF ion-exchange chromatography and affinity purification. The recombinant alpha-AMY showed activity on hydrolysis of starch.