Constitutive expression of barley α-amylase in Pichia pastoris by high-density cell culture
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- 作者：Z. W. Liu (1)
H. X. Yin (1)
X. P. Yi (1)
A. L. Zhang (1) zhangailian6@yahoo.com.cn
J. X. Luo (2)
T. Y. Zhang (2)
C. Y. Fu (3)
Z. H. Zhang (1)
J. C. Shen (1)
L. P. Chen (1) - 关键词：α ; Amylase – Constitutive expression – Pichia pastoris – High ; density cell culture – Bioactivity
- 刊名：Molecular Biology Reports
- 出版年：2012
- 出版时间：May 2012
- 年：2012
- 卷：39
- 期：5
- 页码：5805-5810
- 全文大小：369.9 KB
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- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Life Sciences
Animal Anatomy, Morphology and Histology
Animal Biochemistry - 出版者：Springer Netherlands
- ISSN：1573-4978
文摘
α-amy gene amplified from barley genome was cloned into MCS of pGAP9K to generate pGAP9K-α-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 30°C. Under the control of GAP promoter (pGAP), α-amy gene was constitutively expressed. At the end of the fermentation, the α-AMY expression reached 125 mg/l, while the biomass growth was 186 as measured by absorption of 600 nm. The secreted α-AMY was purified to 97.5% by SP-Sepharose FF ion-exchange chromatography and affinity purification. The recombinant α-AMY showed activity on hydrolysis of starch.