Constitutive expression of barley α-amylase in Pichia pastoris by high-density cell culture.
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.
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, Hainan, China.
Z W
ZW
Liu
H X
HX
Yin
X P
XP
Yi
A L
AL
Zhang
J X
JX
Luo
T Y
TY
Zhang
C Y
CY
Fu
Z H
ZH
Zhang
J C
JC
Shen
L P
LP
Chen
D018533:Biomass; D018929:Cell Culture Techniques / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation / Q000187:drug effects; D018628:Gene Dosage / Q000235:genetics; D001467:Hordeum / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006868:Hydrolysis / Q000187:drug effects; D010461:Peptones / Q000494:pharmacology; D010843:Pichia / Q000166:cytology / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification; D013213:Starch / Q000378:metabolism; D014170:Transformation, Genetic / Q000187:drug effects; D000516:alpha-Amylases / Q000302:isolation & purification / Q000378:metabolism*
D010461:Peptones; D011994:Recombinant Proteins; D013213:Starch; D000516:alpha-Amylases