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Screening, Cloning and Overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favorable characteristics
Dong-Min Zhao,Min Wang,Xi-Jun Mu,Ming-Lv Sun,Xiao-Yun Wang
College of Life Science, Shandong Agricultural University
*Correspondence author
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Funding: China Natural Science，Research Fund for Doctoral Program of Higher（No.30471261，20050434008）
Opened online: 9 April 2007
Accepted by: none
Citation: Dong-Min Zhao,Min Wang,Xi-Jun Mu.Screening, Cloning and Overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favorable characteristics[OL]. [ 9 April 2007] http://en.paper.edu.cn/en_releasepaper/content/12053
 
 
An Aspergillus niger spp. which produces extracellular phytase was isolated. The phyA gene was cloned and sequenced. The results show that the coding region comprises 1347 bp and the homology of nucleotide sequence and amino acid sequence with A. niger NRRL 3135 were about 96% and 93.8%, respectively. The coding sequence phyA fragment was cloned into Pichia secretive expression vector pPICZA and then transformed into chromosome of Pichia pastoris GS115 strain by electroporation. After stepwise screenings by antibiotics zeocin, yeast PCR and induction by methanol, one transformant showed high expression. The activity of fermented broth was 30000-fold of original Aspergillus niger spp. phytase (0.008 U•ml-1) and the specific activity was 503 U•mg-1 of protein. The Km value was 0.196 mmoll-1 for sodium phytate and 18.16 mmoll-1 for pNPP. It showed activity at pH range values of 2-6 with the optimum at 5.5. Studies on thermostability showed that the recombinant phytase remained 70% activity after exposure to 90℃ for 5 min and 65% for 30 min. Fluorescence analyses show that when the enzyme was treated at increasing temperatures, a little fluorescence red-shift was observed (4 nm) with an increase in emission intensity, which indicates that the conformation of the enzyme was very stable during the heating process.
Keywords:Phytase; phyA gene; Pichia pastoris; Overexpression; Thermostability
 
 
 

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