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| Construction and Expression Analyse of N-linked Glycosylation Site Mutants of phyA2 Gene from Aspergillus niger 963 |
| YIN Liang1,2,YANG Wen-zhu2,WANG Xin-yu1,ZHOU Yun-zhi2,YAO Bin3,CHEN Ru-mei2,FAN Yun-liu2 |
1.College of Life Science, Lanzhou University, Lanzhou 730000, China
2.Biotechnology Research Institute, Chinese Academy of Agicultural Sciences, Beijing 100081，China
3.Feed Research Institute, Chinese Academy of Agicultural Sciences, Beijing 100081, China |
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Abstract To study N-linked glycosylation of the phytase from Aspergillus niger 963 in the significance of its enzymatic properties, we constructed two N-glycosylation mutants by Megaprimer PCR-mediated gene site-directed mutagenesis techniques , Make the codon of N87 and N102-asparagine was replaced by the codon of glutamine. according to the location ,the two mutants were named N87Q、N102Q, sequencing alignment and analysis showed that site-directed mutagenesis was achieved in the nucleic acid level,we also constructed expression vector pPIC9-N87Q, pPIC9-N102Q,The recombinant protein were expressed in Pichia pastoris GS115 by fermentation,The N87Q retained more than 50% of its initial activity after incubation at 60℃ for 1h, while the activity of N102Q was lost completely after ten minutes,The N87Q retained more than 70% of its initial activity after being incubated under varying pH conditions (pH 1.0～10.0) at 37?C for 1h,however,The N102Q was not active above pH 8.0.
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Received: 10 March 2010
Published: 12 June 2010
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