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《Biotechnology Bulletin》 2012-01
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Fusion Expression of Glucoamylase and Xylanase in Pichia pastoris

Wang Zhi1 Dun Baoqing1 Gu Jingang3 Zhao Xuan1,2 Tian Gu1,2 Lu Ming1 Li Guiying1(1The National Key Facility for Crop Gene Resources and Genetic Improvement of Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081;2College of Life Sciences,Northeast Agricultural University,Harbin 150030;3Agricultural Culture Collection of China,Institute of Agricultural Resources and Agricultural Divisions,Chinese Academy of Agricultural Sciences,Beijing 100081)  
The mature peptide coding sequence of glucoamylase(amyA) gene was amplified by RT-PCR from Talaromyces emersonii total RNA extracts.The result showed that the mature peptide sequence of amyA was consisted of 1 857 bp,and encoded 618 amino acids.The xylanase(xynA) gene was amplified from Paenibacillus sp.H10-3 total DNA extracts.The result showed that the mature peptide sequence of xynA was consisted of 636 bp,and encoded 211 amino acids.Then the mature peptide sequence of amyA gene,sequence of linker and sequence of xynA gene were spliced by overlap extension PCR(SOE-PCR),by which amyA-l-xynA was obtained.The fusion gene was then inserted into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid pPIC9-amyA-l-xynA.By electric shocks,we successfully transformed the recombinant pPIC9-amyA-l-xynA into Pichia pastoris GS115,and ALX2 was obtained.The maximum yield of the recombinant glucoamylase and xylanase in ALX2 culture medium were 10.7 U/mL and 51.8 U/mL,respectively.
【Fund】: 国际先进农业科学与技术项目(农业部“948”项目)(2011-Z09);; 农业部农村能源综合建设项目(2130138-018);; 中国农业科学院作物科学研究所中央级公益性科研院所基本科研业务费专项资助项目(2060302-13)
【CateGory Index】: Q78
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