黑曲霉中葡萄糖氧化酶基因的克隆及其在毕赤酵母中的表达

Cloning and Heterologous Expression of Glucose Oxidase Gene from Aspergillus niger PCTC in Pichia pastoris

DOI：10.3969/j.issn.1673-1689.2013.06.010

中文关键词: 葡萄糖氧化酶 黑曲霉 异源表达 甲醇毕赤酵母

英文关键词: glucose oxidase Aspergillus niger heterologous expression Pichia pastoris

基金项目:国家自然科学基金项目(21176105);中央高校基本科研业务费专项资金项目(JUSRP111A24)

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中文摘要:

为了提高葡萄糖氧化酶的生产能力,提取和纯化了黑曲霉Aspergillus niger PCTC的基因组DNA,以此为模板进行PCR扩增获得葡萄糖氧化酶基因,经测序,所得基因全长1 772 bp,编码含有589个氨基酸的蛋白质。将目的基因和表达载体pPIC9K连接经电转化导入毕赤酵母GS115中,在甲醇诱导和α信号肽的转运下,将葡萄糖氧化酶分泌到胞外。经G418梯度抗性平板和显色平板的初筛以及摇床复筛,获得了一株产葡萄糖氧化酶活力较高的菌株,该株菌在30℃、180 r/min的培养条件下,经0.5%的甲醇诱导发酵4 d可获得0.342 U/mL的酶活。对该菌株进行了摇瓶产酶条件优化,其最佳发酵条件为:200 r/min、pH 5、接种体积分数50%、25℃下经1.5%甲醇诱导7 d,酶活达到25 U/mL。实验结果表明:葡萄糖氧化酶基因已经成功转入毕赤酵母,并获得相当的产量。

英文摘要:

The aim of this study was to improve the production capacity of glucose oxidase.A gene of glucose oxidase(GOD) from Aspergillus niger TCPC was cloned and sequenced.The whole open reading frame(ORF) consisted of 1 772 bp bases and encoded a putative protein of 589 amino acids.The gene was fused to the pPIC9k plasmid and expressed in Pichia pastoris GS115.The recombinant GOD(rGOD) was secreted into the culture under the control of methanol and α factor signal peptide.After screening by G418 gradient resistance plate,color plate and shaker culture,one strain named GOD2-14 was obtained,which gave the higest enzyme activity(0.342 U/mL) after 4day induction by methanol.Shake flask experiments were conducted to optimize the culture conditions for the enhanced production of GOD.Highest enzyme activity of 25 U/mL was achieved in the shake flask by induction for 7 days under the optimal conditions(200 r/min,pH 5,22 ℃,inoculation amount of 50% and 1.5% methanol).The result showed that GOD gene has been successfully transferred and expressed in the Pichia pastoris GS115。

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