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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (21): 183-188.doi: 10.11924/j.issn.1000-6850.2011-3666

所属专题： 生物技术； 园艺

• 生物技术科学 • 上一篇    下一篇

重组葡萄糖氧化酶毕赤酵母高产菌株的高通量快速筛选方法

周利伟 郜赵伟 张宇宏 张伟   

  • 收稿日期:2011-12-06 修回日期:2012-02-22 出版日期:2012-07-25 发布日期:2012-07-25
  • 基金资助:

    中央级公益性科研院所基本科研业务费

A Rapid and High Throughput Method for Screening Pichia pastoris with Recombinant Glucose Oxidase

  • Received:2011-12-06 Revised:2012-02-22 Online:2012-07-25 Published:2012-07-25

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870

摘要：

根据葡萄糖氧化酶的反应原理和毕赤酵母诱导表达的机制，本研究建立了葡萄糖氧化酶毕赤酵母表达菌株筛选方法。首先在MM平板中诱导重组子表达葡萄糖氧化酶，然后添加显色液，阳性酵母菌株周围可见显色圈。经过与传统液体培养筛选方法的比较验证，结果表明，该方法灵敏度高，稳定性和重复性好，筛选结果与传统方法一致。当葡萄糖氧化酶活力介于0–0.1 U/ml的范围内，显色圈的绝对面积与酶活力相关系数可达0.63。该平板筛选方法可以从大量毕赤酵母重组子中快速筛选出表达葡萄糖氧化酶的阳性菌株，并可方便地判断出各菌株之间表达水平的差异。同时，该方法也可为筛选其他重组酶毕赤酵母菌株提供参考。

关键词: 开花机理, 开花机理

Abstract:

The aim was to establish a convenient and high throughput method for screening Pichia pastoris with high-level recombinant glucose oxidase (GOD). Recombinant P. pastoris clones were cultured on the MM plate and induced by methanol for 2 days, then the plate were covered with color solution. After 30 min reaction at room temperature, measure brown circle area around the colony, then calculate correlation coefficient between the color area and GOD activity. Brown circle appeared clearly when the sample GOD activity higher than 0.0005 U. This new screening method showed high sensitivity, good stability and repeatability, and considerable reliability compared with the traditional screening method. The correlation coefficient between color area and GOD activity reached up to 0.63 when GOD activities lower than 0.1 U/mL. A rapid and high throughput method for screening of P. pastoris with recombinant glucose oxidase was established. At the same time, it may provide a reference for screening of P. pastoris clones with other recombinant enzyme.