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  • ISSN 1006-3080
  • CN 31-1691/TQ

米曲霉产米黑根毛霉脂肪酶高通量筛选方法的建立

熊志岳 许逗 郭元昕 田锡炜 储炬

熊志岳, 许逗, 郭元昕, 田锡炜, 储炬. 米曲霉产米黑根毛霉脂肪酶高通量筛选方法的建立[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20210415002
引用本文: 熊志岳, 许逗, 郭元昕, 田锡炜, 储炬. 米曲霉产米黑根毛霉脂肪酶高通量筛选方法的建立[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20210415002
XIONG Zhiyue, XU Dou, GUO Yuanxin, TIAN Xiwei, CHU Ju. Establishment of a High-Throughput Screening Method for Rhizomucor miehei Lipase Production by A. oryzae[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20210415002
Citation: XIONG Zhiyue, XU Dou, GUO Yuanxin, TIAN Xiwei, CHU Ju. Establishment of a High-Throughput Screening Method for Rhizomucor miehei Lipase Production by A. oryzae[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20210415002

米曲霉产米黑根毛霉脂肪酶高通量筛选方法的建立

doi: 10.14135/j.cnki.1006-3080.20210415002
基金项目: 国家重点研发计划(2019YFA0904803)
详细信息
    作者简介:

    熊志岳(1996—),男,湖北荆门人,硕士生,主要研究方向为微生物育种。E-mail:908831275@qq.com

    通讯作者:

    田锡炜,E-mail:xiweitian@ecust.edu.cn

    储炬,E-mail:juchu@ecust.edu.cn

  • 中图分类号: TS201.3

Establishment of a High-Throughput Screening Method for Rhizomucor miehei Lipase Production by A. oryzae

  • 摘要: 为了快速、高效筛选出高产米黑根毛霉脂肪酶(RML)的米曲霉菌株,本研究利用常温常压等离子体技术对米曲霉孢子进行诱变,并且优化了高通量检测RML酶活的双指示剂法,建立了24孔板米曲霉高通量培养体系。通过对诱变后的1200孔(36000~48000个孢子)进行高通量筛选,最终筛选出4株米曲霉高产突变株。经由摇瓶验证,突变株Ⅰ7-D6-7,Ⅱ4-4B-5,Ⅲ3-5C-1,Ⅴ7-6C-3酶活分别达到了265.98、240.90、253.52、293.50 U/mL,比出发菌株(176.52 U/mL)分别提高了50.68%,36.47%,43.61%,66.27%。

     

  • 图  1  孔板中不同孔吸光度随预热时间的变化

    Figure  1.  Variation of absorbance of different wells in plate with preheating time

    图  2  孔板中不同孔初始吸光度的差异

    Figure  2.  Difference of initial absorbance of different wells in plate

    图  3  不同计算方式下标准曲线R2的差异((a)是利用反应15 min后的吸光度绘制的标准曲线,(b)是利用预热5 min后与反应15 min后的吸光度差值绘制的标准曲线)

    Figure  3.  Difference of standard curve’s R2 value under different calculation methods ((a) is the standard curve drawn by absorbance after reaction for 15 min, and (b) is the standard curve drawn by absorbance difference between preheating 5 min and reaction 15 min)

    图  4  不同培养条件下米曲霉24孔孔板生长情况对比

    Figure  4.  Comparison of the growth of 24-well plate of A. oryzae under different culture conditions

    图  5  米曲霉的ARTP致死率曲线

    Figure  5.  Lethality curve of A. oryzae by ARTP

    图  6  部分突变株初步筛选结果(红线为出发菌株酶活)

    Figure  6.  Preliminary screening results of some mutant strains(Red line is the lipase activity of the original strain)

    图  7  第2轮复筛结果

    Figure  7.  Results of the second round of re-screening

    图  8  突变株摇瓶验证

    Figure  8.  Shaking flask verification of mutant strains

    表  1  双指示剂法与p-NPP法测量一致性的比较

    Table  1.   Comparison of double indicator method and p-NPP method on measuring consistency

    StrainEnzyme activity/ (U·mL−1)
    p-NPP methodDouble indicator method
    Ⅲ3-5C-131.58±7.3646.88±7.49
    Ⅰ7-D6--729.36±0.9942.08±10.89
    Ⅱ4-4B-535.10±3.2554.03±5.54
    V7-6C-330.04±2.2540.63±6.37
    Original strain23.81±1.7837.47±5.02
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出版历程
  • 收稿日期:  2021-04-15
  • 网络出版日期:  2021-06-29

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