食品与发酵工业 >

2023 , Vol. 49 >Issue 20: 73 - 80

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.034079

研究报告

游动放线菌产阿卡波糖高通量筛选模型的建立及优化

  • 周健 ,
  • 李翔飞 ,
  • 李闯 ,
  • 刘坤 ,
  • 赵世光 ,
  • 薛正莲
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  • 1(安徽工程大学 生物与食品工程学院,安徽 芜湖,241000)
    2(安徽省工业微生物分子育种工程实验室,安徽 芜湖,241000)
第一作者：硕士研究生(薛正莲教授为通信作者,E-mail:xuezl@ahpu.edu.cn)

收稿日期: 2022-10-24

  修回日期: 2022-12-12

  网络出版日期: 2023-11-20

基金资助

国家自然科学基金项目(31471615;31871781)

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Establishment and optimization of high-throughput screening model for acarbose production by Actinoplanes sp. SE50

  • ZHOU Jian ,
  • LI Xiangfei ,
  • LI Chuang ,
  • LIU Kun ,
  • ZHAO Shiguang ,
  • XUE Zhenglian
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  • 1(College of Biological and Food Engineering, Anhui Polytechnic University, Wuhu 241000, China)
    2(Anhui Engineering Laboratory for Industrial Microbiology Molecular Beeding, Wuhu 241000, China)

Received date: 2022-10-24

  Revised date: 2022-12-12

  Online published: 2023-11-20

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摘要

Actinoplanes sp. SE50是Ⅱ型糖尿病治疗药物阿卡波糖的主要生产菌株,但是目前阿卡波糖高效检测方法仍存在瓶颈问题。该文采用酶标仪检测技术和孔板发酵构建阿卡波糖高通量检测模型,显著增强阿卡波糖高产菌株的筛选效率。首先,酶标仪检测结合孔板发酵结果表明,24孔板发酵与摇瓶发酵的相关性(R2=0.881 61)优于48孔板发酵(R2=0.833 74)。24孔板发酵实验结果表明,孔板发酵存在边缘效应即阿卡波糖产量中间孔1区＜长边缘孔2区＜短边缘孔3区,对边缘孔填充无菌水可有效消除边缘效应。随后,为减少种子液培养工序,通过正交优化培养基中黄豆饼粉、甘油、碳酸钙的添加量,得出黄豆饼粉3%,甘油1.5%,碳酸钙0.4%(均为质量分数)配比最佳。最后,在最优条件下孔板发酵168 h,阿卡波糖产量可达到(2 183.66±6.60) μg/mL。该文介绍了一种孔板发酵结合酶标仪检测筛选方法,大幅缩短了摇瓶发酵液相检测所需工作量及时间,为快速筛选阿卡波糖高产菌株奠定了基础。

关键词： 阿卡波糖; Actinoplanes sp.SE50; 高通量; 孔板发酵; 发酵优化

本文引用格式

周健 , 李翔飞 , 李闯 , 刘坤 , 赵世光 , 薛正莲 . 游动放线菌产阿卡波糖高通量筛选模型的建立及优化[J]. 食品与发酵工业, 2023 , 49(20) : 73 -80 . DOI: 10.13995/j.cnki.11-1802/ts.034079

Abstract

Actinoplanes sp. SE50 is the main producing strain of acarbose, a therapeutic drug for type 2 diabetes mellitus. But there are still bottlenecks in the efficient detection of acarbose. In this study, the high-throughput detection model of acarbose was constructed using enzyme detection technology and culture in microwell plate, so as to significantly enhance the screening efficiency of acarbose-producing strains. The results of combined orifice plate fermentation detected by enzyme labeling instrument showed that the correlation between 24-well plate fermentation (R2=0.881 61) and shake flask fermentation was better than that of 48-well plate fermentation (R2=0.833 74). The results of 24-well plate fermentation showed that there was edge effect in 24-well plate fermentation, that was, acarbose yield middle well 1 < long edge well 2 < short edge well 3, which could effectively eliminate the edge effect by filling the edge well with sterile water. Then, to further reduce the process of seed liquid culture, through the orthogonal optimization of the addition amount of soybean cake powder, glycerol and calcium carbonate in the culture medium, the best ratio of 3% soybean cake powder, 1.5% glycerol and 0.4% CaCO3 was obtained. Finally, under the optimal conditions, the yield of acarbose could reach (2 183.66 ±6.60) μg/mL after 168 h of plate fermentation. This study introduces a method of detection and screening of microwell plate culture combined with microplate reader, which greatly shortens the workload and time needed for liquid phase detection of shake flask fermentation, and lays a foundation for screening acarbose-producing strains.

Key words： acarbose; Actinoplanes sp. SE50; high-throughput screening; culture in microwell plate; fermentation optimization

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