食品科学 ›› 2020, Vol. 41 ›› Issue (14): 131-137.doi: 10.7506/spkx1002-6630-20190517-191

• 生物工程 • 上一篇    下一篇

ARTP-DES连续诱变选育高产ε-聚赖氨酸突变株

席志文,黄林娜,翟一畅,惠丰立   

  1. (南阳师范学院生命科学与技术学院,河南 南阳 473061)
  • 出版日期:2020-07-25 发布日期:2020-07-29
  • 基金资助:
    国家自然科学基金面上项目(31570021)

Sequential Mutagenesis Treatment with Atmospheric and Room Temperature Plasma and Diethyl Sulfate for Enhanced Microbial Production of ε-Poly-L-lysine

XI Zhiwen, HUANG Linna, ZHAI Yichang, HUI Fengli   

  1. (College of Life Science and Technology, Nanyang Normal University, Nanyang 473061, China)
  • Online:2020-07-25 Published:2020-07-29

摘要: 为提升小白链霉菌(Streptomyces albluus)产ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)能力,在常压室温等离子体(atmospheric and room temperature plasma,ARTP)、硫酸二乙酯(diethyl sulfate,DES)单因子诱变的基础上,采用ARTP-DES连续诱变。连续诱变条件:ARTP工作功率110 W,照射距离2 mm,照射时间为30、60、105 s;DES诱变体积分数0.6%,反应时间20 min。并改进一种快速筛选方法,结合链霉素抗性、亚甲基蓝透明圈及48 微孔板培养,酶标仪快速测定ε-PL含量。结果表明:ARTP-DES连续诱变结合链霉素抗性正突变率可达40.8%,同时,获得1 株遗传性能稳定的链霉素抗性突变株S. albulus AD-9,其ε-PL摇瓶产量为2.1 g/L,是出发菌株的2.1 倍。通过研究高产菌株的生理特性,发现其在营养需求、发酵过程中菌球形态等都发生了变化。ARTP-DES连续诱变选育高产ε-PL菌株是一种高效选育手段。

关键词: 小白链霉菌, ε-聚赖氨酸, 连续诱变, 快速筛选, 生理特性

Abstract: To enhance the production of ε-poly-L-lysine (ε-PL) by Streptomyces albluus, we mutagenized this strain by sequential treatment with atmospheric and room temperature plasma and diethyl sulfate (ARTP-DES). The optimized treatment conditions were determined as follows: ARTP power 110 W, exposure distance 2 mm, mutagenesis time 30, 60, and 105 s, DES concentration 0.6% (V/V), and reaction time 20 min. A rapid screening method was established by streptomycin resistance combined with methylene blue transparent circles. ε-PL content was determined by enzyme-linked immunosorbent assay (ELISA) with a 48-well microplate. The results showed that the positive mutation rate for streptomycin resistance was up to 40.8%. Furthermore, one strain designated AD-9 with good genetic stability was selected. It produced 2.1 g/L ε-PL in shake-flask fermentation, which was 2.1 times as high as that produced by the parent strain. Studies of the physiological characteristics indicated that the mutant strain AD-9 showed changes in nutritional requirements, pellet morphology and nutrient consumption during fermentation. This study indicates that continuous ARTP-DES mutagenesis is an efficient breeding method for increased production of ε-PL.

Key words: Streptomyces albluus, ε-poly-L-lysine, continuous mutation, rapid?screening, physiological properties

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