气液相等离子体诱变选育安普霉素高产菌株的研究  

Gas-Liquid Phase Plasma Mutagenesis Breeding and High-Yield Apramycin Mutant Strain Screening

作　　者：丁强 向继武 周明 程波 谭小芳 闫雪 沈洁 DING Qiang;XIANG Jiwu;ZHOU Ming;CHENG Bo;TAN Xiaofang;YAN Xue;SHEN Jie(Yichang Sanxia Pharmaceutical Co.,Ltd,Yichang 443000,China;School of Environmental Ecology and Biological Engineering,Wuhan Institute of Technology,Wuhan 430205,China;Yichang Humanwell Pharmaceutical Co.,Ltd,Yichang 443000,China;Hefei Institutes of Physical Science,Chinese Academy of Sciences,Hefei 230000,China)

机构地区：[1]宜昌三峡制药有限公司,湖北宜昌443000 [2]武汉工程大学环境生态与生物工程学院,湖北武汉430205 [3]宜昌人福药业有限责任公司,湖北宜昌443000 [4]中国科学院合肥物质科学研究院,安徽合肥230000

出　　处：《武汉工程大学学报》2023年第5期510-516,共7页Journal of Wuhan Institute of Technology

摘　　要：以宜昌三峡制药有限公司保藏的黑暗链霉菌(Streptomyces tenebrarius)菌株A-0为出发菌种,采用改进后的气液相等离子体诱变法,选育安普霉素稳定高产突变菌株。诱变后挑取单孢子获得生长平板后,采用24孔板快速筛选法初筛后摇瓶复筛,高效液相色谱法进行安普霉素含量检测及成品质量分析,同时结合杯碟法(枯草芽孢杆菌(bacillus subtitles)为指示菌)进行生物效价测定。结果显示:气液相等离子体诱变对安普霉素菌株突变效果较显著,处理75 s后,致死率为78.6%时正突变率达28.9%。孔板初筛得到5株效价增幅5%~30%的突变株,复筛后获得1株安普霉素稳定突变株A-4,其发酵水平达到0.375 mg/mL,生物效价达到3420 U/mL,较出发菌株生物效价提高了32.6%。经5代传代,发酵水平变化低于5%。结果表明:诱变用的活性离子导致细胞DNA损伤和破坏,在其自我修复的过程中形成了不完全修复的突变,最终获得了稳定遗传的突变菌株。The original strain Streptomyces tenebrarius A-0,preserved by Yichang Sanxia Pharmaceutical Co.,Ltd,was subjected to mutation using an improved gas-liquid phase plasma mutagenesis method.The objective was to obtain a mutant strain of Streptomyces tenebrarius A-0 with a high yield of apramycin.After mutation,single spores were picked and cultured on agar plates.A preliminary screening was conducted with a 24-well plate,followed by a secondary screening via shake flask fermentation.The assay of apramycin yield and analysis of finished sample quality were conducted using a high performance liquid chromatography.Additionally,the bioassay test was performed using the cylinder plate method with Bacillus subtilis as the test organism.The results indicated that gas-liquid phase plasma mutagenesis has a positive impact on the apramycin strain,with a mutation rate of 28.9%observed when the lethality rate is 78.6%after 75 s of treatment.Five mutant strains with 5%-30%increase in bioassay activity were identified during the preliminary screening.Following the secondary screening,a stable and high-yield apramycin mutant strain named A-4 was obtained,exhibiting a fermentation level of 0.375 mg/mL and a bioassay activity of 3420 U/mL.This represents a 32.6%increase in bioassay activity compared to the original strain.After 5 generations of passage,the fermentation level of the strain A-4 remains stable with less than 5%change.These findings suggested that the active ions utilized in the mutagenesis process induce DNA damage and cell disruption,resulting in incomplete repair mutations during the self-repair mechanism.Finally,a genetically stable mutant strain was obtained.

关 键 词：安普霉素 黑暗链霉菌 气液相等离子体诱变 筛选 

分 类 号：Q812[生物学—生物工程]