摘要
为解决化学合成法生产间苯三酚环境污染严重的问题,本研究设计2对不同的扩增间苯三酚合成关键基因phlD的引物,对前期分离得到的270余株假单胞菌进行筛选。采用LC-MS法对筛选出的目标菌株进行发酵产物检测,并对菌株进行种属鉴定及系统发育树构建;采用HPLC法测定其间苯三酚的产量,再利用正交试验优化菌株的发酵培养条件。结果显示,引物2在筛选产间苯三酚菌株方面效果较好,经LC-MS检测验证,筛选得到的海洋生境假单胞菌LBF-96发酵产物为间苯三酚,其经生理生化鉴定及16S核糖体RNA(16S rRNA)基因序列分析,确定为芸薹假单胞菌(Pseudomonas brassicacearum)。该菌株在28 ℃、150 r/min条件下发酵培养至72 h时,间苯三酚的产量为0.23 g/L,经正交试验优化发酵培养条件后,菌株在30 ℃、pH 7.0、200 r/min条件下间苯三酚的产量最高,比优化前提高了39%。这是关于芸薹假单胞菌产间苯三酚的首次报道,为间苯三酚的生物合成提供了新的种质资源。
Two pairs of different primers for amplifying the key gene phlD for synthesis of phloroglucinol were designed to screen more than 270 Pseudomonas strains isolated in the earlier stage,which was in order to solve the problem of serious environmental pollution in the production of phloroglucinol by chemical synthesis.The LC-MS method was used to detect the fermentation products of the selected target strains,identify the strains and construct the phylogenetic tree.The HPLC method was used to determine the output of phloroglucinol,and the fermentation conditions of the strain were optimized by orthogonal experiment.The results showed that primer 2 was effective in screening of phloroglucinol producing strains.The LC-MS test verified that the fermentation product of Pseudomonas LBF-96 screened in marine habitat was phloroglucinol,and the strain was identified as Pseudomonas brassicacearum through physiological and biochemical identification and 16S ribosomal RNA(16S rRNA) gene sequence analysis.When the strain was cultured at 28 ℃ and 150 r/min for 72 h,the yield of phloroglucinol was 0.23 g/L.After optimizing the fermentation conditions by the orthogonal experiment,the strain produced the highest amount of phloroglucinol under the conditions of 30 ℃,pH 7.0 and 200 r/min,which increased by 39% compared with that before optimization.The production of phloroglucinol by Pseudomonas brassicacearum was reported for the first time in this paper,which provided a new germplasm resource for the biosynthesis of phloroglucinol.
作者
马湘君
李银塔
王祥法
田黎
MA Xiangjun;LI Yinta;WANG Xiangfa;TIAN Li(Weihai Ocean Vocational College,Rongcheng 264300;Ocean University of China,Qingdao 266000;Disha Pharmacenutical Group,Weihai 264200;First Instiute of Oceanography,MNR,Qingdao 266100)
出处
《中国医药工业杂志》
CAS
CSCD
北大核心
2021年第3期352-358,共7页
Chinese Journal of Pharmaceuticals
基金
国家自然科学基金项目(51804021)
2019年山东省高等学校青创人才引育计划(133)
2018年度山东省高校科研计划项目(J18KB062、J18KB083)。
