紫外分光光度法快速测定庆大霉素C_(1a)含量在高通量筛选中的应用
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摘要
目的探索庆大霉素C_(1a)含量的快速检测方法,实现诱变菌株的高通量筛选。方法以紫外分光光度法作为检测庆大霉素C_(1a)含量的手段,对庆大霉素C_(1a)单组分生产菌株进行ARTP、LiCl、微波和ARTP+LiCl诱变,通过高通量筛选获得高产菌株,并在5L发酵罐中对高产稳定性菌株进行验证。结果紫外分光光度法和高效液相色谱法对发酵液中庆大霉素C_(1a)含量进行测定,最大相对误差为3.98%;筛选获得了10株高产菌株,其中AL324菌株与出发菌株相比,摇瓶效价提高了52%;通过稳定性传代实验,获得了4株高产稳定性菌株。5L发酵罐验证实验表明AL324与出发菌株相比效价提高了81.3%。结论紫外分光光度法可以快速检测发酵液中庆大霉素C_(1a)含量,该方法应用于高通量筛选中,获得了高产菌株,筛选结果表明ARTP+LiCl复合诱变方法的正突变率较高,是一种有效的庆大霉素C_(1a)高产菌株诱变方式。
Objective To explore the rapid detection method for the content of gentamicin C_(1a) during high throughput screening. Methods In this paper, a rapid and simple detection method for GM C_(1a) in the broth was established and integrated into the high throughput screening model. Four different mutation methods of atmospheric room temperature plasma(ARTP), microwave, LiCl, and ARTP+LiCl with above mentioned high throughput cultivating and detecting systems were used. Furthermore, the high yield strain AL324 was tested in the 5 L fermentation. Results Comparing to the results of high performance liquid chromatography(HPLC) detection, a maximum deviation of 3.98% was found in this rapid detection method. Among them, a mutant of AL324 could enhance the titer by 52% in the shake flask, compared to the parent strain. After the following rescreening and genetic stability verification processes, only 4 mutants still had better fermentation performances. Moreover, the titer improvement further reached to 81.3% in the 5 L fermentation. Conclusion This rapid detection method can be used to detect the GM C_(1a) titer and integrated into the high throughput screening model. The screening results illuminate the ARTP+LiCl is an effective method for producing high yield strains of GM C_(1a).
Objective To explore the rapid detection method for the content of gentamicin C_(1a) during high throughput screening. Methods In this paper, a rapid and simple detection method for GM C_(1a) in the broth was established and integrated into the high throughput screening model. Four different mutation methods of atmospheric room temperature plasma(ARTP), microwave, LiCl, and ARTP+LiCl with above mentioned high throughput cultivating and detecting systems were used. Furthermore, the high yield strain AL324 was tested in the 5 L fermentation. Results Comparing to the results of high performance liquid chromatography(HPLC) detection, a maximum deviation of 3.98% was found in this rapid detection method. Among them, a mutant of AL324 could enhance the titer by 52% in the shake flask, compared to the parent strain. After the following rescreening and genetic stability verification processes, only 4 mutants still had better fermentation performances. Moreover, the titer improvement further reached to 81.3% in the 5 L fermentation. Conclusion This rapid detection method can be used to detect the GM C_(1a) titer and integrated into the high throughput screening model. The screening results illuminate the ARTP+LiCl is an effective method for producing high yield strains of GM C_(1a).
引文
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[16]范新蕾,肖成建,顾秋亚,等.ARTP诱变选育葡萄糖氧化酶高产菌株及发酵条件优化[J].工业微生物,2015,45(1):15-19.
[17]陈红,王春魁,郭娜,等.微波诱变法选育四环类抗生素菌株的探索和应用[J].中国抗生素杂志,2008,33(11):706-707.
[18]刘本发,吴兆亮,郝冬霞,等.紫外分光光度法快速测定庆大霉素发酵液中庆大霉素浓度及其机理[J].中国抗生素杂志,2002,27(3):166-169.
[19]王风芹,原欢,谢慧,等.丁醇高产菌株诱变育种及发酵条件优化研究[J].中国酿造,2011,30(5):84-86.
[20]郑佳.粟酒裂殖酵母编码Trna3'末端加工酶基因trzl回复突变基因的研究[D].南京:南京师范大学,2012.
[2]丁碧粉,张韬,李继安,等.庆大霉素C1a生产过程中不同样品的HPLC法检测[J].中国医药工业杂志,2016,47(3):310-315.
[3]李家泰,刘健,张华,等.新氨基糖苷类抗生素89-07体外抗菌作用研究[J].中国抗生素杂志,1995,20(6):407-411.
[4]张淑华,欧珍蓉,赵敏,等.抗生素89-07的体内抗菌作用[J].中国抗生素杂志,1995,20(6):420-424.
[5]李兴启,方耀云,孙建和,等.抗生素89-07、庆大霉素和阿米卡星的耳毒性比较[J].中国抗生素杂志,1995,20(6):442-447.
[6]李培忠,郑得琪,沈加弟,等.抗生素89-07多次给予大鼠的毒性[J].中国抗生素杂志,1995,20(6):459-463.
[7]Hermann R,Lehmann M,Buchs J.Characterization of gasliquid mass transfer phenomena in microtiter plates[J].Biotechnol Bioeng,2003,81(2):178-186.
[8]Suchland R J,Stamm W E.Simplified microtiter cell culture method of rapid immunotyping of Chlamydia trachomatis[J].J Clin Microbiol,1991,29(7):1333-1338.
[9]Anaissie E,Paetznick V,Bodey G.P.Fluconazole susceptibility testing of candida albicans:Microtiter method that is independent of inoculum size,temperature,and time of reading[J].Antimicrob Agents Chemother,1999,35(8):1641-1646.
[10]Anandi M,Mirtha C,Portaels F,et al.Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs:rapid,simple,and inexpensive method[J].Antimicrob Agents Chemother,2003,47(11):3616-3619.
[11]国家药典委员会编.中华人民共和国药典[S].北京:中国医药科技出版社.二部.2015:1325-1326.
[12]侯英敏.等离子体诱变生产1,3-丙二醇菌种的研究[D].大连:大连理工大学,2006.
[13]郑明英,蔡友华,陆最青,等.常压室温等离子体快速诱变筛选高脯氨酸产率突变株[J].食品与发酵工业,2013,39(1):36-40.
[14]温利雪,张翼,穆军,等.产孢海洋真菌等离子体诱变活性突变株筛选改进[J].大连交通大学学报,2014,35(4):87-93.
[15]李永泉,翁醒华.微波诱变结合化学诱变选育酸性蛋白酶高产菌[J].微生物学报,1999,39(2):181-184.
[16]范新蕾,肖成建,顾秋亚,等.ARTP诱变选育葡萄糖氧化酶高产菌株及发酵条件优化[J].工业微生物,2015,45(1):15-19.
[17]陈红,王春魁,郭娜,等.微波诱变法选育四环类抗生素菌株的探索和应用[J].中国抗生素杂志,2008,33(11):706-707.
[18]刘本发,吴兆亮,郝冬霞,等.紫外分光光度法快速测定庆大霉素发酵液中庆大霉素浓度及其机理[J].中国抗生素杂志,2002,27(3):166-169.
[19]王风芹,原欢,谢慧,等.丁醇高产菌株诱变育种及发酵条件优化研究[J].中国酿造,2011,30(5):84-86.
[20]郑佳.粟酒裂殖酵母编码Trna3'末端加工酶基因trzl回复突变基因的研究[D].南京:南京师范大学,2012.
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