马来西亚链霉菌ECO 00002产Azalomycin F发酵条件优化
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摘要
【目的】通过优化马来西亚链霉菌ECO 00002菌株的发酵培养基和发酵条件,以提高阿扎霉素F产量,为其产业化开发奠定基础。【方法】在摇瓶水平上,采用Plackett-Burman(PB)试验考察葡萄糖、淀粉、酵母膏、大豆粉、氯化钠、磷酸氢二钾等6个因素对阿扎霉素F HPLC效价的影响,并通过Response-Surface-Analysis(RSA)试验确定各因素最大响应值;进一步通过梯度试验,筛选适宜的初始pH、发酵瓶装液量、接种量、发酵温度、发酵时间等发酵条件;最后利用优化的发酵条件在50 L全自动发酵罐中进行放大应用试验,验证优化条件。【结果】发酵培养基中主要的影响因素为大豆粉和葡萄糖,其最大响应值分别为2.90%和1.59%;优化发酵条件为初始pH 7.5,装液量100 mL/500 mL,接种量为10%,发酵温度28℃,发酵时间72 h。【结论】在优化的发酵培养基和发酵条件下,摇瓶水平上阿扎霉素F HPLC效价由原来的637.59μg/mL上升到1950.26μg/mL,HPLC效价提高率为205.88%(P<0.01);50 L发酵罐阿扎霉素F的HPLC效价为2094.12μg/mL,优化效果显著。
【Objective】The fermentation medium and fermentation conditions of Streptomyces malaysiensis ECO 00002 strain were optimized to improve the yield of Azalomycin F and lay the foundation for its industrial development. 【Method】At the shaking flask level, Plackett-Burman(PB) test was used to investigate the effects of six factors including glucose, starch, yeast extract, soy flour, NaCl, and K_2HPO_4 on the yield of Azalomycin F. The maximum response value of each factor was determined by Response Surface Analysis(RSA) test. The suitable initial pH, fermentation liquid volume, inoculum volume, fermentation temperature, fermentation time were screened by gradient test. The optimization conditions were verified in a 50 L automatic fermentation tank by amplification application test. 【Result】The results showed that the main influencing factors in the fermentation medium were soybean flour and glucose, and the maximum response values were 2.90 % and 1.59 %, respectively. The fermentation conditions were optimized as follows: initial pH 7.5, liquid volume 100 mL/500 mL, inoculum volume 10 %, fermentation temperature 28 ℃, fermentation time 72 hours. 【Conclusion】The titer of Azamycin F increased from 637.59 μg/mL to 1950.26 μg/mL under the optimized fermentation medium and fermentation conditions, and the HPLC titer increasing rate was 205.88 %(P<0.01). The titer of Azamycin F was 2094.12 μg/mL in 50 L fermentation tank, and the optimization effect was remarkable.
【Objective】The fermentation medium and fermentation conditions of Streptomyces malaysiensis ECO 00002 strain were optimized to improve the yield of Azalomycin F and lay the foundation for its industrial development. 【Method】At the shaking flask level, Plackett-Burman(PB) test was used to investigate the effects of six factors including glucose, starch, yeast extract, soy flour, NaCl, and K_2HPO_4 on the yield of Azalomycin F. The maximum response value of each factor was determined by Response Surface Analysis(RSA) test. The suitable initial pH, fermentation liquid volume, inoculum volume, fermentation temperature, fermentation time were screened by gradient test. The optimization conditions were verified in a 50 L automatic fermentation tank by amplification application test. 【Result】The results showed that the main influencing factors in the fermentation medium were soybean flour and glucose, and the maximum response values were 2.90 % and 1.59 %, respectively. The fermentation conditions were optimized as follows: initial pH 7.5, liquid volume 100 mL/500 mL, inoculum volume 10 %, fermentation temperature 28 ℃, fermentation time 72 hours. 【Conclusion】The titer of Azamycin F increased from 637.59 μg/mL to 1950.26 μg/mL under the optimized fermentation medium and fermentation conditions, and the HPLC titer increasing rate was 205.88 %(P<0.01). The titer of Azamycin F was 2094.12 μg/mL in 50 L fermentation tank, and the optimization effect was remarkable.
引文
[1]杨佩文,赵江源,李铭刚,等.马来西亚链霉菌ECO 00002产生的阿扎霉素f3、阿扎霉素f4和尼菲霉素:分离纯化·结构解析及其抗植物病源真菌活性[A].全国新农药创制学术交流会[C]. 2009:352-362.
[2]白亭亭,杨群辉,李铭刚,等.植保抗生素阿扎霉素对白菜根肿病的防治效果[J].植物保护, 2018(1):210-214.
[3]Arai M. Azalomycins B and F, two new antibiotics. I. Production and isolation[J]. Journal of Antibiotics, 1960, 13(214):46-50.
[4]Arai M, Hamano K. Isolation of three main components. F3, F4 and F5, from azalomycin F-complex[J]. Journal of Antibiotics, 1970, 23(3):107-12.
[5]G Yuan, P Li, W Pan, et al. The relative configurations of azalomycins F 5a, F 4a, and F 3a[J]. Journal of Molecular Structure, 2013, 1035(10):31-37.
[6]马艳玲,刘富来,张敏,等. 阿扎霉素F产生菌链霉菌211726基因转移系统的建立[J].生物技术通报, 2016, 32(4):198-202.
[7]徐鞾.阿扎霉素F聚酮骨架生物合成途径研究[D]. 武汉:武汉大学, 2017.
[8]刘绍仁,沈佐锐.浅议农药毒性分级[J]. 农药科学与管理, 2004(5):33-36.
[9]王磊,顾学斌,徐文平,等.长川霉素发酵条件的优化[J].农药, 2004, 43(10):445-447.
[10]曹鹏,胡栋,张君,等.基于比较代谢组学的理性优化方法提高阿维菌素产量[J].微生物学报, 2017, 57(2):281-292.
[11]Takesako K, Nakamura T, Obayashi A, et al. Demalonyl derivatives of azalomycin F4 and scopafungin[J]. Journal of Antibiotics, 1986, 39(5):713-716.
[12]Tanaka Y, Omura S. Agroactive Compounds of Microbial Origin[J]. Annual Review of Microbiology, 1993, 47(1):57.
[13]Chandra A, Nair M G. Azalomycin F complex from Streptomyces hygroscopicus, MSU/MN-4-75B[J]. Journal of Antibiotics, 1995, 48(8):896-898.
[14]Mukhopadhyay T, Vijayakumar E K, Nadkarni S R, et al. 2-Demethylazalomycins F4a and FSa, two new anti-fungal metabolites from Actinomycete sp. HILY 9120362[J]. Journal of Antibiotics, 1995, 48(11): 1350-1352.
[15]Namikoshi M, Iwasaki S, Sasaki K, et al. Okuda S. Studies on macrocyclic lactone antibiotics. III. Skeletal structure of azalomycin F4a[J]. Chemical & Pharmaceutical Bulletin, 2008, 30(5):1669-1673.
[16]Iwasaki S, Namikoshi M, Sasaki, et al. Studies on Macrocyclic Lactone Antibiotics. V. The Structures of Azalomycins F3a and F5a[J]. Chemical & Pharmaceutical Bulletin, 2008, 30(11):4006-4014.
[17]Ganjun Yuan, Kui Hong, Haipeng Lin, et al. New Azalomycin F Analogs from Mangrove Streptomyces sp. 211726 with Activity against Microbes and Cancer Cells[J]. Marine Drugs, 2013, 11(3):817-829.
[18]马艳玲.阿扎霉素F_(3a)生物合成相关基因的克隆和功能分析[D]. 武汉:武汉大学, 2014.
[19]严淑玲,黄为一.阿扎霉素B(Azalomycin B)研究进展[J].微生物学通报, 2002, 29(5):103-107.
[20]罗少娥,解修超,曾庆飞,等. 阿扎霉素F5a抗南方根结线虫和香蕉枯萎病菌活性研究[J]. 热带农业科学, 2016, 36(3):34-38.
[21]杨建文. 金霉素发酵过程软测量建模及优化控制策略研究[D].北京:北京理工大学, 2015.
[22]谭之磊,王洪翠,魏彧翘,等. 碳源和氮源对5-酮基-葡萄糖酸生成的影响[J]. 生物工程学报, 2014, 30(1):76-82.
[23]Chen C Y, Ho S H, Liu C C, et al. Enhancing lutein production with Chlorella sorokiniana Mb-1 by optimizing acetate and nitrate concentrations under mixotrophic growth[J]. Journal of the Taiwan Institute of Chemical Engineers, 2017, 79: 88-96.
[2]白亭亭,杨群辉,李铭刚,等.植保抗生素阿扎霉素对白菜根肿病的防治效果[J].植物保护, 2018(1):210-214.
[3]Arai M. Azalomycins B and F, two new antibiotics. I. Production and isolation[J]. Journal of Antibiotics, 1960, 13(214):46-50.
[4]Arai M, Hamano K. Isolation of three main components. F3, F4 and F5, from azalomycin F-complex[J]. Journal of Antibiotics, 1970, 23(3):107-12.
[5]G Yuan, P Li, W Pan, et al. The relative configurations of azalomycins F 5a, F 4a, and F 3a[J]. Journal of Molecular Structure, 2013, 1035(10):31-37.
[6]马艳玲,刘富来,张敏,等. 阿扎霉素F产生菌链霉菌211726基因转移系统的建立[J].生物技术通报, 2016, 32(4):198-202.
[7]徐鞾.阿扎霉素F聚酮骨架生物合成途径研究[D]. 武汉:武汉大学, 2017.
[8]刘绍仁,沈佐锐.浅议农药毒性分级[J]. 农药科学与管理, 2004(5):33-36.
[9]王磊,顾学斌,徐文平,等.长川霉素发酵条件的优化[J].农药, 2004, 43(10):445-447.
[10]曹鹏,胡栋,张君,等.基于比较代谢组学的理性优化方法提高阿维菌素产量[J].微生物学报, 2017, 57(2):281-292.
[11]Takesako K, Nakamura T, Obayashi A, et al. Demalonyl derivatives of azalomycin F4 and scopafungin[J]. Journal of Antibiotics, 1986, 39(5):713-716.
[12]Tanaka Y, Omura S. Agroactive Compounds of Microbial Origin[J]. Annual Review of Microbiology, 1993, 47(1):57.
[13]Chandra A, Nair M G. Azalomycin F complex from Streptomyces hygroscopicus, MSU/MN-4-75B[J]. Journal of Antibiotics, 1995, 48(8):896-898.
[14]Mukhopadhyay T, Vijayakumar E K, Nadkarni S R, et al. 2-Demethylazalomycins F4a and FSa, two new anti-fungal metabolites from Actinomycete sp. HILY 9120362[J]. Journal of Antibiotics, 1995, 48(11): 1350-1352.
[15]Namikoshi M, Iwasaki S, Sasaki K, et al. Okuda S. Studies on macrocyclic lactone antibiotics. III. Skeletal structure of azalomycin F4a[J]. Chemical & Pharmaceutical Bulletin, 2008, 30(5):1669-1673.
[16]Iwasaki S, Namikoshi M, Sasaki, et al. Studies on Macrocyclic Lactone Antibiotics. V. The Structures of Azalomycins F3a and F5a[J]. Chemical & Pharmaceutical Bulletin, 2008, 30(11):4006-4014.
[17]Ganjun Yuan, Kui Hong, Haipeng Lin, et al. New Azalomycin F Analogs from Mangrove Streptomyces sp. 211726 with Activity against Microbes and Cancer Cells[J]. Marine Drugs, 2013, 11(3):817-829.
[18]马艳玲.阿扎霉素F_(3a)生物合成相关基因的克隆和功能分析[D]. 武汉:武汉大学, 2014.
[19]严淑玲,黄为一.阿扎霉素B(Azalomycin B)研究进展[J].微生物学通报, 2002, 29(5):103-107.
[20]罗少娥,解修超,曾庆飞,等. 阿扎霉素F5a抗南方根结线虫和香蕉枯萎病菌活性研究[J]. 热带农业科学, 2016, 36(3):34-38.
[21]杨建文. 金霉素发酵过程软测量建模及优化控制策略研究[D].北京:北京理工大学, 2015.
[22]谭之磊,王洪翠,魏彧翘,等. 碳源和氮源对5-酮基-葡萄糖酸生成的影响[J]. 生物工程学报, 2014, 30(1):76-82.
[23]Chen C Y, Ho S H, Liu C C, et al. Enhancing lutein production with Chlorella sorokiniana Mb-1 by optimizing acetate and nitrate concentrations under mixotrophic growth[J]. Journal of the Taiwan Institute of Chemical Engineers, 2017, 79: 88-96.
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