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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (7): 22-31    DOI: 10.13523/j.cb.2103050
研究报告     
盐增强培养对弗氏链霉菌产新霉素的影响
王珊1,薛正莲1,2,3,*(),孙俊峰1,王芳1,周健1,刘艳1,2,3,王洲1,2,3
1 安徽工程大学生物与化学工程学院 芜湖 241000
2 微生物发酵安徽省工程研究中心 芜湖 241000
3 安徽省工业微生物分子育种工程实验室 芜湖 241000
Effect of Salt-enhanced Culture on the Production of Neomycin by Streptomyces fradiae
WANG Shan1,XUE Zheng-lian1,2,3,*(),SUN Jun-feng1,WANG Fang1,ZHOU Jian1,LIU Yan1,2,3,WANG Zhou1,2,3
1 College of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu 241000, China
2 Anhui Engineering Technology Research Center of Microbial Fermentation, Wuhu 241000, China
3 Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, Wuhu　241000, China
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摘要：

目的: 弗氏链霉菌(Streptomyces fradiae)作为氨基糖苷类抗生素新霉素的主要生产菌株,其新霉素B具有抗菌活性强、抗癌、抗HIV等作用,提高新霉素B的效价具有重要意义。方法: 在满足微生物正常生长所需盐离子的条件下,通过盐增强培养的方式向培养基中添加不同种类、浓度无机盐来改变细胞壁附近的理化特性、渗透压以及培养基中的碳氮比。结果: 不同无机盐对新霉素B效价影响程度由高到低为(NH4)2SO4、NaCl、KCl、K2SO4;当添加60 mmol/L(NH4)2SO4时新霉素B的效价达到最高为15 864 U/mL,而NaCl在80 mmol/L浓度时产量最高为7 429.7 U/mL,相比未添加无机盐分别提高3.8倍和0.82倍。在含有80 mmol/L的NaCl的发酵培养基中添加不同浓度的(NH4)2SO4并定时取样检测其中氨基氮、还原糖、pH、TG、菌浓以及新霉素B效价的变化,发现60 mmol/L(NH4)2SO4浓度下氨基氮、还原糖、TG的消耗速率加快,菌体比生长速率μ最大为0.097/h,新霉素B的合成加快且产量提高为17 399 U/mL,同时菌丝呈现出聚集变短的形态且产孢提前。结论: 盐增强培养方式既可以作为一种形态学工程手段,通过改变弗氏链霉菌的微观形态来促进次级代谢产物新霉素B产量的提高,同时也可以作为培养基优化策略来提高新霉素B的产量,这为进一步提高弗氏链霉菌中次级代谢产物产量的研究奠定了基础。
关键词： 弗氏链霉菌;  盐增强培养;  新霉素    
Abstract:

Objective: Streptomyces fradiae is the main production strain of the aminoglycoside antibiotic neomycin. Its neomycin B has strong antibacterial activity, anti-cancer and anti-HIV effects, and it is of great significance to increase the titer of neomycin B.Methods: We added different kinds and concentrations of inorganic salts to the medium to change the physical and chemical properties near the cell wall, osmotic pressure and C/N ratio of the medium under the condition of satisfying the salt ions required for the normal growth of microorganisms.Results: The degree of influence of different inorganic salts on the titer of neomycin B from high to low is (NH4)2SO4, NaCl, KCl and K2SO4 from high to low, and the titer of neomycin B reached the highest value of 15 864 U/mL when 60 mmol/L (NH4)2SO4 was added, the maximum yield of NaCl at 80 mmol/L concentration was 7 429.7 U/mL, which was 3.8 times and 0.82 times higher than that without adding inorganic salts. Then we added different concentrations of (NH4)2SO4 to the culture medium containing 80 mmol/L NaCl and sampled them regularly to detect the changes of amino nitrogen, reducing sugar, pH, TG, bacterial concentration and neomycin B titer, it was found that the consumption rate of amino nitrogen, reducing sugar and TG was increased at the concentration of 60 mmol/L (NH4)2SO4, the maximum specific growth rate of mycelium was 0.097 /h, the synthesis of neomycin B was accelerated and the yield was increased to 17 399 U/mL, at the same time, the mycelium was shortened and the sporulation was advanced.Conclusion: The salt-enhanced culture could be used as a morphological engineering method to improve the yield of neomycin B by changing the microscopic morphology of Streptomyces fradiae. It can also be used as a medium optimization strategy to increase the yield of neomycin B, which laid the foundation for further studies on increasing the yield of Streptomyces secondary metabolite.
Key words: Streptomyces fradiae    Salt enhanced culture    Neomycin
收稿日期: 2021-03-21 出版日期: 2021-08-03
ZTFLH:  Q819  
通讯作者: 薛正莲     E-mail: xuezl@ahpu.edu.cn
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引用本文:

王珊,薛正莲,孙俊峰,王芳,周健,刘艳,王洲. 盐增强培养对弗氏链霉菌产新霉素的影响[J]. 中国生物工程杂志, 2021, 41(7): 22-31.

WANG Shan,XUE Zheng-lian,SUN Jun-feng,WANG Fang,ZHOU Jian,LIU Yan,WANG Zhou. Effect of Salt-enhanced Culture on the Production of Neomycin by Streptomyces fradiae. China Biotechnology, 2021, 41(7): 22-31.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2103050    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I7/22

图1  不同浓度无机盐下新霉素B的效价
图2  不同(NH4)2SO4浓度下的比生长速率和新霉素B效价
图3  不同(NH4)2SO4浓度下的pH
图4  不同(NH4)2SO4浓度下的TG、氨基氮和残糖
图5  60 mmol/L (NH4)2SO4添加下的发酵过程参数
图6  无(NH4)2SO4添加下的发酵过程参数
发酵过程参数 (NH4)2SO4浓度
0 mmol/L 60 mmol/L
最大比生长率(1/h) 0.06 0.097
新霉素B效价(U/mL) 7 429.7 17 399
TG含量(mmol/gprot) 1.13 0.335
残糖含量(g/L) 14.01 7.33
氨基氮含量(mg/100mL) 66.223 74.219
最大新霉素B效价增长率[U/(mL·h)] 70.743 259.09
最大还原糖消耗速率[g /(L·h)] 0.789 1.228
最大TG消耗速率[mmol/(gprot·h)] 0.021 0.066
表1  两种(NH4)2SO4浓度下的发酵过程参数
图7  零添加和60 mmol/L(NH4)2SO4添加下的弗氏链霉菌在不同发酵时间的菌丝形态
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