质粒pSET152对小单孢菌40027菌株产福堤霉素A的影响
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摘要
小单孢菌(Micromonospora)是一类能产生结构类型多样的抗生素的稀有放线菌。就小单孢菌近年来所产生的生物活性物质的数目,小单孢菌已超过链霉菌而位居首位。因此,小单孢菌属作为寻找新抗生素和其它新生物活性物质的一种新菌源受到越来越多的关注和重视,显示出巨大的研究潜力。
本文以小单孢菌40027原始菌株和小单孢菌40027::pSET152菌株为研究对象,通过比较小单孢菌40027原始菌株和小单孢菌40027::pSET152菌株在发酵过程的生物活性与发酵产物中的生物活性,研究了质粒pSET152所携带的外源基因(acc(3)IV)对小单孢菌40027菌株所产生的次级代谢产物-福堤霉素A的产量、结构、功能之间的影响。
采用超声波细胞破碎法和反复冻融法对小单孢菌40027菌株的菌丝体进行破碎,研究表明超声波细胞破碎法破碎小单孢菌40027菌株的菌丝体效果比反复冻融法好,且经验证小单孢菌40027菌株菌丝体内所产生的少量的抗菌活性物质包含有福堤霉素A,但是胞外所产的生物活性物质要高于胞内。
通过对小单孢菌40027菌株发酵工艺的研究,结果表明二级种子液制备至12h和发酵液制备至42h时,这2个时间分别为小单孢菌40027菌株种子培养期及发酵培养期的最佳时间。
比较小单孢菌40027原始菌株和小单孢菌40027::pSET152菌株在相同培养时期的抑菌活性,研究发现在菌种活化时期、发酵培养时期,小单孢菌40027菌株抑菌活性比小单孢菌40027::pSET 152的抑菌活性强。
对小单孢菌40027菌株的发酵液分离纯化工艺进行研究,结果表明:小单孢菌40027菌株发酵所产福堤霉素A的饱和吸附量为6倍树脂体积,0.5N的氨水洗脱液的用量为3倍树脂体积。提取的粗产物采用TCL检测,确定了氯仿-甲醇-浓氨水为1:2:1是小单孢菌40027菌株发酵产物的粗提物薄层层析的最佳展层系统。
对1mg/mL福堤霉素A标准品进行色谱分析,采用示差折光检测器和蒸发光散射检测器进行检测,表明了蒸发光散射检测器检测福堤霉素A的效果要优于前者的检测效果,且确定蒸发光散射检测器的检测条件为:0.2 mol/L三氟乙酸溶液与甲醇比例为95:5;流速为1.0mL/min;漂移管温度为40℃;进样量为20μL;雾化气体压力为350kPa。在此条件下1mg?mL福堤霉素A标准品保留时间是7.20min。
对小单孢菌40027::pSET152菌株和小单孢菌40027菌株的发酵纯化产物经高效液相色谱分离,确定了小单孢菌40027::pSET152菌株和小单孢菌40027菌株的发酵所产福堤霉素A的保留时间分别为7.31、7.35 min,峰面积所占比例分别为11.9%、12.3%,表明了二者发酵产物中含有基本等量的福堤霉素A。结果暗示了二者之间抑菌活性的差别不是由于二者所产的福堤霉素A的含量不同造成的,而是由于质粒pSET152所包含的acc(3)IV对福堤霉素A的结构有不同程度的钝化,引起福堤霉素A结构变化,从而导致了福堤霉素A抑菌活性的降低。
The Micromonospora which can produce antibiotics with Various types of structures belongs to the rare actinomycetes. In terms of out-put of new antibiotics, Micromonospora is more than Streptomyces for the moment. so, Micromonospora, as Searching for new antibiotics and other bioactive substances people pays more attention to it.It also has an enormous research potential.
The article mainly talked about the Micromonospora sp. 40027 strain and Micromonospora sp. 40027:: pSET152 strain. By comparing of bioactive of their fermentation culture and fermentation product, this paper studied the effects of plasmid pSET 152 with heterologous genes acc(3)IV on production, structure and function of fortimicin A.
Mycelium is disrupted by ultrasonic method and freezing- thawing method. The results show that ultrasonic method is much better for mycelium disruption, and bioactive substances of Micromonospora sp. 40027 strain intracellular contain fortimicin A by TLC detection. The bioactive substances of extracellular is higher than intracellular.
According to the study on the fermentationin broth of Micromonospora sp. 40027 strain, the results show that the 12th hour and 42th hour are the best time of seed culture and fermentation culture for Micromonospora sp 40027 strain.
Bacteriostatic activity of Micromonospora sp. 40027 strain and Micromonospora sp.40027: : pSET 152 strain are compared during active strains and fermentation period. The results show that Micromonospora sp. 40027 strain has higher bacteriostatic activity than Micromonospora sp. 40027: : pSET 152 strain.
Separation and purification of the active component from the fermentation broth of Micromonospora sp.40027 stains were studied too.The results indicated that the maximum absorption capacity of fotimicin A from Micromonospora sp. 40027 stains are 6 times to the volume of ion exchange resin; 0.5N ammonia eluent is 4 times to the volume of ion exchange resin. Theirs extracts are basically determined on the basis of analysis of the Thin Layer Chromatographic (TLC).The most effectly developing solvent is composed of chloroform, methanal and concentrated ammonia(1:2:1).
Using refractive index detector(RID) and evaporative light scattering detector(ELSD) detect standard of fortimicin A for chromatographic analysis.The results show that ELSD is more effective than RID. The condition of ELSD determination: the mobile phase is the mixture of 0.2 mol/L trifluoroacetic acid and methanol(95: 5) at the rate of 1.0mL/min with the temperature of the drift tube 40℃and the injection volume of 20μL, high pure nitrogen atomizing gas pressure of 350 kPa. Under these conditions, 1mg/mL fotimicin A standard retention time is 7.20min.
The extras of fotimicin A of Micromonospora sp. 40027:: pSET152 strain and Micromonospora sp. 40027 strain are detected by HPLC,Their respective retention time is 7.31、7.35 min, the proportion of peak area of fotimicin A are 11.9%、12.3%. It shows that their fermentation product contains the same amount of fotimicin A. The results show the difference between the antibacterial activity is not only produced by fotimicin A content , but also the plasmid pSET152 with acc(3)IV had a passive effect on activities of fotimicin A. so it lead to decrease antibacterial activity.
本文以小单孢菌40027原始菌株和小单孢菌40027::pSET152菌株为研究对象,通过比较小单孢菌40027原始菌株和小单孢菌40027::pSET152菌株在发酵过程的生物活性与发酵产物中的生物活性,研究了质粒pSET152所携带的外源基因(acc(3)IV)对小单孢菌40027菌株所产生的次级代谢产物-福堤霉素A的产量、结构、功能之间的影响。
采用超声波细胞破碎法和反复冻融法对小单孢菌40027菌株的菌丝体进行破碎,研究表明超声波细胞破碎法破碎小单孢菌40027菌株的菌丝体效果比反复冻融法好,且经验证小单孢菌40027菌株菌丝体内所产生的少量的抗菌活性物质包含有福堤霉素A,但是胞外所产的生物活性物质要高于胞内。
通过对小单孢菌40027菌株发酵工艺的研究,结果表明二级种子液制备至12h和发酵液制备至42h时,这2个时间分别为小单孢菌40027菌株种子培养期及发酵培养期的最佳时间。
比较小单孢菌40027原始菌株和小单孢菌40027::pSET152菌株在相同培养时期的抑菌活性,研究发现在菌种活化时期、发酵培养时期,小单孢菌40027菌株抑菌活性比小单孢菌40027::pSET 152的抑菌活性强。
对小单孢菌40027菌株的发酵液分离纯化工艺进行研究,结果表明:小单孢菌40027菌株发酵所产福堤霉素A的饱和吸附量为6倍树脂体积,0.5N的氨水洗脱液的用量为3倍树脂体积。提取的粗产物采用TCL检测,确定了氯仿-甲醇-浓氨水为1:2:1是小单孢菌40027菌株发酵产物的粗提物薄层层析的最佳展层系统。
对1mg/mL福堤霉素A标准品进行色谱分析,采用示差折光检测器和蒸发光散射检测器进行检测,表明了蒸发光散射检测器检测福堤霉素A的效果要优于前者的检测效果,且确定蒸发光散射检测器的检测条件为:0.2 mol/L三氟乙酸溶液与甲醇比例为95:5;流速为1.0mL/min;漂移管温度为40℃;进样量为20μL;雾化气体压力为350kPa。在此条件下1mg?mL福堤霉素A标准品保留时间是7.20min。
对小单孢菌40027::pSET152菌株和小单孢菌40027菌株的发酵纯化产物经高效液相色谱分离,确定了小单孢菌40027::pSET152菌株和小单孢菌40027菌株的发酵所产福堤霉素A的保留时间分别为7.31、7.35 min,峰面积所占比例分别为11.9%、12.3%,表明了二者发酵产物中含有基本等量的福堤霉素A。结果暗示了二者之间抑菌活性的差别不是由于二者所产的福堤霉素A的含量不同造成的,而是由于质粒pSET152所包含的acc(3)IV对福堤霉素A的结构有不同程度的钝化,引起福堤霉素A结构变化,从而导致了福堤霉素A抑菌活性的降低。
The Micromonospora which can produce antibiotics with Various types of structures belongs to the rare actinomycetes. In terms of out-put of new antibiotics, Micromonospora is more than Streptomyces for the moment. so, Micromonospora, as Searching for new antibiotics and other bioactive substances people pays more attention to it.It also has an enormous research potential.
The article mainly talked about the Micromonospora sp. 40027 strain and Micromonospora sp. 40027:: pSET152 strain. By comparing of bioactive of their fermentation culture and fermentation product, this paper studied the effects of plasmid pSET 152 with heterologous genes acc(3)IV on production, structure and function of fortimicin A.
Mycelium is disrupted by ultrasonic method and freezing- thawing method. The results show that ultrasonic method is much better for mycelium disruption, and bioactive substances of Micromonospora sp. 40027 strain intracellular contain fortimicin A by TLC detection. The bioactive substances of extracellular is higher than intracellular.
According to the study on the fermentationin broth of Micromonospora sp. 40027 strain, the results show that the 12th hour and 42th hour are the best time of seed culture and fermentation culture for Micromonospora sp 40027 strain.
Bacteriostatic activity of Micromonospora sp. 40027 strain and Micromonospora sp.40027: : pSET 152 strain are compared during active strains and fermentation period. The results show that Micromonospora sp. 40027 strain has higher bacteriostatic activity than Micromonospora sp. 40027: : pSET 152 strain.
Separation and purification of the active component from the fermentation broth of Micromonospora sp.40027 stains were studied too.The results indicated that the maximum absorption capacity of fotimicin A from Micromonospora sp. 40027 stains are 6 times to the volume of ion exchange resin; 0.5N ammonia eluent is 4 times to the volume of ion exchange resin. Theirs extracts are basically determined on the basis of analysis of the Thin Layer Chromatographic (TLC).The most effectly developing solvent is composed of chloroform, methanal and concentrated ammonia(1:2:1).
Using refractive index detector(RID) and evaporative light scattering detector(ELSD) detect standard of fortimicin A for chromatographic analysis.The results show that ELSD is more effective than RID. The condition of ELSD determination: the mobile phase is the mixture of 0.2 mol/L trifluoroacetic acid and methanol(95: 5) at the rate of 1.0mL/min with the temperature of the drift tube 40℃and the injection volume of 20μL, high pure nitrogen atomizing gas pressure of 350 kPa. Under these conditions, 1mg/mL fotimicin A standard retention time is 7.20min.
The extras of fotimicin A of Micromonospora sp. 40027:: pSET152 strain and Micromonospora sp. 40027 strain are detected by HPLC,Their respective retention time is 7.31、7.35 min, the proportion of peak area of fotimicin A are 11.9%、12.3%. It shows that their fermentation product contains the same amount of fotimicin A. The results show the difference between the antibacterial activity is not only produced by fotimicin A content , but also the plasmid pSET152 with acc(3)IV had a passive effect on activities of fotimicin A. so it lead to decrease antibacterial activity.
引文
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