庆大霉素高产菌的选育及其发酵条件优化研究
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摘要
采用实验室保藏的产庆大霉素的棘孢小单胞菌进行发酵试验,首先对所得酵液进行了分析方法方面的研究,主要针对TLC检测体系、HPLC以及离交纯化方法进行了研究。通过较全面的优化分析得到最佳TLC检测体系为:将含羧甲基纤维素钠0.2%,磷酸二氢钠5%,和乙二胺四醋酸二钠0.1%的溶液,与硅胶粉按照3:1的比例混合研磨0.5 h后铺板,采用氯仿:甲醇:氨水(25%)=5:4:3,静置分层,上相饱和,下相展开,碘蒸气显色,检测限为1.0μg。将发酵液简单浓缩后在该体系下检测,可检测到发酵液中的10个左右的组分,为微量组分的研究奠定基础。另外,该体系下检测到的GM C斑点规整,根据直径和颜色可以快速估算出GM C的组分含量情况,与HPLC检测结果进行比较发现两种方法存在较好的一致性;在HPLC检测体系对比分析中,结果表明采用甲醇:水:甲酸=74:21:5配制庚烷磺酸钠溶液(4.0 g/L)为流动相效果更优;采用大孔弱酸DK110型树脂纯化GM,洗脱峰更集中,较732型树脂更便于发酵液的纯化和浓缩操作。
为得到优良的庆大霉素(GM)高产菌,以Me07为出发菌,经UV、硫酸二乙酯(DES)和微波诱变处理,以自身代谢终产物为筛子,采取抗性平板以及摇瓶动态选育两种方式,结合TLC及HPLC检测进行优良突变菌株的筛选。筛选得到的产C1组分比例较高的优良突变菌株Me07-1D15,生物效价为1400 U/mL,较出发菌株(680 U/ml)提高了2倍,其发酵液中C1含量最终检测达到47.59%,并且具有良好的遗传稳定性。
通过Plackett-Burman实验设计筛选得到影响发酵水平的显著因子为可溶性淀粉、豆饼粉、CoCl2、CaCO3。通过单因素分析及正交分析得到显著因子最佳浓度为:淀粉60 g/L,豆饼粉30 g/L,氯化钴0.008 g/L, CaCO3 4 g/L。对主要发酵条件进行优化研究,得出最佳发酵条件为:种龄选择2 d,接种量为12 mL,发酵培养基初始pH为7.2,最佳装液量为40 mL(500 mL摇瓶),培养温度34℃,摇瓶转速为250 r/min,培养周期8 d,最佳条件下发酵水平达到1824 U/mL。
过程控制研究从降低基质浓度和非营养刺激两方面考察,结果表明适当减少培养基的营养成分可以使产抗期提前;在发酵96h时进行水浴45℃处理可以使GM产量达到1685 U/mL;在144小时添加0.6%的氯化钠溶液,GM产量达到1715 U/mL,是对照试验的1.20倍;在96 h添加4%乙醇,庆大霉素的产量达到1696 U/ml,而当乙醇浓度达到5～6%时,对庆大霉素的合成有副作用,乙醇的添加抑制了棘孢小单孢菌的生长。
By perfecting the operating condition of TLC, the optimum detecting system was obtained, and the detection limit and reproducibility were studied. Based on the optimum system, we successfully separated the main components (C1,C1a,C2) of GM and effectively detected 10 components of GM fermentation broth which was simply concentrated. In addition, the system was used to detect the component ratio combining with titer test, which showed good consistency with the results of HPLC. This method is simple, rapid and reliable. About HPLC system, contrast study of mobile phase showed that methanol: water:formic acid = 74:21:5 solution (sodium heptanesulfonate 4.0 g / L) used as the mobile phase was better. Studies on separation and purification showed that the weak acid DK110 macroporous resin was better (eluting peaks were more focused) than 732 resin.
To get high-producing gentamiacin (GM) strains, which was resistant to the material produced by itself, Me07 was taken as the initial strain and was treated by UV, DES and microwave mutagenesis. The experimental results showed that DES treatment was the best way. Then, the GM production mutants was screened by two breeding ways of resistance plates and dynamic flasks combining with TLC analysis. The high-yield strain Me07-1D15 was obtained and compared with Me07, the yield of gentamicin in fermention broth was increased by 2.0 fold from 680 U/ml to 1400 U/ml,and C1 content was as high as 47.59%.
The fermentation medium of gentamicin was optimized,and the results showed that starch was the best carbon source, the bean powder 1# was the most suitable nitrogen source. The results of Plackett-Burman test indicated that starch, soybean powder CoCl2 and CaCO3 were significant factors influencing the fermentation level. Then the single factor experiments and orthogonal combination experiments were did, and the results showed that the optimal concentration of significant factors were: starch 60 g/L, soybean powder 30 g/L, CoCl2 0.008 g/L, CaCO3 4 g/L.
The optimization of fermentation conditions were studied, and the best fermentation conditions were obtained:seed age was 2 days, liquid volume in 500 mL flask was 40 mL, culture temperature was 34℃, the shaking speed was 250 r/min, the fermentation time was 8 days. Research on the fermentation process showed that rationally reducing the nutritional content of the fermentation medium could greatly shorten the fermentation cycle and maintain a high production level of GM. Besides, Application of different stress conditions like heat shock, feeding high ethanol and high NaCl concentrations during fermentation has found to be effective for the increased production of gentamicin. Production of gentamicin was increased to 1715 U/mL in medium supplemented with 0.6% NaCl to 144 hrs old culture. Production of gentamicin was increased to 1696 U/mL in medium supplemented with 4% ethanol to 96 hrs old culture, while, when the ethanol concentration was 5～6%, the synthesis of gentamicin was inhibited.
为得到优良的庆大霉素(GM)高产菌,以Me07为出发菌,经UV、硫酸二乙酯(DES)和微波诱变处理,以自身代谢终产物为筛子,采取抗性平板以及摇瓶动态选育两种方式,结合TLC及HPLC检测进行优良突变菌株的筛选。筛选得到的产C1组分比例较高的优良突变菌株Me07-1D15,生物效价为1400 U/mL,较出发菌株(680 U/ml)提高了2倍,其发酵液中C1含量最终检测达到47.59%,并且具有良好的遗传稳定性。
通过Plackett-Burman实验设计筛选得到影响发酵水平的显著因子为可溶性淀粉、豆饼粉、CoCl2、CaCO3。通过单因素分析及正交分析得到显著因子最佳浓度为:淀粉60 g/L,豆饼粉30 g/L,氯化钴0.008 g/L, CaCO3 4 g/L。对主要发酵条件进行优化研究,得出最佳发酵条件为:种龄选择2 d,接种量为12 mL,发酵培养基初始pH为7.2,最佳装液量为40 mL(500 mL摇瓶),培养温度34℃,摇瓶转速为250 r/min,培养周期8 d,最佳条件下发酵水平达到1824 U/mL。
过程控制研究从降低基质浓度和非营养刺激两方面考察,结果表明适当减少培养基的营养成分可以使产抗期提前;在发酵96h时进行水浴45℃处理可以使GM产量达到1685 U/mL;在144小时添加0.6%的氯化钠溶液,GM产量达到1715 U/mL,是对照试验的1.20倍;在96 h添加4%乙醇,庆大霉素的产量达到1696 U/ml,而当乙醇浓度达到5～6%时,对庆大霉素的合成有副作用,乙醇的添加抑制了棘孢小单孢菌的生长。
By perfecting the operating condition of TLC, the optimum detecting system was obtained, and the detection limit and reproducibility were studied. Based on the optimum system, we successfully separated the main components (C1,C1a,C2) of GM and effectively detected 10 components of GM fermentation broth which was simply concentrated. In addition, the system was used to detect the component ratio combining with titer test, which showed good consistency with the results of HPLC. This method is simple, rapid and reliable. About HPLC system, contrast study of mobile phase showed that methanol: water:formic acid = 74:21:5 solution (sodium heptanesulfonate 4.0 g / L) used as the mobile phase was better. Studies on separation and purification showed that the weak acid DK110 macroporous resin was better (eluting peaks were more focused) than 732 resin.
To get high-producing gentamiacin (GM) strains, which was resistant to the material produced by itself, Me07 was taken as the initial strain and was treated by UV, DES and microwave mutagenesis. The experimental results showed that DES treatment was the best way. Then, the GM production mutants was screened by two breeding ways of resistance plates and dynamic flasks combining with TLC analysis. The high-yield strain Me07-1D15 was obtained and compared with Me07, the yield of gentamicin in fermention broth was increased by 2.0 fold from 680 U/ml to 1400 U/ml,and C1 content was as high as 47.59%.
The fermentation medium of gentamicin was optimized,and the results showed that starch was the best carbon source, the bean powder 1# was the most suitable nitrogen source. The results of Plackett-Burman test indicated that starch, soybean powder CoCl2 and CaCO3 were significant factors influencing the fermentation level. Then the single factor experiments and orthogonal combination experiments were did, and the results showed that the optimal concentration of significant factors were: starch 60 g/L, soybean powder 30 g/L, CoCl2 0.008 g/L, CaCO3 4 g/L.
The optimization of fermentation conditions were studied, and the best fermentation conditions were obtained:seed age was 2 days, liquid volume in 500 mL flask was 40 mL, culture temperature was 34℃, the shaking speed was 250 r/min, the fermentation time was 8 days. Research on the fermentation process showed that rationally reducing the nutritional content of the fermentation medium could greatly shorten the fermentation cycle and maintain a high production level of GM. Besides, Application of different stress conditions like heat shock, feeding high ethanol and high NaCl concentrations during fermentation has found to be effective for the increased production of gentamicin. Production of gentamicin was increased to 1715 U/mL in medium supplemented with 0.6% NaCl to 144 hrs old culture. Production of gentamicin was increased to 1696 U/mL in medium supplemented with 4% ethanol to 96 hrs old culture, while, when the ethanol concentration was 5～6%, the synthesis of gentamicin was inhibited.
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