转化右旋磷霉素菌株的诱变及培养条件优化

发布时间：2018-01-01 07:34

本文关键词：转化右旋磷霉素菌株的诱变及培养条件优化　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：磷霉素是一种结构简单且分子量小的新型广谱抗生素,对革兰阳性和阴性菌均有杀菌作用,被国际药学界列为二十一世纪新型抗生素。目前工业上采用化学合成法生产磷霉素的总收率不足35%,其中50%左旋磷霉素有抗生素活性,另一半的右旋磷霉素为无活性物质。这样不但浪费了资源,提高了生产成本,同时也污染了环境。因此转化右旋磷霉素、提高其有效利用,对工业化生产磷霉素具有重要意义。微生物转化法具有高度专一性,发生反应的条件温和,工艺简单,成本较低等特点,能够完成化学方法难以进行的反应。采用微生物转化法,利用具有转化能力的生产菌株,可将右旋磷霉素中的一部分转化为有效的左旋磷霉素。本研究在实验中筛选出1株解淀粉芽孢杆菌,并对其进行理化复合诱变。通过试验确定了紫外线对解淀粉芽孢杆菌的最佳诱变时间是90s,经紫外诱变后,选育出1株突变株UV2,对其进行发酵培养,发现突变菌株UV2的转化能力有所提高,其转化率为11.76%;UV2相较于原始菌株,转化率升高3.84%。然后以UV2为出发菌株,选择硫酸二乙酯再进行化学诱变,确定诱变的最佳时间是40min,经过诱变后选育出1株突变菌株U-D2,对其进行发酵培养,发现突变菌株的转化能力有所提高,其转化率为13.2%,与UV2相比转化率提高了 1.44%。U-D2与原始菌株解淀粉芽孢杆菌相比转化率提高了5.25%。对突变菌株U-D2进行了 6代传代培养,以金黄色葡萄球菌为指示菌,通过发酵液的抑菌性能测定,结果表明突变菌株U-D2具有良好的遗传稳定性。采用单因素试验结合响应面设计法中的CCD中心组合试验设计方法,对U-D2的培养基营养成分进行优化。确定最优培养基浓度为:牛肉膏3.62g/L、葡萄糖12.39g/L、蛋白胨7.19 g/L、硫酸镁0.42 g/L、磷酸氢二钾0.85 g/L。突变菌株U-D2以优化的培养基培养,其培养液通过抑菌试验,测得抑菌圈直径为37.4mm,U-D2的转化率为15.51%,与培养基优化前相比转化率提高了 2.31%。
[Abstract]:Fosfomycin is a new broad-spectrum antibiotics has the advantages of simple structure and low molecular weight, the gram positive and negative bacteria have bactericidal effect, by the international pharmaceutical industry listed as twenty-first Century. New antibiotic is widely used in industrial chemical synthesis, the total yield of production of phosphonomycin is less than 35%, of which 50% levo fosfomycin is known as antibiotic activity right, the other half of fosfomycin non active substances. It is not only a waste of resources, improve the cost of production, but also pollutes the environment. So the transformation of dextrofosfomysin, improve the effective use of hormone has important significance for the industrial production of fosfomycin. Microbial transformation method has high specificity, reaction conditions a mild, simple process, low cost, able to complete the chemical method is difficult to analyze reaction. By using microbial transformation method, using the production strain has the ability of converting the dextrofosfomysin in a Part of the transformation is left-handed fosfomycin effective. The study in the experiment screened 1 strains of Bacillus amyloliquefaciens, and its chemical compound mutagenesis. Through the test the UV is 90s on the best mutation time of Bacillus amyloliquefaciens, by UV induced mutation breeding, 1 strains of mutant strain UV2 that the fermentation, found that the transformation ability of mutant strain UV2 increased, the conversion rate is 11.76%; UV2 compared with the original strain, the conversion rate of 3.84%. increased and then with UV2 as the starting strain, choose two ethyl sulfate by chemical mutagenesis, to determine the optimum time of mutagenesis is 40min, after 1 after mutation breeding mutant strain U-D2, the fermentation culture, found that transformation ability mutants increased, the conversion rate is 13.2%, compared with the UV2 conversion rate was increased by 1.44%.U-D2 and the original strain of Bacillus amyloliquefaciens compared with the conversion rate was increased by 5.25% . the mutant strain U-D2 was the 6 generation of subculture, Staphylococcus aureus as the indicator bacteria, the antibacterial properties of fermentation liquid were measured, results showed that the mutant U-D2 has good genetic stability. By single factor test and response surface design method in CCD central composite experimental design method of medium nutrients optimization of the U-D2. To determine the optimal concentration of culture medium was beef extract 3.62g/L, glucose 12.39g/L, peptone 7.19 g/L, Magnesium Sulfate 0.42 g/L, two potassium hydrogen phosphate 0.85 g/L. mutant strain U-D2 to optimize the culture medium, the medium through the antibacterial test, measured the diameter of inhibition zone was 37.4mm, the conversion rate of U-D2 to 15.51%, compared with the optimal medium before the conversion rate increased by 2.31%.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93

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