富马酸生产菌米根霉的高效选育

文档编号:SW160  文档字数:10028,页数:30

 摘 要：本文以实验室自行保存的富马酸生产菌米根霉ZJU作为初始菌株，通过传统的紫外诱变，结合简单有效的平板筛选，辅以培养基的优化，大幅度提高了富马酸生产菌的产酸能力。
 建立了适合于产酸丝状真菌诱变选育的变色圈平板初筛法，大大降低了诱变育种后高产菌株选育的工作量。以实验室储藏的米根霉作为出发菌株，初始富马酸产量为29.8g/l，经UV反复诱变处理后，应用上述方法筛选得到了一株富马酸高产菌株－米根霉，在出发菌株的最佳发酵条件下富马酸产量为50.9g/l,比原始菌株提高了70.8％。对突变菌株的培养基进行了优化，发现突变菌株和原始菌株最佳培养基组成相似，最佳培养温度为35℃，初始摇床转速100rpm，到大量产酸时转速升至200rpm。培养基中需要加入0.6g碳酸钙以控制pH值。
关键词：富马酸；米根霉；紫外诱变；制霉菌素

Abstract: A systematic investigation was carried out to improve the productivity of fumaric acid producing strain by UV irritation and bioprocess optimization. During the selection procedure, a simple way of screening for high-yield fumaric acid producing strain was developed and evaluated, in which, area ratio of area of colored ring to that of colony was designed as an indicator.
 Strain improvement has been conventionally achieved through mutation and selection. A fumaric acid over-producing strain mutant, Rhizopus oryzae , was isolated by mutagenizing the parent strain by UV mutagenesis. Starting with an optimized concentration of 29.8 g/l glucose, the mutant Rhizopus oryzae ZJU11 produced 50.9 g/l fumaric acid in shaking-flask fermentation , which were 70.8% higher than that of the parent strain produced respectively.
 The influences of nitrogen sources, carbon sources, concentration of CaCO3 and its addition method CaCO3 in culture medium on fumaric acid production by the mutant Rhizopus oryae ZJU11were investigated.
Keywords：Fumaric acid; Rhizopus oryzae; UV irritation; optimization; Nystatin

目  录
中文摘要 I
英文摘要 Ⅱ
目录 Ⅲ
1. 绪论 1
 1.1  富马酸的理化性质 1
 1.2　富马酸的应用 2 
 1.3　富马酸的生产进展 2
 1.4　米根霉发酵生成富马酸的代谢途径 3
 1.5　诱变育种 4
 1.6  本文档主要研究内容 7
2. 实验部分 8
 2.1  实验材料 8
 2.2  实验方法 9
 2.3  分析方法 9
 2.4  主要试剂 10
 2.5  主要实验设备与仪器 11
 2.6  富马酸高产菌株的选育 11
3. 结果与讨论 13
 3.1   获得单菌落 13
 3.2　指示剂的选择 13
 3.3　KMnO4滴定结果 17
 3.4　出发菌株产富马酸测定 18
 3.5　诱变剂的剂量选择 18
 3.6　米根霉在摇瓶中发酵 19
 3.7　筛选方法评估 19
 3.8　诱变菌株的产酸量测定 19
 3.9　小结 21
4．总结与展望 22
 4.1  结论与创新点 22
 4.2  展望 23
致谢 24
参考文献 25

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