【作者】 彭彦峰；

【导师】 吴兆亮；

【作者基本信息】 河北工业大学 ， 化学工艺， 2003， 硕士

【摘要】 D-核糖是遗传物质核酸的构成成分，也是若干辅酶和维生素的组成部分，具有重要的生理作用。近年来利用D-核糖生产抗癌和抗病毒药物也显示出了强大的生命力。目前发酵法生产D-核糖是最有效的生产方法，使得发酵法生产D-核糖得到了广泛的研究。本文针对D-核糖发酵的性质，对D-核糖发酵过程中一些参数的测定方法进行了研究，并对本发酵体系的菌株进行了自然选育和诱变育种，采用不同数学统计方法对发酵培养基进行优化，并进行了比较，建立了发酵的动力学模型。 1．研究了分光光度法测定发酵液中D-核糖的方法，得出发酵液中非葡萄糖组分对D-核糖测定没有影响，葡萄糖对测定有显著影响。建立了分光光度法测定微生物发酵液中D-核糖浓度的有效方法。其最佳操作条件为：测定波长670nm：加热时间25min；反应氢离子浓度8mol·L^-1。建立了发酵液中有其它影响因素存在时的计算公式。 2．研究了细胞生长曲线（包括种子液和发酵液）的分光光度快速测定方法，排除了测定过程中的干扰因素（包括色素和不溶物质的影响），得出种子液测定的优化方法为直接测定，发酵液测定的优化方法为稀释10倍后测定，并分别关联了种子液和发酵液测定中吸光度与细胞干重间关系。 3．对实验菌株采用自然选育和紫外诱变育种进行了筛选，以自然选育和紫外诱变选育相比较，紫外诱变选育可以获得高产量的突变株，而自然选育一般只能作为纯化菌种的方法来保存菌种。得出本实验的合适诱变剂量是30s。 4．对本D-核糖发酵体系的操作条件和培养基进行了优化，得出其最佳接种量为15％，合适的摇床转速为240r／min，合适的装液量为500mi三角瓶装35ml未接种发酵液。在培养基优化中分别采用了均匀设计法和部分因子与响应曲面结合的组合方法，得出在微生物发酵这种操作条件不易严格控制的情况下，部分因子与响应曲面结合的组合方法优化效果强于均匀设计法。并以组合优化方法得出了优化的培养基组合为：葡萄糖：146g／1；玉米浆13.4g／1；硫酸氨7g／1；硫酸锰0.05g／4；碳酸钙15g／1，对应的D-核糖产量为44.1g／1。 5．在优化的发酵培养基的基础上建立了摇瓶的发酵动力学模型，模型曲线分析表明模型能较好的反映D-核糖发酵过程。初步进行了发酵罐的放大发酵操作，并建立了其发酵动力学模型，模型能较好的反映其发酵过程。 因此，本文通过对D-核糖发酵过程中参数分析方法的研究及一些工艺的研究，为提高发酵法生产D-核糖的产量进一步提供了理论与实践基础。更多还原

【Abstract】 D-ribose, which is an important constituent of DNA, some coenzymes and vitamins, has extensive usages in foodstuff and medicine field. In recent years, D-ribose, a kind of raw material for new medicines of anticancer and antivirus, shows good perspective for application. Interest in fermentation by microorganism has recently increased as a result of its little byproducts, low cost and high yield. In this paper, according to D-ribose fermentation properties, the measurement methods of parameters in fermentation process were studied, the cell strain of fermentation was screened, the different statistical methods of optimizing media was adopted and compared, and dynamics model was constructed.1. The method of measurement of D-ribose concentration in the fermentation broth by spectrophotometry has been studied. According to the principle of D-ribose measurement by spectrophothmetry, the main factors affecting correct measurement of D-ribose concentration in the fermentation broth were obtained. The optimum measured wavelength is 670 nm, the heating time 25 min and the hydrogen ion concentration 8 mol . L-1. Absorbance A shows remarkable linear relationship to D-ribose concentration in the range of 10-30 ug.ml-1. Effect caused by the non-glucose element in the fermentation broth can be ignored and glucose significantly influences the measurement of D-ribose. The effect of glucose concentration on A was obtained through experiments. An effective method was established to measure D-ribose concentration in microbial fermentation broth by spectrophotometry.2. A quick, accurate and simply absorbance measurement method for the cell growth curve (including seed solution and broth) was investigated, and the function relation between the cellular concentration and absorbance was established, which provides the foundation for the measuring online of cellular concentration in industry and metabolism control of producing D-ribose by fermentation.3. Natural screening and UV-mutation were applied to experimental cell strain. UV-mutationcould obtain higher yield strain than natural screening and the latter was only suitable to conserve cell strain. According to the usual standard that 70% death ration is suitable, 30s is the appropriate mutation quantum.4. D-ribose fermentation conditions were studied, and the optional D-ribose fermentation conditions were 240r/min, 35ml broth volume in 500ml tri-angle bottle and 15% inoculation volume. In media optimization process, uniform design and multi-method joining fractional factorial design and response surface optimization design were adopted respectively, and the experimental results showed that multi-method is better than uniform design on the condition that operation conditions cannot be controlled strictly. The optional media is obtained by using multi-method: glucose 150g/l, corn steep liquor 134 g/1, (NH4)2SO4 7g/l, MnSO4 0.05g/l, CaCO3 15g/l, and the corresponding D-ribose yield is 44.1g/1.5. Based on the optimal fermentation media, ratable bottle fermentation dynamics model was constructed. The magnifying fermentation on reactor was processed, and its dynamics model was constructed.So, in this paper, the theoretical and practical basement of increasing D-ribose fermentation further is provided by studying analytical method of parameters and studying fermentation process.更多还原