【作者】 董惠钧；

【导师】 贾士儒；

【作者基本信息】 天津科技大学 ， 发酵工程， 2003， 硕士

【摘要】 ε-聚赖氨酸（ε-Poly-L-Lysine）是由微生物发酵合成的一种由赖氨酸单体通过ε-酰胺键形成的多肽。ε-PL具有优良的抑菌作用，对革兰氏阳性菌和阴性菌、酵母和一些病毒都有抑制作用，而且兼有水溶性好、热稳定性高等特点。目前，ε-PL已广泛用于食品加工和储藏的防腐保鲜、可降解生物材料、药物包被物、靶向药物载体以及医学研究中。 本文以白色链霉菌（Streptomyces albulus）为ε-PL的生产菌株，以提高ε-PL产量，优化发酵工艺，研究ε-PL分批发酵方法的规律以及预测ε-PL的结构为主要研究目标，研究内容包括：①ε-PL生产菌株的形态和生理生化特征鉴定以及ε-PL生产菌株的选育；②培养基和培养条件的优化；③分批发酵中搅拌和pH对ε-PL发酵的影响；④ε-PL二级结构和部分性质的预测。 主要研究结果如下： 1．确定了初筛抗性标记AEC和甘氨酸以及相应的临界浓度，分别是6mg/mL和3mg/mL，选用抑菌圈法作为第一次复筛方法，通过ε-PL对酵母菌、大肠杆菌、乳酸菌和枯草芽孢杆菌的抑制试验，证明枯草芽孢杆菌对ε-PL最敏感，H/C值最大。 2．用紫外线（UV）和硫酸二乙酯（DES）分别对白色链霉菌Streptomyces albulus TF-1进行诱变处理。首先研究了紫外线诱变中不同照射剂量对细胞存活率的影响，作出了诱变曲线，同时根据诱变的普遍规律，即高突变率出现在高剂量的原则，确定紫外线诱变25S为最佳诱变剂量，紫外诱变后，经初筛和两次复筛得到一株产量为0.51g/L的突变株，比野生型菌株的ε-PL产量提高37.8％。 以紫外诱变获得的突变株为出发菌株，用化学诱变剂DES进行了再次诱变。首先根据诱变剂用量和诱变时间对细胞存活率和突变率的影响，作出诱变曲线，得出最佳诱变条件为：1％（体积百分比）和25min。对突变株进行化学诱变后，经初筛和两次复筛得到一株产量为0.79g/L的突变株，产量提高了54.9％。 对上述突变株进行了遗传稳定性实验。经5次传代后的发酵实验结果表明突变株性状稳定。 3．不同的碳源和氮源对ε-PL产生有明显的不同，葡萄糖和硫酸铵分别是最佳的碳源和氮源。对组合氮源进行了研究，发现硫酸铵和酵母粉复合使用更有利于ε-PL发酵。在发酵液中添加无机盐对ε-PL发酵有不同的影响，其中K₂HPO₄、KH₂PO₄和MgSO₄对发酵有一定促进作用，而ZnSO₄和FeSO₄几乎没有影响。 采用中心组合设计对影响发酵产量的培养基主要成分葡萄糖、硫酸铰和酵母粉的含量进行了忧化，得出了忧化模型；优化后培养基组成为；葡萄糖5％，硫酸铰l．5％，酵母粉1％；KZHPO。0．l％；MgSO。0．l％；KH。PO。0．15％，卜聚赖氨酸的预测产率为＊石gg／L。 对培养条件的研究表明：当装液量为 50mL，初始 pH为 6刀，接种量 8％，培养温度30C时，卜干L的发酵产量最高，为1．83g／L。4．采用 SL小型发酵罐，对。PL分批发酵中搅拌速率和 pH的影响进行了研究。发现搅拌速率提高对菌体生长和。PL的合成有显著的促进作用；但搅拌速率过高会将菌体打碎，导致细胞死亡，。PL产量下降。实验证明当搅拌速率维持在 350rpm左右时，产物得率最高为 0．0688 g．卜PL乍．sub。 pH值对菌体生长和。干L合成旦有明显影响，当pH维待45以上，有利于菌体生长：当pH低于3．5时，对菌体生长有抑制作用；但州对。PL合成的影响Z时B反，pH为 40时，促进卜干L的合成，而 pH为 6刀时，没有。干L的合成。 在最佳搅拌速率和 pH的控制条件下进行卜聚赖氨酸发酵，菌体量为9．刀g儿，。－队产量达至2．9＊叭，产物得率为0刀62 g。－巩／sub。5．用高效液相色谱对聚赖氨酸标准品和发酵样品进行了分析检测，标准品色谱图为单一色谱峰，保留时间为8．4们m山；发酵样品在8刀5加 处有一强色谱峰，与标准品色谱峰的保留时间桐同。6．利用在线网络预测了。－聚赖氨酸的二级结构、亲水性和空间结构。从数据分析显示，。－聚赖氨酸主要的二级结构单元为a一螺旋结构，还有部分无规卷曲结构。有较强的亲水性，暴露残基数多于内部残基数，多肽链呈非球形空间结构。更多还原

【Abstract】 ε-Poly-L-Lysine is comprised of lysine linked by amide bond through microbial fermentation. It has excellent antibacterial activity against Gram-positive and Gram-negative bacteria, yeast and some kinds of viruses. In additional ε-Poly-L-Lysine is soluble and attractive heat stable. At present ε -Poly-L-Lysine have been used as natural food preservative mainly, degradable biological matreria, coat of drug, carrier of target drug and medical research.In the essay Streptomyces albulus was considered as tool of production, in order to improving the productivity of ε-PL. optimizing the process of fermentation, making out the rules of batch fermentation and predicting the secondary structure of ε -PL, works were done as following. (1) morphological and physiological -biochemical characteristics of Streptomyces albulus, and the breeding of the strain producing ε -PL. (2) the optimization of culture media and conditions (3) the effect of stir velocity and pH on the synthesis of E -PL in batch fermentation (4) the prediction of ε-PL’s secondary structure and biochemical attributes.AEC and glycine were choose as resistant label, their corresponding concentration was 6mg/mL and 3mg/mL respectively. Meanwhile the elective method of inhibition circle was be used as the second limiting condition during the breed of mutant. Four bacterial species such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus were tested, the results showed that Bacillus subtilis was the most sensitive among those microbe.Streptomyces albulus TF-1 was treated by UV and DBS in order. Firstly we studied the effect of UV on the survival rate of cells, then drew a plot of mutation depending on the results. Meanwhile the law about mutation was considered, in according to the above conclusion the optimum irradiation time was 25S. After mutation, initial selection and repeat selection I got a mutant which could produce 0.51g/L.I chose the UV mutant as the initial strain of DES treatment. The effect of the usage and time of DES treatment on the survival rate of cell were studied firstly, the optimum value of usage and time were 1 one percent and 25min. After mutation, initial selection and repeat selection I got a mutant which could produce 0.79g/L.In order to investigate the genetic stability, the mutant was test after five continuous inoculation. The results showed that the mutant could transfer the genetic information to the son generation.Different carbon and nitrogen resource had conspicuous difference in the synthesis of ε -PL, the test indicated that glucose and ammonium sulfate benefited obviously the synthesis of ε -PL. furthermore we studied the function of the mixture of distinctive nitrogen resource. When we blended the yeast extraction to ammonium sulfate, the yield of ε-PL increased obviously. In additional we added various inorganic salts such as K.2HPO4, KH2PO4, MgSO4, ZnSO4 and FeSO4 to investigate the effects. The conclusion told me that except ZnSO4 and FeSCU other three inorganic salts presented limiting promotion. So we determined to select glucose, ammonium sulfate and yeast extraction to conduct the mixture plan with SAS software. The software helped give me the predictive answer which was five percent of glucose, one and half percent of ammonium sulfate, 1 percent of yeast extraction, by the way the optimum results indicated that there was interaction between glucose and ammonium sulfate.We further studied what condition were the best culture conditions. After contrasting test the results showed that there increased in the yield of ε-PL only under the following conditions such as volume of 50mL in 500mL flask, initial pH 6.0, feed inoculation of 8 percent, culture temperature of 30℃.The researches showed that the stir velocity and pH play an important roles in the fermentation of ε-PL in 5L automatic fermentation jar. High stir speed would improve the growth of cells and the synthesis of ε-PL, however over high stir speed could break the mycelium so as to get the cells更多还原