【作者】 姜俊云；

【导师】 贾士儒；

【作者基本信息】 天津科技大学 ， 发酵工程， 2004， 硕士

【摘要】 本文以白色链霉菌（Streptomyces albulus）为ε-聚赖氨酸的生产菌株，以提高ε-聚赖氨酸产量，优化ε-聚赖氨酸发酵工艺主要研究目标，研究内容包括：①ε-聚赖氨酸生产菌株的选育；②分批发酵中搅拌转速、pH、通风比和温度对ε-聚赖氨酸发酵的影响；③流加补料发酵条件的研究以及ε-聚赖氨酸空间结构的预测。 1．用紫外线（UV）对白色链霉菌Streptomyces albulus 9-5进行诱变处理。首先确定紫外线诱变最佳诱变剂量为45S，紫外诱变后，经初筛、复筛得到一株产量为1.085g／L的突变株UV3-9（AEC^r），比出发菌株9-5的ε-聚赖氨酸产量提高15.2％，遗传性状稳定。采用5L自动发酵罐发酵结果表明突变株UV3-9要比原始菌株9-5的发酵性能有所提高，主要体现在：菌株生长的延滞期缩短，对数期延长，菌体适应能力增强，生长速度快，ε-聚赖氨酸的合成能力比原始菌株9-5提高13％。 向培养基中适量添加生物素（200 μg／L～500μg／L）分别可提高菌株ε-聚赖氨酸产量35％。 2．采用5L自动发酵罐，对ε-聚赖氨酸分批发酵中搅拌速率、pH、通风比和温度的影响进行了研究。发现搅拌速率提高对菌体生长和ε-聚赖氨酸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，ε-聚赖氨酸产量下降。实验证明当搅拌速率维持在350r／min左右时，Y_p／s最高为0.0688g．ε-PL／g.sub。 pH值对菌体生长和ε-聚赖氨酸合成具有明显影响，当pH维持4.5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pH对ε-聚赖氨酸合成的影响却相反，pH为4.0时，促进ε-聚赖氨酸的合成，而pH为6.0时，没有ε-聚赖氨酸的合成。 一定通风比下ε-聚赖氨酸产量与通风比成正比，通风比为1.25vvm时，ε-聚赖氨酸产物得率和比生成速率最高，分别为0.072g．ε-PL／g．sub和0.007g．ε-PL／g．cell·h；当超过1.25vvm后，随着通风比增加DO提高；通风比为2.67vvm时，DO维持60％以上，有利于菌体生长，Y_x／s达到0.393g．cell／g．sub，但葡萄糖消耗速率降低，至96h残糖仍为1.05％，ε-聚赖氨酸的产量下降，发酵时间延长。 28℃时对细胞生长非常有利，菌体得率Y_x／s达到0.429g．cell／g．sub，发酵结束时菌体量最高为10.07g／L，但ε-聚赖氨酸产量仅0.79／L。30℃下菌体量较高，且ε-聚赖氨酸的产量和ε-聚赖氨酸产物得率Y_p／s远高于前两个控制条件，分别为2.44g／L和0.073g．ε-PL／g．sub。摘要 优化条件下（搅拌转速350r/min，30℃，初期通风比0.svvm，当DO降至30%后，调节通风比为1.25vvm，初始pH6.8，自然降至pH4.o后以10%氨水控制pH4.0）进行发酵实验，实验所得菌体产量、“一聚赖氨酸产量、聚赖氨酸相对葡萄糖得率和比生成速率:分别为10.84留L、3.02岁L、0.0759.“一PUg.sub和0，0169.卜PUg.eell·h。 3，采用了三种不同流加方式对流加培养发酵￡一聚赖氨酸进行了研究，分别是有反馈控制的间歇流加培养和变速流加培养，以及恒速流加培养。前两种流加方式通过建立流加数学模型进行控制，间歇流加的数学模型为VlxCsl+F X FS，=V:X CsZ;变速流加的数学模型为F=dQ/dt=K（t一ta）（t一tL）平「，Q二cK（c为常数）。流加基质为葡萄糖和硫酸钱，流加阶段葡萄糖浓度控制在10g/L。 流加发酵实验表明，变速流加优于其他两种流加方式，其￡一聚赖氨酸产量和菌体量分别达到6.939/L和19.80留L，恒速流加的“一聚赖氨酸产量为5.29留L。另外间歇流加方式的发酵结果最不理想，c一聚赖氨酸产量仅为3.77留L。但是从发酵动力学参数考察变速流加和恒速流加方式，后者要优于前者，因此还需进一步对这两种流加方式做深.入研究。 4.借助生物信息学和网络预测服务，对具有抑菌功能的异型短肤￡一聚赖氨酸进行了二级结构和空间结构预测。考察了链长对￡一聚赖氨酸结构的影响，基于相似蛋白的结构模拟了20AA、30AA和4OAA￡一聚赖氨酸的三级结构;同时依照Shoji Shima等人关于￡一聚赖氨酸一级结构与其活性的关系，初步推断出。螺旋及其在整个肤链中的比例对￡一聚赖氨酸的抑菌活性有重要作用。更多还原

【Abstract】 In the essay Streptomyces albulus was considered as tool of production, in order to improve the productivity of e -PL, optimize the process of fermentation, make out the rules of batch fermentation and fed-batch fermentation of -PL, works were done as follows: (1) the breeding of the strain producing e -PL. (2)the effect of stir velocity, pH, aeration and temperature on the synthesis of -PL in batch fermentation (3)the fed-batch fermentation was studied and the secondary and space structure of -PL was predicted by using of bioinformatics and internet prediction serve.Streptomyces albulus 9-5 was treated by UV, the optimum irradiation time was 45S. After mutation, initial selection and repeat selection, a mutant UV3-9(AECr) producing -PL 1.085g/L was obtained. The main findings that the capability of fermentation of UV3-9 was higher than 9-5 are as follows: the procrastinate period of UV3-9 was decreased, and the logarithm growth period of UV3-9 was increased; the strains grew rapidly and the capability of e -PL-producing was increased, which is 13% higher than original strain9-5. 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.By adding D-Biotin(200~500 g/L) , the capability of -PL-producing can also be increased 35 %.The researches showed that the stir velocity, pH, aeration ratio and temperature play on important roles in the fermentation of -PL in 5-L automatic fermentation jar. High stir speed would improve the growth of cells and the synthesis of -PL, however over high stir speed could break the mycelium so as to get the cells to death. The test showed that at about 350r/min of stir speed the yield of -PL fermentation was the highest. When pH was controlled under low value, which could inhibit the growth of cells. In reverse when pH was higher than 4.5, which could promote the metabolic ability of cells. But high pH would not benefit the synthesis of -PL, the reason was not clarified until now.The field of -PL was enhanced companying with the increasing of aeration ratio. The highest yield of glucose to -PL and productivity were 0.072 g. -PL/g.sub and 0.007g. -PL/g.cell-h under aeration ratio 1.25vvm.Under aeration ratio 2.67vvm dissolved oxygen could maintain over 60%,glucose was consumed slowly and still had 1.05% at 96h, Yx/s was 0.393 g.cell/g.sub, but the 拢 -PL producing lever was fell and the fermentation circle was protracted.The temperature for Streptomyces albulus to grow was 28 C,the yield of glucose to Streptomyces albulus was 0.429 g.cell/g.sub, the field of Streptomyces albulus was 10.07g/L after the batch fermentation, the field of 拢 -PL was only 0.79g/L.30 掳C was fit for the 拢 -PL producing, at which the field of e -PL and the yield of glucose to 拢 -PL, higher than those at 28 C or 32 C,were 2.44g/L and 0.073g. E -PL/g.sub, respectively.,Batch fermentation of e -PL in 5.0L bioreactor was carried out at suitable culture conditions of : agitation speed 350r/min,temperature 30 C, aeration 0.8vvm and adjust aeration 1.25vvm when DO was 30%,pH6.8,and control pH4.0 by adding 10% ammonia as soon as pH was under 4.0.The field of Streptomyces albulus, E -PL concentratin, the yield of glucose to E -PL and volumetric productivity were 10.84g/L,3.02g/L,0.075g. 拢 -PL/g.sub and 0.016 g. 拢 -PL/g. cell-h, respectively.Three different kinds of feed-batch cultures were designed in order to investigate the effect taken by each on e -PL fermentation. These ways included span feed-batch and varible speed feed-batch, as well as constant speed feed-batch which were controlled by substrate feedback based on feed math models. The math model used by the span feed fermentation was Vi X Csi + F X F Si = V2 XCS2, the other was F=dQ/dt=K(t-ta)(t - tL) and Q=cK (c is constant number) . Here the feedback signal was the concentration of glucose which was maintained around lOg/L. Once the concentration of glucose during fermentation process decre更多还原