【作者】 宋文军；

【导师】 张克旭；

【作者基本信息】 天津科技大学 ， 发酵工程， 2004， 博士

【摘要】 本论文根据代谢控制发酵理论，研究了L-异亮氨酸高产菌选育及其发酵条件。主要研究内容和结果如下： (1) 应用“纸层析—色斑洗脱比色法”和“化学比色法”对发酵液中L-异亮氨酸进行了定量分析研究，确立了L-异亮氨酸定量测定条件和计算方法。以高速氨基酸分析仪测定结果为参比，用化学比色法测定结果的准确度比纸层析一色斑洗脱比色法高，且应用化学比色法测定L-异亮氨酸的含量较为方便。 (2) 以黄色短杆菌HL41为出发菌株，通过硫酸二乙酯(DES)和紫外线诱变处理，经摇管初筛、摇瓶复筛、遗传标记验证、单菌落分离和连续传代，筛选出一株L-异亮氨酸生产菌株ISW330(Met~-+AEC~r+α-AB~r)。该菌株在未经优化的条件下可积累L-异亮氨酸9.18g／L。 (3) 确定了亲株ISW330和AS1.495原生质体形成和再生及进行原生质体融合的最佳条件，采用原生质体融合技术最终筛选出一株带有目的遗传标记的L-异亮氨酸高产菌SW0370(Leu~-+Met~-+AEC~r+α-AB~r)。该菌株在未优化条件下可产L-异亮氨酸140.27g／L。经验证，菌株SW0370的遗传性能稳定。 (4) 研究了菌株SW0370的摇瓶分批发酵条件。应用均匀设计和MATLAB软件获得该菌株优化的种子培养基和发酵培养基配比，并对种子培养条件和发酵培养条件进行了研究。在优化条件下，菌株SW0370摇瓶分批发酵96h，可产L-异亮氨酸20.7g／L。 (5) 以摇瓶分批发酵最优条件为基础，对菌株SW0370进行了7L发酵罐分批发酵试验。以7L罐分批发酵试验数据为依据，利用遗传算法对L-异亮氨酸分批发酵动力学进行了研究，建立了菌体生长、产物形成和底物消耗的动力学模型，拟合模型能较好地反映L-异亮氨酸分批发酵过程。 (6) 以分批发酵最优条件为依据，对菌株SW0370进行了补料分批发酵研究。摇瓶补料分批发酵的初糖浓度为80g／L，于发酵过程第24h、48h、60h、80h分四次补加补料液，发酵96h产L-异亮氨酸21.96g／L，比摇瓶分批发酵提高了7.29％。在此基础上进行了7L发酵罐补料分批发酵试验。由于采用补糖补氨工艺，发酵96h产L-异亮氨酸24.82g／L。 本论文在L-异亮氨酸代谢控制发酵研究领域的创新之处在于，成功地将原生质体融合技术应用于L-异亮氨酸高产菌的选育，并且选育出一株带有双缺标记Leu~-+Met~-)的L-异亮氨酸高产菌SW0370，采用补料分批发酵工艺，发酵96h可产L-异亮氨酸24.82g／L，达到国内先进水平。确立了测定发酵液中L-异亮氨酸的“化学比色法”，此法快速高效准确，在工业化生产L-异亮氨酸的定量测定中具有重要的指导意义。以发酵过程溶氧变化为依据，结合动力学分析，提摘要出了分阶段供认的拧制模式，将遗传算法用于动力学模型的构建，并对所建模型进行了较宽初糖范围适用性的研究，对发酵法工业生产L一异亮氨酸的过程控制优化具有较强的于片导意义更多还原

【Abstract】 According to the theory of metabolic control fermentation, the dissertation focuses on the breeding of L-isoleucine high-yielding strain and its fermentation conditions. The main research contents and results are as follows:(1) L-isoleucine concentration in fermented broth was quantitatively determined with methods of paper chromatography-mottle elution colorimetry and chemical colorimetry. The quantitative determination condition and computing method were decided. Compared with high-speed amino acid analyzer, the determination value of L-isoleucine concentration with chemical colorimetry is more accurate than with paper chromatography-mottle elution colorimetry.(2) Brevibacterium flavum HL41 as a starting strain was stepwise mutated with protoplast UV and Diethyl sulfate. One L-isoleucine high-producing strain ISW330 with genetic marker was screened through shake tube primary screening, shake flask compound screening, genetic marker test and single clone isolation and continuous passage cultivation This strain could produce L-isoleucine 9.18g/L after fermentation under the fermentation condition without being optimized. Genetic character of strain ISW330 was very stable.(3) The protoplast formation rate and regeneration and fusion condition of strain ISW330 and AS 1.495 was studied in this dissertation. One L-isoleucine high-pro.ducing strain SW0370 was screened through protoplast fusion, shake flask compound screening, genetic marker test and single clone isolation and continuous passage cultivation. This strain could produce L-isoleucine 20.7g/L after fermentation under the fermentation condition without being optimized. Genetic character of strain SW0370 was very stable.(4)The batch fermentation conditions of strain SW0370 in shake flask were studied in this dissertation. The proportions of seed medium and fermentation medium components were optimized by uniform design experiment and MATLAB software .The seed culture conditions and fermentation conditions were also studied.Under the optimum conditions, strain SW0370 could produce L-isoleucine 20.7g/L after fermentation for 96 hours by flask-shaking batch fermentation.(5) Based on the above optimum condition, the batch fermentation was performed with strain SW0370 in 7-liter fermentor. The L-isoleucine batch fermentation kinetics was studied by genetic algorithms based on the experimental data from 7-liter fermentor’s batch fermentation. Three kinetic models were constructed which could reflect the regularity of growth, product formation and substrate consumption in the process of batch fermentation.(6) Fed batch fermentation was studied with SW0370 according to the optimum condition of batch fermentation. The optimum initial glucose concentration was 80g/L in shake flask fed batch fermentation. The intermittent feeding was performed at the 24th, 48lh, 60th, 80th hour respectively. The production of L-isoleucine reached 21.96g/L after fermentation for 96 hours. Based on the above experiment, fed batch fermentation in 7-liter fermentor was performed. The production was improved by 7.29% than that of fermentation in shake flask fed batch.Because of the technology of low glucose, low amino and fed batch, production of L-isoleucine reached 24.82g/L after fermentation for 96 hours.The innovation of this dissertation in the research field of L-isoleucine metabolic control fermentation is that protoplast fusion technique was successfully applied to breed L-isoleucine high-yielding strain.which has two defective mutations(Leu"+Met") An L-isoleucine high-yielding strain SW0370 was obtained, which could produce L-isoleucine 24.82g/L after fed batch fermentation in 7-liter fermentor for 96 hours, which has reached the advanced technique level of the same research in China. The chemical colorimetric method was built to detect L-isolecuine,which was quick , efficient and .In addition ,this method has importantly instrumental function in industrial production of L-isoleucine to assay its content更多还原