【作者】 黄国昌；

【导师】 印培民；

【作者基本信息】 南昌大学 ， 微生物学， 2007， 硕士

【摘要】 本文以白色链霉菌(Streptomyces albulus)为ε-聚赖氨酸(ε-poly-L-Lysine，简称为ε-PL)的生产菌，以提高ε-PL的产量为目的，对ε-PL发酵条件和提取工艺进行了研究。研究的主要内容包括：①通过摇瓶发酵对ε-PL生产条件进行初步探索；②在50 L罐上进行分批发酵条件的优化；③补料分批发酵方式的研究；④ε-PL分离提取工艺的初步探讨。主要结果如下：对ε-PL摇瓶发酵过程中温度、pH和装液量的影响进行研究，为50 L罐分批发酵条件优化奠定基础。对结果进行分析发现，30℃为菌体生长和ε-PL合成最适温度，28℃和30℃对摇瓶发酵结果影响不大，但32℃不利于ε-PL的合成；pH对ε-PL的合成影响显著，pH控制时ε-PL产量从没有控制时的0.89 g/L提高到1.59 g/L；装液量为100 mL时，发酵液中的溶氧量既有利于菌体生长，又有利于ε-PL的合成，而装液量为150 mL时，最终ε-PL的合成量最小。在50L罐中进行发酵条件的优化，菌体生长和ε-PL合成的最适温度为30℃，32℃时菌体生长缓慢，ε-PL的产量不高；pH3.0时，葡萄糖消耗速度较慢，最终残糖浓度仍为20.1 g/L，既不适合菌体生长，也不利于ε-PL的合成，pH4.0为ε-PL合成的最佳pH，最终ε-PL的产量和产率分别为3.05 g/L和0.062 g.ε-PL／g.sub，pH5.0时菌体生成量最大，为12.88 g/L，是菌体生长的最适pH，但pH5.0时ε-PL会出现降解现象；随着搅拌转速提高，溶氧水平也相应提高，有利于菌体生长和ε-PL合成，但发酵前期200 rpm时的溶氧水平足以维持菌体的正常生长，而搅拌转速400 rpm时，由于剪切力的作用会损伤菌丝，导致ε-PL合成受阻。优化后的条件为：温度30℃，初始pH6.8，通气量1 200 L／h，发酵前期搅拌转速为200 rpm，pH自然下降至4.0，发酵后期搅拌转速为300 rpm，控制pH4.0，经过84.5 h发酵后，ε-PL的产量和得率分别为3.21 g／L和0.065 g.ε-PL/g.sub，ε-PL产量较优化前的0.78 g/L提高了310％，产率也有较大提高。采用葡萄糖浓度反馈控制间歇补料培养和pH间接反馈控制流加培养对ε-PL补料分批发酵进行研究，实验结果表明，补料分批发酵明显优于分批发酵，说明补料分批发酵是一种适合S.albulus发酵生产ε-PL的培养方式。此外，pH反馈控制变速流加培养方式要略优于葡萄糖反馈控制间歇补料培养，两种补料培养最后的ε-PL产量、ε-PL得率分别为7.36 g／L和6.53 g/L、0.072 g.ε-PL/g.sub和0.068 g.ε-PL/g.sub。而且发酵过程中，由于变速流加葡萄糖对培养基中的基质浓度影响较小，不会造成发酵环境的剧烈变化，能为菌体生长和ε-PL合成代谢提供持续稳定的环境，而间歇补料时由于一次性加入的营养物质浓度和体积较大，对发酵环境的影响较大，不利于细胞内各种代谢的正常进行。对ε-PL提取工艺进行初步研究发现，发酵液预处理过程中，过滤可能会导致ε-PL的损失；选择大孔弱酸性丙烯酸系阳离子交换树脂D152对ε-PL进行粗提取，洗脱剂为0.1 N HCl溶液，洗脱速度为2.5 mL／min时，ε-PL的浓缩倍率为2.29倍，回收率为78.83％；选择活性炭进行脱色，既能达到脱色的目的，又能使ε-PL因吸附作用而导致的损失降低。更多还原

【Abstract】 In order to enhanceε-poly-L-Lysine(ε-PL) production by Streptomyces albulusfermentation, the fermentation conditions and extraction technology ofε-PL fromculture broth were inverstigated as following:①explored the conditions ofε-PLfermentation in shake flask;②optimization of batch fermentation conditions ofε-PLproduction in 50 L autocontrol fermentor;③compare of different fed-batch methods;④the primarily study of the sepatation and extraction ofε-PL from culture broth.The effects of temperature, pH and culture medium amount onε-PL productionin shake flask were studied, it was a foundation work for optimization of batchfermentation in 50 L fermentor. The results indicated that the suitable temperature forS. albulus growth andε-PL polymerization is 30℃, the situation at 28℃wassimilar to 30℃, but the accumulation ofε-PL was lowest at 32℃; The productionofε-PL with pH control was enhanced to 1.59 g/L from 0.89 g/L without pH control;The culture medium volume of 100 mL in 500 mL shake flask was favorable for thegrowth of cells and synthesis ofε-PL.The researches showed that temperature, pH and stirrer speed played importantroles on the batch fermentation ofε-PL in 50 L autocontrol fermentation jar. Theoptimal temperature for S. albulus growth andε-PL synthesis was 30℃, the same asin shake flask, the low yield ofε-PL was accompanied by the low rate of cells growthat 32℃; The residual glucose concentration was still 20.1 g/L at pH3.0, low pHwould benefit neither cells growth norε-PL synthesis, the high accumulation(3.05 g/L)ofε-PL was achieved at pH4.0, and the high dry cell weight(12.88 g/L) wasobtained at pH5.0, however, when pH shifts to 5.0 or higher, the accumulatedε-PLwas depolymerized; High stirrer speed accompanying high dissolved oxygen wouldimprove the growth of cells and synthesis ofε-PL, and the dissolved oxygen wassufficient to meet the requirement of cells growth at 200 rpm, however over highstirrer speed such as 400 rpm would damage the mycelium so as to decreaseε-PLproduction.Under the optimized condition: 30℃, initial pH6.8, aeration 1 200 L/h, stirrer speed 200 rpm at prophase of culture, at anaphase, stirrer speed 300 rpm, pHcontrolled at 4.0, after 84.5 h culture, the yield and productivity ofε-PL was 3.21 g/Land 0.065 g.ε-PL/g.sub respectively, the yield ofε-PL was increased by 310% from0.78 g/L.Two different kinds of fed-batch culture were investigated, the results showedthat fed-batch culture was fitter forε-PL production with S.albulus than batch culture.In addition, pH control and variable speed fed-batch culture was superior to glucoseconcentration control and span fed-batch culture, both the yield and productivity ofε-PL with former tactic were higher than the latter, they were 7.36 g/L and 6.53 g/L,0.072 g.ε-PL/g.sub and 0.068 g.ε-PL/g.sub, respectively. The reason was likelyattributed to that the latter fed-batch culture way would bring a tremendous change ofculture system due to the addition of great volume nutriment with high concentration,and lead to the disorder of metabolism, but the former way could keep a continual andstabile condition for cells growth andε-PL synthesis.Then, the separation and extraction ofε-PL from broth was pilot studied. In theprocess of broth pretreatment, filtration was likely to leadε-PL losing; The weak acidcation-exchange resin D152 was choosed to separateε-PL from pretreated broth, theeluent was 0.1 N hydrochloric acid, velocity of flow was 2.5 mL/min, theconcentration ofε-PL was condensed by 229%, coefficient of recovery reached78.83%; decolorized with activated charcoal could decrease the loss ofε-PL.更多还原