【作者】 徐艳萍；

【导师】 李华钟；

【作者基本信息】 江南大学 ， 环境工程， 2004， 硕士

【摘要】 聚?-谷氨酸(?-PGA)是由微生物发酵合成的一种由D-和L-谷氨酸通过?-谷氨酰键,在合成酶系的作用下形成的一种胞外氨基酸高聚物。聚?-谷氨酸具有增稠、乳化、凝胶、保湿和粘结等功能特性,是一种生物相容性和可完全降解的新型天然高分子材料。在食品、日化、石油、农业、制药及生物工程领域具有广泛的应用前景。本论文以地衣芽孢杆菌(Bacillus licheniformis WBL-3)为聚??谷氨酸的生产菌株,通过诱变筛选出高产菌株,研究了摇瓶中的营养条件和环境条件对聚?-谷氨酸的影响,5L台式小型发酵罐上不同溶氧对聚?-谷氨酸的影响,以及碳源的代谢机制。主要研究结果如下:1.通过亚硝基胍和60Co诱变,筛选到一株遗传性能稳定的高产突变株Bacillus licheniformis C9,其聚?-谷氨酸的产量由诱变前的9.44g/L提高到19.76g/L,提高109%。2.考察了各种摇瓶条件对Bacillus licheniformis C9发酵合成聚?-谷氨酸的影响。在单因素实验的基础上,采用L9(34)正交表安排发酵培养基中碳源、氮源及其浓度的正交优化实验,得出培养基中碳源的浓度为:柠檬酸12g/L,甘油80g/L,L-谷氨酸23g/L,氯化铵7.0g/L。在此实验基础上,又对磷酸氢二钾、硫酸镁、三氯化铁、氯化钙、硫酸锰、钼酸氨进行了L25(56)正交实验。正交实验的结果为:三氯化铁0.06g/L,氯化钙0.40g/L,钼酸氨0.20g/L,硫酸镁0.60g/L,磷酸氢二钾0.60g/L,硫酸锰0.104g/L。进一步获得了聚?-谷氨酸摇瓶发酵较优的环境条件组合为:初始pH7.0,装液量50ml/250ml,接种量5%,温度37℃。3.在5L搅拌式发酵罐中分批培养生产聚?-谷氨酸,培养温度为37℃,通气量1.17vvm的情况下,不控制pH时,发酵液pH迅速下降到5.3,48h细胞干重达到6.22g/L,发酵64h,聚?-谷氨酸产量最高为26.82g/L。pH恒定控制在6.5时,40h细胞干重达7.48g/L,发酵48h,聚?-谷氨酸产量最高为29.16g/L。4.在5L搅拌式发酵罐中,pH恒定为6.5,培养温度37℃,通气量1.17vvm情况下,不同溶氧对聚?-谷氨酸分批发酵的影响有较大差异。考察了不同的搅拌转速及添加2%正十二烷对菌体生长、聚?-谷氨酸合成及消耗碳源的影响。研究发现,溶氧对菌体生长和聚?-谷氨酸合成形成明显的影响。添加2%正十二烷时,聚?-谷氨酸产量为38.06g/L,比转速为600r/min时提高了30.5%,说明添加2%正十二烷是一种有效的方式。5.通过改变发酵培养基的碳源,考察了不同碳源的作用。柠檬酸、谷氨酸是合成聚?-谷氨酸的碳源。柠檬酸经过TCA循环到达?-酮戊二酸,进而转化为谷氨酸,合成聚?-谷氨酸。对发酵液进行高压液相分析,发现有乙醇、乙酸、丙酮酸等代谢产物生成。由于培养基中甘油的添加量较大,甘油进入EMP途径,产生ATP,为微生物活动提供大量能量。更多还原

【Abstract】 ?-Poly(glutamic acid)(??-PGA) is a water-soluble macromolecular peptide that consists of D-and L-glutamic acids and is polymerized by??-glutamyl bonds.??-PGA is produced by a variety of Bacillus species and is freely excreted into the culture broth.It holds thickeners、humectants、substained release materials、form films and fibers.The polymer has been expected to be a new industrial biodegradable resources,which will solve some of the world-wide environmental problems,abandoning of waste plastic.Moreover,its use in the fields of food,cosmetics,or medicine has been proposed,and biodegradable fibers and hydrogels have already been made from ?-PGA.The polymer firmly stands as a potential candidate for replacing currently used nonbiodegradable water-soluble polymers. In this study,a high productive mutant of ?-PGA was obtained from Bacillus licheniformis WBL-3 with mutagenesis。The mutritional and environmental conditions were investigated。The effects of dissolved oxygen on the batch production of ?-PGA in a 5 L stirred fermentor by Bacillus licheniformis C9was studied。 Metabolic mechanism of carbon source of the production strain was studied。Main results are as follows:A high productive mutant of ?-PGA was obtained from Bacillus licheniformis WBL-3 with mutagenesis by NTG and 60Co。The productivity of ?-PGA was increased from 9.44g/L to 19.76g/L,the mutant showed genetic stability。The effects of shake flask conditions of Bacillus licheniformis C9 on the ?-PGA were investigated。According to the single-factor experiments,the L9(34) orthogonal experiments were carried out to optimize the concentrations of the nutrients。Aiming at achiving high ?-PGA concentration,the optimal mass concentrations of the nutrients were gained as following:citric acid 12.0g/L,glycerol 80.0g/L,L-glutamic acid 23.0g/L,ammonium chloride 7.0g/L. According to the single-factor experiments,the L25(56) orthogonal experiments were carried out to optimize the concentrations of the nutrients.The optimal mass concentrations of the nutrients were gained as following:FeCl3?6H2O 0.06g/L,CaCl2?2H2O 0.40g/L,(NH4)6Mo7O24?4H2O 0.20g/L,MgSO4 0.60g/L,K2HPO4 0.60g/L,MnSO4?H2O 0.104g/L.The environmental conditions of ?-PGA production were also investigated and an optimal combination was developed:an initial pH of 7.0,a medium content of 50 mL medium in 500mL flask,and an inoculum size of 5%,temperature 37℃.The mode of pH control is important factor for batch ?-PGA fermentation.At suitable culture conditions of temperature 37℃,aeration 1.17vvm.When Bacillus licheniformis C9 was cultivated without pH control,cell growth and ?-PGA production were inhibited to a certain extent due to the fact that pH decreased dramatically.At fermentation time 48h,DCW reached <WP=7>6.22g/L,???h ?-PGA concentration reached 26.82g/L.At pH6.5 and fermentation time 40h,DCW reached 7.48g/L,???h ?-PGA concentration reached 29.16g/L.Batch fermentation of ?-PGA in 5.0L bioreactor was carried out.The effect of dissolved oxygen(DO)concentration on the batch production of ?-PGA in a 5L stirred fermentor by Bacillus licheniformis C9 was studied.It was found that DCW and ?-PGA production were greatly influenced by dissolved oxygen.When add 2%dodecane,??-PGA concentration higher than agitation rate 600. At fermentation time 48h,??-PGA concentration reached 38.06%,which increased 30.5% comparison to agitation rate 600.We undertook a set of experiments in which certain carbon sources were removed from medium in order to examine the role of each carbon source in terms of growth and ?-PGA production.It is presumed that L-glutamic acid is produced from citric acid via isocitric acid and ??keto glutaric acid in the TCA cycle.The presence of ethanol、acetic acid、pyruvic acid is indicative with HPLC.Glycerol was transforms to ATP via EMP cycle,which provides a great deal of energies for microorganism.更多还原