摘要
γ-聚谷氨酸(Poly γ-glumatic acid,γ-PGA)是通过微生物合成的阴离子型均聚氨基酸。由于具有良好的生物相容性、生物降解性和高吸水性,γ-PGA及其衍生物可广泛用于食品、化妆品、医药和水处理等领域。目前通常采用微生物液体发酵实现γ-PGA的规模化生产,但在发酵后期发酵液黏度增加会导致产物积累的速率明显下降。
本文首先采用气相色谱研究γ-PGA发酵过程中的主要代谢副产物,确定了2,3-丁二醇和3-羟基-2-丁酮为积累量最大的碳代谢副产物。然后在摇瓶发酵实验中考察了这两种副产物对细胞生长和γ-PGA合成的影响,结果显示,两种副产物对发酵不同阶段的细胞生长和产物合成都存在抑制作用。进一步研究了三种不同pH值下发酵过程中主要胞外代谢产物的积累情况,通过对γ-PGA相关碳代谢途径的代谢通量分析,发现最佳发酵过程pH值为6.5。在这一最适pH下,发酵代谢副产物积累总量最少,而细胞生物量、底物转化率和产物生成的速率均为最优。
为了寻找经济价廉碳源,本文筛选获得了能够利用木糖和葡萄糖高效生产γ-PGA的枯草芽孢杆菌HB-1。该菌株能够以稀酸水解处理秸秆得到的六碳糖和五碳糖混合物为培养基碳源发酵生产γ-PGA,批次发酵结果表明该生产菌能够有效利用这一粗原料和前体谷氨酸,γ-PGA产量达到24.38g/L,并能通过流加秸秆水解糖将γ-PGA的产量提高到28.15g/L。最后,开展了发酵液微滤除菌、超滤浓缩、沉淀脱盐和喷雾干燥等工艺参数优化研究,确定了一条高效节能的中试工艺路线。
本论文对微生物发酵生产γ-PGA的代谢过程、发酵工艺和分离纯化进行了深入研究,探索了副产物积累和粗原料利用等新的途径和工艺,具有应用于γ-PGA工业化生产的潜力。
Poly (y-glutamic acid)(y-PGA) is an extracellular anionic polymer produced by microbial fermentation. Possessing several beneficial characteristics such as water-soluble, biocompatible, biodegradable and non-toxic, y-PGA and its derivatives can be wildly used in food, cosmetic, medicine and water treatment industry fields. Although microbial fermentation is a common approach producing y-PGA, the broth at the end stage usually becomes very viscous and severely affects the biosynthesis of y-PGA.
Firstly, the present work applied GC analysis to confirm that acetoin and2,3-butanediol were the main byproducts during the fed-batch fermentation and their effects on the cell growth and y-PGA biosynthesis were further investigated in shake flasks. The outcome indicated that both acetoin and2,3-butanediol showed clear impairment on y-PGA production of B. subtilis ZJU-7. Moreover, the extracellular metabolites profiles of fermentation under three different pH values were acquired and the metabolic flux redistribution of pathways related to y-PGA biosynthesis was calculated from the collected data. As a result, the metabolic flux favored to distribute toward glycolytic pathway at pH6.5, in which the contents of byproducts were lower, the ingestion rate of extracellular glutamic acid was higher, and the subsequent y-PGA biosynthesis was enhanced.
The increasing applications of y-PGA have motivated us to exploit cheap and renewable carbonsources as the major fermentation feedstock. The strain Bacillus subtilis HB-1was screened out with the capacity of producing y-PGA efficiently using xylose and glucose. The corncob fibers were hydrolyzed with0.25mol/L HCl and turned into syrup containing both hexoses and pentoses. Then the corncob fibres hydrolysate (CFH) has been validated to support efficient production of y-PGA by HB-1. In batch fermentation, this strain could efficiently convert the raw substrate and glutamate into24.38g/L y-PGA. In the fed-batch experiments using CFH, the titer of y-PGA was increased up to28.15g/L. At last, the operating parameters were optimized to improve the performance of y-PGA isolation process including micro-filtration, ultrafiltration, desalination, and spray drying. An efficient and energy-economic process was proposed for recovery and purification of y-PGA at pilot scale.
This thesis presented a series of research work about the metabolic profiling, fermentation process and purification procedure of microbial derived y-PGA, providing some new insights and new approaches into the biosynthesis and scale up production of this biopolymer.
引文
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