Knowledge Management System Of Institute of process engineering,CAS
|Thesis Advisor||赵庆生, 查圣华|
ε-聚赖氨酸（ε poly-l-lysine，简称ε-PL）产自白色链霉菌，为25-35个赖氨酸通过异肽键脱水缩合形成的高分子等聚物抗菌肽。作为一种天然绿色安全的生物防腐剂，成为近年来研究的热点，其产量经过多年菌种选育和发酵工艺不断优化有了显著的提高，但后续分离纯化工艺的研究不足，导致ε-PL纯度和回收率较低。自然界目前天然同型氨基酸聚合物仅有ε-PL和γ-PGA两种，其中ε-PL结构和抑菌机制还未曾探索清楚，限制了其在国内的开发和利用。本文以ε-PL为研究对象，进行了分离纯化工艺优化、放大生产试验以及动态结构和抑菌活性等研究，为降低ε-PL生产成本和产品应用开发奠定了基础。本论文得到的主要研究成果如下： （1）分离纯化研究。总体分为固液分离-离子交换-脱盐干燥三个阶段。在固液分离阶段联合离心与10 KDa聚砜超滤膜，能最大程度分离杂质，减少对后续离子吸附树脂的污染导致交换能力降低。离子交换阶段使用单弱酸性阳离子柱，节省酸碱和水用量，加入40%中性乙醇洗脱除杂质，IRC50弱酸性阳离子树脂吸附量达到0.17 g ε-PL/g（干重）。脱盐脱色阶段采用二级3000 Da聚砜超滤膜，解决了原来活性炭和大孔吸附树脂用于ε-PL脱色回收率低和纳滤膜脱盐效率低的问题。喷雾干燥产品微观结构为圆球状，表面均匀。优化后的分离纯化工艺可使ε-PL纯度和回收率最终分别达到98.7%和90%。采用板框过滤替代蝶式离心机，降低了用水量，完成了8 m3放大生产验证试验。（2） 动态结构研究。对ε-PL在溶液中和固体粉末的构象进行研究，通过动态光散射（DLS）测定ε-PL在pH 8.0时左右平均粒径最大达到800 nm，常态下ε-PL带阳离子电荷会与聚砜膜静电吸附以及与杂质或自身形成较大的聚集体，沉积在膜表面；通过圆二色光谱（CD）和侧链氨基甲氨酰化修饰表明ε-PL在酸性溶液中呈现静电扩张构象，当溶液pH高于等电点，氨基电荷消失，不再抑制分子间氢键的形成，ε-PL构象会转变为β-折叠构象，形成稳定结构；通过红外光谱拟合计算出酸性ε-PL溶液喷雾干燥过程中会有一小段肽链结构转变为β-折叠；二维核磁共振表明常态下ε-PL在溶液中氢原子间的空间距离大于0.5 nm。（3）抑菌性能研究。对ε-PL抑菌活性和机制展开初步研究，发现冷冻干燥相对于喷雾干燥能更好地保留ε-PL的抑菌活性，枯草芽孢杆菌最小抑菌浓度为1 mg/mL；ε-PL抑菌活性受到pH影响较小，在中性条件下活性最强，阳离子静电吸附在抑菌过程中促使ε-PL与细胞膜表面磷脂结合速率加快。（4）结构改性探索。通过烷基化改性后活性与氨基甲氨酰化改性后活性对比，证明引入疏水基团可以略微增强ε-PL抑菌活性，后者活性的消失表明ε-PL主链可能在ε-PL与细胞膜结合使其通透性改变的过程中起到作用。;ε-poly-L-lysine (ε-PL) consists of 25-35 lysine residues which are linked by isopeptide bonds formed by dehydration condensation of α-carboxyl and ε-amino groups, having good antibacterial activity and broad-spectrum inhibition range. As a natural and safe biological preservative, it has become a research hotspot in recent years, and its output has been significantly increased after years of strain breeding and fermentation strategies optimization. However, insufficient research on subsequent separation and purification processes has resulted in lower purity and recovery of ε-PL; at the same time, the structure and antibacterial mechanism of ε-PL, which is one of the only two homologous amino acid polymers in nature, have not been revealed clearly. In this study, ε-PL was taken as the object of the study to optimize its industrial green short-process production, study the dynamic secondary structure and antibacterial activity, which laid a foundation for reducing production cost and product application development of ε-PL. The main research results obtained in this paper are as follows:Research on separation and purification. The whole production is divided into three stages: solid-liquid separation-ion exchange-desalination drying. The 10 KDa polysulfone ultrafiltration membrane was used in the solid-liquid separation stage, which separated impurities to the greatest extent and reduced the contamination of subsequent ion adsorption resins, resulting in a decrease in exchange capacity. In the ion exchange stage, the secondary anion column was removed, and 40% neutral ethanol was added to elute and remove impurities. The purity of the product reached 98%, and the amount of acid, alkali and water was decreased. IRC50 adsorption capacity reached 0.17 g ε-PL/g resin (dry weight). 3000 Da polysulfone ultrafiltration membrane was used in the decolorization and desalination stage, which solved the problems of low recovery rate of activated carbon and macroporous adsorption resin and low desalination efficiency of the nanofiltration membrane; The microstructure of the spray-dried product was spherical and the surface was uniform. The ε-PL purity and recovery of in the optimized separation and purification process reached 98.7% and 90%, respectively. The plate and frame filtration was used instead of butterfly centrifuge, resulting in the decrease of the water consumption; and 8m3 scale-up production verification test was completed.Research on dynamic structure. The conformation of ε-PL in solution and solid powder was studied. Through dynamic light scattering (DLS), the average particle size of ε-PL was as high as 800 nm with pH 8.0. Under normal conditions, ε-PL had cationic charges, it interacted with the polysulfone membrane and impurities, was electrostatically adsorbed and accumulated on the membrane surface. Through circular dichroism spectroscopy (CD) and side chain carbamoylation modification, ε-PL exhibited an electrostatically expanded conformation in the acidic solution. When the pH of the solution was higher than the isoelectric point, the amino charge disappeared, the formation of intermolecular hydrogen bonds was no longer inhibited, and the conformation of ε-PL changed to the β-sheet conformation, forming a stable structure. The infrared spectrum fitting calculation showed that part of the conformation of the acidic ε-PL solution was converted into β-sheet during the spray drying process. Two-dimensional nuclear magnetic resonance showed that under normal conditions, the spacing between ε-PL hydrogen atoms in the solution was greater than 0.5 nm. Research on the antimicrobial activity and mechanism of ε-PL. It was found that freeze-drying could retain its antibacterial activity better than spray drying. The minimum antibacterial concentration of Bacillus subtilis was 1mg/mL; ε-PL had the strongest activity under neutral conditions, but the change of pH value had no particularly significant effect on the antibacterial activity of ε-PL. Electrostatic adsorption of cations accelerated the binding rate of ε-PL to phospholipids on the surface of the cell membranes during the antibacterial process.Exploration on structural modification. We compared the activity after the alkylation modification with the activity after the carbamoylation modification, revealing that the introduction of hydrophobic groups slightly enhanced the antibacterial activity of ε-PL. The disappearance of the latter activity indicated that the ε-PL main chain might plays a role in the process of combining with the cell membrane to change its permeability.
|刘家宁. ε-聚赖氨酸分离纯化、动态结构及抑菌活性研究[D]. 中国科学院大学,2020.|
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