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离子液体溶解角蛋白过程及机理研究
张振磊
学位类型硕士
导师聂毅 ; 张锁江
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业化学工程
关键词角蛋白 二硫键 离子液体 溶解条件 水含量
摘要

角蛋白作为一种丰度高、来源广的可再生天然高分子,由于其良好的材料力学性能及生物相容性,在可降解材料、医用品及其他功能性材料方面具有广泛用途。利用角蛋白的前提是溶解角蛋白,然而角蛋白中复杂的二硫键交联结构导致其较难被传统溶剂溶解。离子液体作为一种绿色高效溶剂在溶解角蛋白等生物高分子方面的应用逐渐受到关注,其可用于溶解废弃羊毛、羽毛等提取角蛋白,或者以离子液体为溶解介质进一步制备角蛋白基复合功能材料。清晰认识离子液体溶解角蛋白机理,特别是离子液体对二硫键的影响是设计与筛选功能化离子液体溶解角蛋白的基础;此外,通过离子液体阴阳离子种类与溶解条件可调控角蛋白的溶解量及再生角蛋白结构与性能,获得这些变量对再生角蛋白二硫键含量等性质的影响规律可为优化离子液体溶解角蛋白工艺提供基础数据支持;最后,离子液体回收问题是其工业化应用的技术瓶颈之一,水常作为角蛋白再生的凝固浴,如何较大规模地从凝固浴中回收循环利用离子液体的问题也亟待解决。针对上述三个问题,围绕对羊毛角蛋白溶解具有重要影响的二硫键,本论文结合实验、谱学表征与模拟计算从不同尺度上研究获得离子液体与角蛋白的相互作用机制和构-效关系的共性关键科学问题,为设计高效离子液体溶解角蛋白以及离子液体溶解角蛋白的工业化应用提供科学基础。论文的创新性及主要研究成果如下:(1)采用含二硫键模型化合物氧化型谷胱甘肽,考察离子液体对二硫键影响。研究发现,模型化合物再生后特征峰消失,分子量降低,并检测到硫化氢生成,首次证明离子液体可以还原断裂二硫键。进一步使用14种离子液体,1-丁基-3-甲基咪唑卤盐 ([Bmim]Cl/[Bmim]Br)、1-乙基-3-甲基咪唑氯盐 ([Emim]Cl)、1-己基-3-甲基咪唑氯盐 ([Hmim]Cl)、1-丁基-3-甲基吡啶氯盐 ([Bpy]Cl)、1,2,3,4-四丁基季铵氯盐 ([N4444]Cl)、1,2,3,4-四丁基季磷氯盐 ([P4444]Cl)、1-烯丙基-3-甲基氯化咪唑 ([Amim]Cl)、1-丁基-3-甲基咪唑醋酸盐([Bmim]OAc)、1-乙基-3-甲基咪唑醋酸盐 ([Emim]OAc)、1-乙基-3-甲基咪唑磷酸二甲酯 ([Emim]DMP)、1-乙基-3-甲基咪唑磷酸二乙酯 ([Emim]DEP)、1-丁基-3-甲基咪唑磷酸二丁酯([Bmim]DBP)、1-丁基-3-甲基咪唑磷酸二氢盐([Bmim]H2PO4)),溶解羊毛角蛋白考察离子液体种类对再生角蛋白中二硫键含量及微观结构的影响,同样地,在溶解过程中检测到硫化氢生成,并且发现离子液体对角蛋白中二硫键具有差异化的破坏能力,咪唑醋酸类离子液体可断裂角蛋白中90 %以上的二硫键,而季铵或季磷类离子液体对二硫键基本无破坏能力。并且利用13C-NMR分析再生角蛋白二级结构信息发现,离子液体的二硫键断裂能力越强,再生角蛋白中α-helix含量越低,β-sheet与无定型结构含量越高。通过分子模拟研究发现离子液体断裂二硫键能力越强,其与胱氨酸的相互作用能越强,离子液体在胱氨酸周围的分布密度越大。(2)在相同溶解条件下,对比离子液体阴阳离子种类对其破坏二硫键性能的影响。通过再生角蛋白二硫键含量表征发现,阴离子断裂二硫键的能力:[Emim]+系列 [OAc]- > Cl- > [DEP]- > [DMP] –;[Bmim]+系列 [OAc]- > Cl- > Br- > [DBP]- > [H2PO4]-。阳离子断裂二硫键的能力为[Emim]+ > [Amim]+ ≈[Bmim]+ > [Hmim]+ > [BPy]+ > [P4444]+ > [N4444]+。此外,在离子液体阴离子相同,阳离子含有咪唑环的情况下,咪唑环上侧链长度对离子液体断裂二硫键能力的影响并不显著。进一步,分别考察溶解温度、时间及气氛对再生角蛋白中二硫键含量、胱氨酸含量、硫元素含量以及结晶度的影响。研究发现:随着溶解温度、时间的增加,再生角蛋白中二硫键含量、胱氨酸含量、硫元素含量均不断降低;在N2气氛下溶解角蛋白,可减少再生角蛋白中二硫键断裂量。升高溶解温度及延长溶解时间均会导致再生角蛋白结晶度下降,但温度的影响更加显著。(3)水分子与离子液体形成氢键作用,通过分子动力学模拟研究发现,随水含量增加,离子液体[Emim]DMP阴阳离子之间的相互作用被削弱。通过表征再生角蛋白二硫键含量,发现水含量增加导致离子液体二硫键断裂能力逐渐降低,溶解角蛋白所需时间逐渐延长。为保证离子液体对角蛋白具有较好溶解能力,水含量(摩尔比,H2O/IL)应小于0.8。进一步设计开发了小试规模回收离子液体的降膜蒸发装置,经回收后离子液体水含量可低至4000 ppm左右,回收后仍能够实现对角蛋白的高效溶解。

其他摘要

Wool keratin is a kind of naturally abundant fibrous protein which exhibits excellent biocompatibility and mechanical properties within wide temperature range and unfolds beyond high limits. These properties have led to the remarkable development of keratin-based materials with applications in biodegradable materials, biomedicine and other functional materials. Achieving the dissolution of keratin is premise to use it, however, complicated disulfide bond network in keratin is an obstacle for its dissolution by traditional solvents. In recent years, applying ionic liquids (ILs) to dissolve keratin has aroused the attention of researchers. It has potentially broad application to use ILs extracting keratin or serve ILs as dissolving medium to further prepare keratin-based composite materials. The foundation to screen and design functional ILs dissolving keratin is to fully clarify dissolution mechanism of keratin in ILs, especially ILs’effects on disulfide bond.Further, solubility of keratin in ILs and properties of regenerated keratin could be manipulated by changing types of cations and anions or dissolution conditions, the rule of these dissolution variables’effects on properties of regenerated keratin would provide basic date for optimizing keratin dissolution parameters. Finally, ILs recycling is the bottle neck for ILs’industrial application, water is often used as antisolvent to regenerate keratin from IL/keratin solution, the large-scale recycling of ILs from these solution is urgently to be dealt with. The breakage of disulfide bonds, which is the key point for keratin dissolution in ILs, the method combining experiments, spectrum characterization and molecular simulation was used to investigate interaction mechanism and structure activity relationship for the dissolution of keratin in ILs. These would supply scientific foundation for designing efficient ILs to dissolve keratin and accelerating its industrial utilization.The innovative results of the dissertation are as follows.(1) Oxidized glutathione (GSSG) was chosen as the disulfide bond-containing model compound to investigate the change of disulfide bond in ILs.After GSSG regenerating from ILs, the molecular weight of GSSG was decreased and H2S was detected,implying that ILs could induce the reductive cleavage of disulfide bond. What’more, 14 kinds of ILs,1-butyl-3-methylimidazolium halide ([Bmim]Cl/[Bmim]Br), 1-ethyl-3-methylimidazolium chloride ([Emim]Cl), 1-hexyl-3-methylimidazolium chloride ([Hmim]Cl),1-butyl-pyridinium chloride ([BPy]Cl),tetrabutylphosphonium chloride ([P4444]Cl), tetrabutyl-ammonium chloride ([N4444]Cl), 1-allyl-3-methylimidazolium chloride ([Amim]Cl), 1-butyl-3-methylimidazolium acetate ([Bmim]OAc, [Emim]OAc), 1-ethyl-3-methylimidazolium dimethylphosphate ([Emim]DMP), 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP), 1-butyl-3-methylimidazolium dibutylphosphate ([Bmim]DBP), and 1-butyl-3-methlimidazolium dihydrogen phosphate ([Bmim]H2PO4), were used to investigate the influence of ILs types on the disulfide bond content and properties of the regenerate wool keratin. During the dissolution process, H2S was also detected, and these ILs showed differentiated capability to cleave disulfide bonds. Imidazolium-based acetate could cleavage 90 % of disulfide in keratin while quaternary ammonium and phosphonium ILs had nearly no capability to cleave disulfide bond. 13C-NMR was used to analysis keratin’s secondary structure distribution, the less content of α-helix structure, more content of β-sheet and disordered structure and less recovery percentage of keratin were observed with increasing disulfide bond cleaving ability of ILs. Through molecular simulation, it can also be found that the ILs’ ability of cleaving disulfide bond was closely linked with interaction energy between cystine and ILs and the distribution density of ILs around cystine.(2) Under same dissolution condition, different cations and anions in ILs can lead to varied cleaving ability of disulfide bond in keratin. For [Bmim]+-based ILs, the order of the ability to cleave -S-S- followed: [OAc]- > Cl- > Br- > [DBP]- > [H2PO4]-. For the ILs incorporating [Emim]+ cation, the order was [OAc]-> Cl- > [DEP]- > [DMP] –. The effectiveness of the cations in cleaving disulfide bond was found to be [Emim]+ > [Amim]+ ≈[Bmim]+ > [Hmim]+ > [BPy]+ > [P4444]+ > [N4444]+. Further, the side chain lengths of imidazole cation have little effect on the ILs’ disulfide bonds cleaving ability. In addition, the effects of dissolution temperature, time and atmosphere on disulfide bond, cystine, sulphur content and degree of crystallinity were studied. With dissolution time and temperature rising, the content of disulfide bond, cystine and sulfur in regenerated keratin decreased. And the dissolution of keratin under N2 could reduce the cleavage of disulfide bond. Either rising dissolution temperature or increasing dissolution time could lead to the decrease of degree of crystallinity of regenerated keratin, however, temperature’s effect was more obvious.(3) Water molecule could interact with ILs to form hydrogen bond, molecular dynamic simulation found that the interaction energy between cation and anion in ILs decreased with water content increasing, which could reduce ILs’capability to cleave disulfide bond and increase its dissolution time for wool keratin. To guarantee the ILs’ dissolving ability for wool keratin, the water content in ILs should be less than 0.8 (mole ratio, H2O/IL). Finally, fall-film evaporator was developed to recycle ILs, and the water content can be reduced to 4000 ppm after evaporating, which could still efficiently dissolve keratin. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/23592
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张振磊. 离子液体溶解角蛋白过程及机理研究[D]. 北京. 中国科学院研究生院,2016.
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