【作者】 张博；

【导师】 康艳红；

【作者基本信息】 沈阳师范大学 ， 分析化学， 2018， 硕士

【摘要】 蚕蛹蛋白因含有大量的羟基、氨基等活性基团,其水解后所得小分子多肽具有生物活性功能等,在生物医药等领域的应用日益广泛。然而,目前工业上蚕蛹蛋白的制备一直延用传统的碱溶酸沉处理法,存在生产流程长,产物收率低,环境污染重等诸多弊端。本课题组报道了以咪唑类离子液体为溶剂将蚕蛹蛋白从蚕蛹中溶解并再生出来,但该方所用离子液体仍具有较高的毒性且不可生物降解,无法达到绿色分离要求。本文以可再生生物质资源利用为背景,以氢键破坏和重构为科学基础,开展以蚕蛹为原料,离子液体为介质,利用可降解胆碱类离子液体进行从蚕蛹中提取蚕蛹蛋白的绿色过程研究,对促进生物质资源的绿色分离过程具有重要的意义。具体内容及研究结论包括:(1)开展了氨基酸胆碱类离子液体溶解蚕蛹分离蚕蛹蛋白的研究。采用酸碱中和法合成八种氨基酸胆碱类离子液体,~1H NMR及FT-IR光谱测试表明其纯度满足蚕蛹溶解实验的要求。光学显微镜观察下,氨基酸胆碱类离子液体很难完全溶解蚕蛹粉,即使反应24 h仍有部分甲壳素等未溶物质存在;改变反应时间和料液比对于[Ch][Gly]溶解蚕蛹粉的溶解率的影响并不显著,提高反应温度可明显降低[Ch][Gly]粘度从而提高离子液体溶解能力。其中,溶解反应时间不宜超过8 h,否则持续高温会造成蚕蛹蛋白结构被破坏且反应24 h时会造成[Ch][Gly]分解;反应温度超过80°C以上时,再生蛋白质易发生变性,而温度大于100°C时[Ch][Gly]分解产生的甘氨酸参与蚕蛹蛋白再生过程造成再生物收率非正常升高。(2)开展了脂肪酸胆碱类离子液体溶解蚕蛹分离蚕蛹蛋白的研究。通过对比离子交换法和酸碱中和法选用酸碱中和法合成了四种脂肪酸胆碱类离子液体,并通过~1H NMR、质谱及FT-IR对其结构及纯度进行表征,以确保可应用于蚕蛹蛋白溶解再生实验。考察四种脂肪酸胆碱类离子液体对蚕蛹蛋白的溶解分离效果,优选[Ch][OAc]作为溶剂并通过对反应时间、温度、料液比的调控确立了[Ch][OAc]溶解分离蚕蛹蛋白的最优实验条件;对最优条件下对所得再生产物进行细致表征,发现经[Ch][OAc]溶解后再生蚕蛹蛋白的形貌、结构及氨基酸组成成分均无明显变化,再生后蚕蛹蛋白的热稳定性稍有提高,但其结晶度略有下降;对最优实验条件进行初步中试放大,证明其具有进行工业化生产的可能。更多还原

【Abstract】 The protein from silkworm chrysalis has been widely used in the field of biological medicine,due to its various hydroxyl group and amino active group and hydrolysis products of it with high biological activity.However,the traditional method of preparation of silkworm chrysalis in the industry is alkaline dissolution and acid precipitation,which is long production process,low product yield and heavy environmental pollution.Our group previously reported imidazole ionic liquids as solvents to dissolve and regenerate of silkworm pupa protein from silkworm pupa.However,the used imidazole ionic liquid was high toxic and non-biodegradable,which was unable to meet the requirements of green separation.In this paper,we used renewable biomass resources as the material,hydrogen bond destruction and reconstruction as the science foundation,and biodegradable choline ionic liquid as the medium to extract protein from silkworm pupa.The extraction process,law and mechanism were studied in the paper,which was important to the biomass green separation.The specific content and research conclusions are listed as follows:(1)The study on the separation of protein from silkworm pupa was carried out by the amino acid choline ionic liquids(AAILs).Eight AAILs were synthesized by acid-base neutralization method.The synthesized AAILs were characterized by ~1H NMR and FT-IR spectra to determine their purity.The results showed that the synthesized AAILs were satisfied with the requirements of the dissolution experiments.Under the observation of optical microscope,the AAILs were difficult to completely dissolve the silkworm pupa powder,even if the experiment time was up to 24 h;the dissolution law was studied with[Ch][Gly]as solvent.The results showed that the experiment time and liquid ratio had little influence on the dissolution rate of silkworm pupa,and the higher temperature was good for protein extraction.The results showed that the optimal experiment conditions were 8 h and 80°C,because the longer time could break protein structure and the higher temperature could cause protein denaturation and[Ch][Gly]degradation.(2)The study on the separation of silkworm chrysalis was carried out by fatty acid choline ionic liquids(FCILs).The aicd-alkali neutralization method was finally used to prepare four FCILs by comparing with the ion exchange method.The synthesized FCILs were characterized by ~1H NMR,MS and FT-IR spectra to determine their purity.The performance of the four kinds of FCILs on protein separation were studied.Finally,[Ch][OAc]was chosen as the optimal solvent to explore experiment factors.The effect of experiment time,temperature,ratio of[Ch][OAc]and silkworm pupa on the protein extraction were studied to determine the optimal conditions.Under the optimal conditions,morphology,structure and amino acid composition of the extracted protein was almost same with the raw material.However,the thermal stability and the crystallinity of extracted protein increased and decreased slightly,respectively.更多还原