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题名:
新型藻胆蛋白的制备及其在生物传感和染料敏化太阳能电池中的应用
作者: 李文军1,2
学位类别: 博士
答辩日期: 2017
授予单位: 中国科学院大学
授予地点: 北京
导师: 秦松
关键词: B-藻红蛋白,高效纯化,重组藻胆蛋白,能量传递,生物传感器,染料敏化太阳能电池
学科分类: 地球科学::海洋科学
中文摘要:  光合作用是一种古老而重要的化学反应,通过捕光体系对光能高效的吸收,能够将光能转化为生物能。藻胆体是红、蓝藻中主要的捕光天线复合物,也是光合放氧生物的两大捕光蛋白复合物类型之一。藻胆体由藻胆蛋白和连接蛋白构成,藻胆蛋白则是由藻胆素色基与脱辅基蛋白通过共价键联接而成。藻胆蛋白通过构成有序的藻胆体,使藻胆体可以吸收不同波长的光能,并且能量在藻胆体内能够以95%以上的效率传递到光反应中心。藻胆蛋白由于具有优异的光学特性,被广泛应用于生物医学等领域。近年来通过基因工程构建的体外重组藻胆蛋白,不仅为研究藻胆蛋白的能量传递提供了新的途径,而且为开发生物传感器的提供了新的途径。本文对天然和基因重组藻胆蛋白的制备、光谱特性以及应用展开了以下几方面的研究:
1.以紫球藻(Porphyridium cruentum)为材料,研究了B-藻红蛋白(B-PE)的高效分离纯化方法。首先利用渗透压法对细胞进行破碎,使B-PE从紫球藻中释放到溶液中,然后分别利用超滤法,硫酸铵盐析法和壳聚糖吸附法对B-PE进行粗提,最后用SOURCE 15Q离子交换层析进行纯化。纯化后得到分析级的B-PE,纯度(A565/A280)可达5.1,回收率高达68.5%,为商业化生产分析级B-PE提供了参考。
SDS-PAGE电泳表明B-PE的α和β亚基分子量为18~20 kDa,γ亚基的分子量约为27 kDa。光谱数据表明,B-PE在545 nm和565 nm有2个吸收峰,在498 nm处有1个肩峰,荧光发射峰在575 nm和620 nm。对B-PE在250~750 nm范围内圆二色谱(CD)数据的解析表明,B-PE在近紫外区260 nm和305 nm有两个CD峰,分别由苯丙氨酸和色氨酸产生,两种芳香族氨基酸可能共同处于疏水的蛋白微环境中。推测B-PE在PEB139α/PEB158β和PEB82α/PEB82β两个位置,形成耦合的激子对,4个耦合分子内以激子分裂的形式进行能量传递,其它色基之间则以福斯特共振进行能量传递,最后对B-PE内能量传递的途径进行了预测。
2.对MAC工程菌株的培养条件进行了优化,进行了3次10 L密度发酵,获得大量表达MAC的菌体。MAC(链霉亲和素-藻蓝蛋白α亚基融合蛋白)的表达量占菌体可溶性蛋白的比率可达43%,菌体密度OD600最大达到12.5,收集到MAC菌体湿重总量达到约400 g。随后对工程菌的破碎条件进行优化,并对MAC的进行了层析纯化。
SDS-PAGE结果表明,纯化后的MAC仅有一个亚基,分子量为在76 kDa附近,与预期的蛋白分子量相符。MAC在近紫外-可见光区共有3处吸收峰,分别位于340 nm和370 nm和625 nm;在575 nm还有一个肩峰,MAC的最大荧光发射峰位于640 nm,圆二色谱中的吸收峰结果与吸收光谱中一致。当对藻胆素或者芳香族氨基酸进行激发时,能够获得640 nm的荧光发射峰,表明能量能够通过藻胆素或者芳香族氨基酸传递至发色团。光谱结果表明MAC有正确的构象,并且具有良好的光学活性。
3.构建基于MF0(基因重组藻蓝蛋白α亚基)和氧化石墨烯(GO)的葡萄糖生物传感器。首先用低分子量壳聚糖(CS)修饰氧化石墨烯,制得CS-GO复合物。GO-CS可以非特异性吸附MF0上的麦芽糖结合蛋白(MBP),造成MF0的荧光淬灭。当体系中存在葡萄糖时,MBP会特异吸附葡萄糖,造成MF0无法再吸附GO-CS,荧光强度增加。通过荧光的强度变化,可以间接对葡萄糖含量进行定性和定量分析。该葡萄糖生物传感器的检测线性范围为0.1~1 mg/mL,最低检测限(LOD)为0.05 mg/mL,具有较高的灵敏度和选择性。
4.选择7种不同特性的藻胆蛋白作为染料敏化二氧化钛光阳极,组装成染料敏化太阳能电池(DSSC)并研究其光电特性。结果表明B-PE能够明显提高DSSC的光电性能,得到DSSC的短路电流、开路电压、填充因子和光电转化效率为分别为0.809 A/cm2、0.545 V、0.569和1%。所构建的胶原蛋白/羧基化碳纳米管/聚丙烯酰胺复合凝胶,不仅利于DSSC封装,同时可增进光电转换效率,提高光电池的短路电流,能够作为准固态电解质,推进染料敏化太阳能电池的实际应用。结合晶体结构和圆二色谱数据,解析了藻胆蛋白染料敏化DSSC的IPCE和ICE光谱,为研究藻胆蛋白的结构和功能提供了新的途径。
 
英文摘要:  Photosynthesis is an ancient and important conversion of solar light to biological energy in photosynthetic organisms. This highly efficient process starts from the light capturing by light-harvesting antenna of photosynthetic RCs. Phycobilisomes system, in cyanobacteria and red algae is the one of two major light-harvesting systems of photosynthetic oxygen organisms. Phycobilisomes are aggregations of water-soluble phycobiliproteins and linker polypeptides. Phycobiliproteins are multi-subunit complex bearing covalently attached open-chain tetrapyrroles, known as phycobilins, orderly assembled into phycobilisomes system and enabling them to harvest light in the visible region of the spectrum. The absorbed energy can be transferred at almost 95% efficiency to the reaction center. Phycobiliproteins have been applied in the field biomedicine and DSSC for their unique molecular structures and spectral characterizations. In recent years, the reconstruction of phycobiliproteins by gene engineering technology,not only provided a new way for the research on the energy transfer process of phycobilisomes, but also provide conditions for the development and application of biosensors. Based on above facts, this study investigated the preparation of natural and recombinant phycobiliproteins,and then study the spectral characteristics and explore the application. Detailed results will be introduced in the following sections:
1. In this thesis, we discussed the isolation and efficient purification of B-phycoerythrin (B-PE) from microalga Porphyridium cruentum. Phycobiliproteins in Porphyridium cruentum was extracted by osmotic shock and initially purified by ultrafiltration, ammonium sulphate precipitation and chitosan adsorbent respectively. Further purification was carried out with a SOURCE 15Q exchange column Analytical grade B-PE was obtained with a purity ratio (absorbance ratio, A545/A280) of 5.1 and a yield of 68.5%. Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level.
The analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed a bulky band between 18 and 20 kDa which could be assigned to subunits α and β and a low intensity band of 27 kDa assigned to γ subunit. It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm, and displayed a fluorescence emission maximum at 580 nm and 620 nm.CD spectrum of B-PE in 250~750 nm was obtained and resolved for the first time. The three CD spectrum peaks of B-PE: 260 nm,305 nm and 330-380 nm, were considered to correlated to Phe, Trp and phycobilin respectively. Phe and Tyr might be in a conservative hydrophobic microenvironment PEB139α/ PEB158β and PEB82α/ PEB82β were consisted as two exciton-coupled bilin pairs. Energy transfer within exciton-coupled pairs was by exciton splitting, while between exciton-coupled pairs by Förster resonance. Potential energy transfer was obtained finally.
2. In this study, we optimized the culture conditions of the engineering strain MAC; 10L fermentation was performed 3 times to collect a large number of cells with recombinant protein of MAC. The expression rate of MAC reached 43% of the total soluble proteins and the final optical cell density of the broth was 12.5,the total wet weight of MAC is around 400 g. The process of ultrasonication was optimized by orthogonal test and then the MAC was purified by chromatography.
The analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed a bulky 76 kDa band as well as expected. It showed three absorption peaks at 628 nm, 340 nm, 370 nm and a shoulder peak at 575 nm, and displayed a fluorescence emission maximum at 640 nm. The CD spectrum peaks was the same with the absorption spectrum. We found that when aromatic amino acids and phycobilins were excited, fluorescence arose in visible region of the spectra, suggesting there is energy transfer pathway from aromatic amino acid residues and phycobilins to chromophores. Spectra results showed that the MAC has the correct conformation and good optical activity.
3. Glucose Biosensor Based on MF0 (α subunit of recombinant phycocyanin) and Graphene Oxide was designed. A novel graphene oxide (GO) nano-material conjugated with low molecular chitosan (CS) was synthesized through a rapid and simple covalent conjugation. Synthesized GO-CS was employed to detect glucose in a low concentration level based on the competitive binding with Maltose-binding protein (MBP) labeled MF0. In this designed biosensor system, the MF0 emission is quenched through the binding between GO-CS and MBP, while, in the present of glucose, MF0 fluorescence released after glucose competitively bind to MBP. Based on fluorescence signal recovery measurement, the target glucose was detected sensitively and selectively with the linear detection range from 0.1 mg/mL to 1 mg/mL. The limit of detection (LOD) for glucose is around 0.05 mg/mL. This biosensor system exhibits sensitivity and specificity properties for glucose.
4. Dye-sensitized solar cells were assembled using 7 different phycobiliprotein as sensitizer, TiO2 thin films as photoelectrodes, and their photoelectrical properties were studied. The result showed that the sensitization of B-PE was superior to other phycobiliprotein, eg could significantly improve the photoelectrical properties of DSSC. The short-circuit current, open circuit voltage, fill factor and photoelectric conversion efficiency of DSSC assembled with TiO2 thin films as photoelectrode were 0.809 A/cm2, 0.545 V,0.569 and 1% respectively. Coupling extended spectral response range and increased the photoelectric conversion efficiency of DSSC. The composite gel was syntheticed and had been proved to be stable and conducive to encapsulate. The composite gel can not only improve the photoelectric conversion efficiency, but also improve the short-circuit current of cell and can be used as an approximate solid electrolyte. The IPCE and ICE provided a new way for the research on the structure and energy transfer process of phycobiliprotein.
 
语种: 中文
内容类型: 学位论文
URI标识: http://ir.yic.ac.cn/handle/133337/22452
Appears in Collections:科研共享资源_学位论文
中科院烟台海岸带研究所知识产出_学位论文

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作者单位: 1.中国科学院大学
2.中国科学院烟台海岸带研究所

Recommended Citation:
李文军. 新型藻胆蛋白的制备及其在生物传感和染料敏化太阳能电池中的应用[D]. 北京. 中国科学院大学. 2017.
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