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

发布时间：2018-06-05 03:09

本文选题：B-藻红蛋白 + 高效纯化　；参考：《中国科学院烟台海岸带研究所》2017年博士论文

【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/m L,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。
[Abstract]:Photosynthesis is an ancient and important chemical reaction that can be used to absorb light energy efficiently and convert light energy into bioenergy. The algal body is the main light trap complex in red and cyanobacteria, and one of the two types of light trap protein complexes of photosynthetic oxygen species. The algal gallbladder is composed of algin and connexin. Phycogallinin (phycogallinin) is formed by a covalent bond linked to the phycogallinic and devalent proteins through a covalent bond. By forming an ordered phycogallbladder, the phycogallbladder can absorb different wavelengths of light energy, and energy can be transferred to the light reaction center with more than 95% efficiency in the algal gallbladder. In recent years, it is widely used in biomedicine and other fields. In recent years, recombinant alginin was constructed by gene engineering, which not only provides a new way to study the energy transfer of alginin, but also provides a new way for the development of biosensors. In this paper, the preparation, spectral characteristics and Application of natural and gene recombinant alginin are introduced in this paper. The following aspects are studied: 1. using Porphyridium cruentum as the material, the efficient separation and purification of B- phycoerythroprotein (B-PE) was studied. First, the cells were broken by osmotic pressure, and B-PE was released from the purple Chlorella to the solution. Then, the ultrafiltration, ammonium sulfate salting out and chitosan adsorption were used to carry out B-PE respectively. Finally, the purified B-PE was purified by SOURCE 15Q ion exchange chromatography. The purified B-PE was obtained, the purity (A565/A280) was up to 5.1, and the recovery rate was up to 68.5%. The reference.SDS-PAGE electrophoresis for commercial production analysis B-PE showed that the alpha and beta subunits of B-PE were 18~20 kDa, and the molecular weight of the gamma subunit was about 27 kDa.. -PE has 2 absorption peaks at 545 nm and 565 nm, with 1 acromion at 498 nm. The fluorescence emission peak is analyzed by 575 nm and 620 nm. for B-PE in 250~750 nm circle two chromatography (CD) data, indicating that B-PE in the near ultraviolet region 260 nm and 305 peaks have two peaks, respectively from phenylalanine and tryptophan, and that two aromatic amino acids may be in common in sparsely. In the protein microenvironment of water, it is speculated that B-PE is in the two positions of PEB139 alpha /PEB158 beta and PEB82 alpha /PEB82 beta, forming a coupled exciton pair, and the energy transfer is carried out in the form of exciton splitting in the 4 coupling molecules, and the energy transfer is carried out by Forster resonance among the other chromaticity. Finally, the way of.2. to MAC engineering is predicted for the pathway of energy transfer in B-PE. The culture conditions of the strain were optimized, and 3 times of 10 L density fermentation were carried out. The expression of.MAC (streptavidin - phycocyanin alpha subunit fusion protein) which expressed a large number of MAC (streptavidin - alpha subunit fusion protein) was up to 43%, the bulk density of OD600 reached 12.5, and the total amount of wet weight of MAC fungus reached about 400 g. subsequently. The crushing conditions of MAC were optimized, and the results of chromatographic purification of.SDS-PAGE showed that the purified MAC had only one subunit, the molecular weight was near 76 kDa, and its molecular weight was in accordance with the expected protein molecular weight. There were 3 absorption peaks in the near ultraviolet visible light region, which were located at 340 nm and 370 nm and 625 nm, and at 575 nm, there was a shoulder peak, M. The maximum emission peak of AC is located at 640 nm, and the absorption peak in circular two chromatography is the same as that in the absorption spectrum. When the algirin or aromatic amino acids are excited, the fluorescence emission peak of 640 nm can be obtained, indicating that the energy can be transmitted to the chromophore through the algirin or aromatic amino acids. The spectral results show that the MAC is correct. A glucose biosensor based on MF0 (recombinant phycocyanin alpha subunit) and graphene oxide (GO) was constructed with good optical activity.3.. First, the modified graphene oxide was modified with low molecular weight chitosan (CS), and the CS-GO complex.GO-CS could nonspecifically adsorb maltose binding protein (MBP) on MF0 and cause the MF0 fluorescence. Light quenching. When glucose exists in the system, MBP can adsorb glucose specifically, causing MF0 to not adsorb GO-CS and increase the fluorescence intensity. Through the change of fluorescence intensity, the glucose content can be qualitatively and quantitatively analyzed. The linear range of the glucose biosensor is 0.1~1 mg/mL, and the minimum detection limit (LOD) is 0.05 mg/m L, with high sensitivity and selective.4. selection of 7 different characteristics of phycogallinin as dye sensitized TiO 2 photoanode, assembled into dye sensitized solar cell (DSSC) and studying its photoelectric properties. The results show that B-PE can obviously improve the photoelectric properties of DSSC, obtain the short-circuit current of DSSC, open circuit voltage, filling factor and light. The efficiency of electric conversion is 0.809 A/cm2,0.545 V, 0.569 and 1%. respectively. The collagen / carboxyl carbon nanotube / polyacrylamide composite gel is not only beneficial to the DSSC packaging, but also improves the photoelectric conversion efficiency and the short circuit current of the photocell. It can be used as the quasi solid state electrosolution and promote the practical application of the dye sensitized solar cell. Using the crystal structure and the circular two chromatographic data, the IPCE and ICE spectra of the algin sensitized DSSC were analyzed, which provided a new way for the study of the structure and function of the algin.
【学位授予单位】：中国科学院烟台海岸带研究所
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TM914.4

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