發(fā)布時(shí)間：2018-06-05 03:09

  本文選題：B-藻紅蛋白 + 高效純化��； 參考：《中國科學(xué)院煙臺(tái)海岸帶研究所》2017年博士論文

【摘要】：光合作用是一種古老而重要的化學(xué)反應(yīng),通過捕光體系對光能高效的吸收,能夠?qū)⒐饽苻D(zhuǎn)化為生物能。藻膽體是紅、藍(lán)藻中主要的捕光天線復(fù)合物,也是光合放氧生物的兩大捕光蛋白復(fù)合物類型之一。藻膽體由藻膽蛋白和連接蛋白構(gòu)成,藻膽蛋白則是由藻膽素色基與脫輔基蛋白通過共價(jià)鍵聯(lián)接而成。藻膽蛋白通過構(gòu)成有序的藻膽體,使藻膽體可以吸收不同波長的光能,并且能量在藻膽體內(nèi)能夠以95%以上的效率傳遞到光反應(yīng)中心。藻膽蛋白由于具有優(yōu)異的光學(xué)特性,被廣泛應(yīng)用于生物醫(yī)學(xué)等領(lǐng)域。近年來通過基因工程構(gòu)建的體外重組藻膽蛋白,不僅為研究藻膽蛋白的能量傳遞提供了新的途徑,而且為開發(fā)生物傳感器的提供了新的途徑。本文對天然和基因重組藻膽蛋白的制備、光譜特性以及應(yīng)用展開了以下幾方面的研究:1.以紫球藻(Porphyridium cruentum)為材料,研究了B-藻紅蛋白(B-PE)的高效分離純化方法。首先利用滲透壓法對細(xì)胞進(jìn)行破碎,使B-PE從紫球藻中釋放到溶液中,然后分別利用超濾法,硫酸銨鹽析法和殼聚糖吸附法對B-PE進(jìn)行粗提,最后用SOURCE 15Q離子交換層析進(jìn)行純化。純化后得到分析級(jí)的B-PE,純度(A565/A280)可達(dá)5.1,回收率高達(dá)68.5%,為商業(yè)化生產(chǎn)分析級(jí)B-PE提供了參考。SDS-PAGE電泳表明B-PE的α和β亞基分子量為18~20 kDa,γ亞基的分子量約為27 kDa。光譜數(shù)據(jù)表明,B-PE在545 nm和565 nm有2個(gè)吸收峰,在498 nm處有1個(gè)肩峰,熒光發(fā)射峰在575 nm和620 nm。對B-PE在250~750 nm范圍內(nèi)圓二色譜(CD)數(shù)據(jù)的解析表明,B-PE在近紫外區(qū)260 nm和305 nm有兩個(gè)CD峰,分別由苯丙氨酸和色氨酸產(chǎn)生,兩種芳香族氨基酸可能共同處于疏水的蛋白微環(huán)境中。推測B-PE在PEB139α/PEB158β和PEB82α/PEB82β兩個(gè)位置,形成耦合的激子對,4個(gè)耦合分子內(nèi)以激子分裂的形式進(jìn)行能量傳遞,其它色基之間則以福斯特共振進(jìn)行能量傳遞,最后對B-PE內(nèi)能量傳遞的途徑進(jìn)行了預(yù)測。2.對MAC工程菌株的培養(yǎng)條件進(jìn)行了優(yōu)化,進(jìn)行了3次10 L密度發(fā)酵,獲得大量表達(dá)MAC的菌體。MAC(鏈霉親和素-藻藍(lán)蛋白α亞基融合蛋白)的表達(dá)量占菌體可溶性蛋白的比率可達(dá)43%,菌體密度OD600最大達(dá)到12.5,收集到MAC菌體濕重總量達(dá)到約400 g。隨后對工程菌的破碎條件進(jìn)行優(yōu)化,并對MAC的進(jìn)行了層析純化。SDS-PAGE結(jié)果表明,純化后的MAC僅有一個(gè)亞基,分子量為在76 kDa附近,與預(yù)期的蛋白分子量相符。MAC在近紫外-可見光區(qū)共有3處吸收峰,分別位于340 nm和370 nm和625 nm;在575 nm還有一個(gè)肩峰,MAC的最大熒光發(fā)射峰位于640 nm,圓二色譜中的吸收峰結(jié)果與吸收光譜中一致。當(dāng)對藻膽素或者芳香族氨基酸進(jìn)行激發(fā)時(shí),能夠獲得640 nm的熒光發(fā)射峰,表明能量能夠通過藻膽素或者芳香族氨基酸傳遞至發(fā)色團(tuán)。光譜結(jié)果表明MAC有正確的構(gòu)象,并且具有良好的光學(xué)活性。3.構(gòu)建基于MF0(基因重組藻藍(lán)蛋白α亞基)和氧化石墨烯(GO)的葡萄糖生物傳感器。首先用低分子量殼聚糖(CS)修飾氧化石墨烯,制得CS-GO復(fù)合物。GO-CS可以非特異性吸附MF0上的麥芽糖結(jié)合蛋白(MBP),造成MF0的熒光淬滅。當(dāng)體系中存在葡萄糖時(shí),MBP會(huì)特異吸附葡萄糖,造成MF0無法再吸附GO-CS,熒光強(qiáng)度增加。通過熒光的強(qiáng)度變化,可以間接對葡萄糖含量進(jìn)行定性和定量分析。該葡萄糖生物傳感器的檢測線性范圍為0.1~1 mg/mL,最低檢測限(LOD)為0.05 mg/m L,具有較高的靈敏度和選擇性。4.選擇7種不同特性的藻膽蛋白作為染料敏化二氧化鈦光陽極,組裝成染料敏化太陽能電池(DSSC)并研究其光電特性。結(jié)果表明B-PE能夠明顯提高DSSC的光電性能,得到DSSC的短路電流、開路電壓、填充因子和光電轉(zhuǎn)化效率為分別為0.809 A/cm2、0.545 V、0.569和1%。所構(gòu)建的膠原蛋白/羧基化碳納米管/聚丙烯酰胺復(fù)合凝膠,不僅利于DSSC封裝,同時(shí)可增進(jìn)光電轉(zhuǎn)換效率,提高光電池的短路電流,能夠作為準(zhǔn)固態(tài)電解質(zhì),推進(jìn)染料敏化太陽能電池的實(shí)際應(yīng)用。結(jié)合晶體結(jié)構(gòu)和圓二色譜數(shù)據(jù),解析了藻膽蛋白染料敏化DSSC的IPCE和ICE光譜,為研究藻膽蛋白的結(jié)構(gòu)和功能提供了新的途徑。
[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.
【學(xué)位授予單位】：中國科學(xué)院煙臺(tái)海岸帶研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TM914.4

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