海洋藻类功能性蛋白质的纯化、鉴定及其应用的研究

使用膨化柱和离子交换或羟基磷灰石柱层析相结合的方法，分别从多管藻、坛紫菜及钝顶螺旋藻中分离纯化了R-藻红蛋白溶液和C-藻蓝蛋白。光谱检测及电泳分析结果证明完全符合经典的藻胆蛋白纯度标准。彭化床最突出的优点是克服了常规分离方法堵塞色谱柱的难题，纯化速度快、产量高、不需要常规色谱方法所要求的填料的平衡及粗提液的预处理，仅需一步操作就可以得到满足一般食品添加剂纯度要求的藻胆蛋白，极大地简化了后续的纯化程序，减少了分离纯化的步骤和时间，而其产率及纯度均高于常规的藻胆蛋白分离方法。这同时也降低了藻胆蛋白分离纯化的成本。 本文通过戊二醛或环氧氯丙烷交联的方法，合成了四种壳聚糖-氨基酸共聚小球。并选取吸附性好的戊二醛交联孔球和戊二醛交联微球系统测定了其对R-藻红蛋白和C-藻蓝蛋白的吸附和缓释性能。 纯化了藓羽藻中与其细胞器团聚密切相关的一种凝集素并进行了部分性质的鉴定。N端前15个氨基酸序列及LC-ESI-MS质谱分析结果证明此凝集素属于一种新的蛋白质族。实验证明，凝血活性与细胞器团聚活性并不完全依赖于此凝集素分子相同的结构域。 通过异双功能试剂SPDP处理藓羽藻凝集素使之衍生化，DTT处理R-PE在其分子内引入外源巯基，然后将活化的R-藻红蛋白与凝集素进行交联反应。交联产物经凝胶过滤纯化并检测，但电泳及荧光显微镜检测结果并不能证明交联探针的成功制备。

Firstly, R-phycoerythrin was isolated and purified from the red alga, Polysiphonia urceolata Grev, or Porphyra haitanensis using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. Then, C-phycocyanin was purified from the Spirulina platensis using the same technology. The absorption spectra and the fluorescence spectra of these pigment protein were consistent with the typical spectra of phycobiliproteins. SDS–PAGE or LC-ESI-MS showed they conform to the published data of phycobiliproteins. The method described here was a scaleable technology that allows large quantity of phycobiliproteins to be recovered from the unclarified alga crude extract. It was a high protein recovery technology while reducing both processing costs and times. The very important point is that using of this new novel method, block of chromatographic column due to the abundance of polysaccharide in alga could be overcomed successfully. One cycle of the isolation process by EBA chromatography, including equilibration (30 min) + loading (60 min) + washing (50 min) + elution (60 min), took only about 3.5 h. Only one step, we can get the fractions could be used as food additives and cosmetics additives. Four different chitosan microspheres (CS-CL) were prepared by suspension crosslinking method and used as carriers of phycobiliproteins. In this study, phycobiliproteins were loaded in the microspheres and released in vitro. The basic loading and releasing behavior for phycobiliproteins of these kind of new microspheres were also investigated. The results showed that all these chitosan microspheres have the ability to controlrelease of phycobiliproteins. A lectin presented in the marine green alga Bryopsis hypnoides was purified using affinity chromatography on Fetuin-agrose column and then partially characterized. N-terminal 15 amino acid sequence and LC-ESI-MS analysis of this lectin did not show sequence significant homology with those lectins or with any other proteins reported in public protein data bases, which indicated that the lectin belongs to a novel protein family. The chemical conjugation between R-phycoerythin and a lectin from a green alga Bryopsis hypnoides was studied. Results showed that there was a fraction possessed good optical properties and hemagglutinating activity. However, electrophoresis assay did not indicate the combination of these two molecules.