南极磷虾抗冻蛋白的结构特性及冰晶生长抑制机理研究

With the good characteristics of ice growth inhibition, Antifreeze proteins (AFPs) has a wide range of application in food and cryobiology.However, the limited production of AFPs limited its application. The exploration of new AFPs derived from the species with extensive sources is necessary and urgent, as well as study on its clear mechanism of ice growth inhibition. Antarctic krill (Euphausia Superba Dana)as the major species in Antarctic sea area is enormous resources, which have a brilliant future of development and utilisation. In adaption to the cold environment in Antarctic waters, Antarctic krill produce some specially antifreeze active substances. Former research had revealed the hydrolysates of Antarctic krill proteins with the antifreeze activity. However, research on the antifreeze activity of Antarctic krill relevent proteins have not been reported until now. Based on our former work on the relevent protein derived from Antarctic krill with the antifreeze activity, the AFPs derived from Antarctic krill had been extracted and further studied. In this project, firstly, we try to get the purer AFPs derived from Antarctic krill by chromatography and specific binding purification. Secondly, technique in mass spectrometry, optical spectrum, chromatography and bioinformatics should be used to resolve its structure, further to compare its homology with the sequence and structure of the known AFPs. Thirdly, the molecular dynamics simulation were used in research on the AFPs derived from Antarctic krill recognization and bind to the ice crystal certain planes. Finally, based on the specific functional groups and the functional structure domains of AFPs derived from Antarctic krill, we would reveal the mechanism of ice growth inhibition of AFPs derived from Antarctic krill which provide the basic theory for its application. The result of the project will enrich the basic theory on AFPs, broad the range in exploration of Antarctic krill proteins and promote the development of the natural antifreeze agents.

抗冻蛋白具有良好的冰晶生长抑制特性，在食品和低温生物学等领域具有广泛的应用。但是，目前现有抗冻蛋白产量有限，无法满足应用需求；亟需发掘来源广泛，抗冻机理明确的新来源抗冻蛋白。南极磷虾是南极海域的优势物种，生物资源量大。研究表明南极磷虾蛋白质酶解物具有抗冻活性，但对南极磷虾抗冻蛋白结构特性及冰晶生长抑制机理的研究尚未见报道。在课题组前期研究发现南极磷虾蛋白质具有抗冻活性的基础上，以南极磷虾抗冻蛋白为切入点，采用特异性亲和吸附法和色谱法分离纯化南极磷虾抗冻蛋白；利用质谱、光谱、色谱和生物信息学方法相结合解析南极磷虾抗冻蛋白的结构特性并分析同源进化关系；通过分子动力学模拟阐明南极磷虾抗冻蛋白与冰晶特定"面"的识别-结合机制，揭示南极磷虾抗冻蛋白的冰晶生长抑制机理。本项目的研究结果有望丰富抗冻蛋白的理论研究，拓展南极磷虾蛋白质应用的新领域和促进天然抗冻剂的开发。因此，具有重要的科学意义和应用前景。

南极磷虾(Euphausia superba)是一种生活在南极海域的小型海洋浮游甲壳类动物，据评估其生物量约为1.25~7.5亿t；南极磷虾蛋白质含量很高，约占其干重的65%，被称为人类未来的蛋白质仓库。抗冻蛋白具有良好的冰晶生长抑制特性，在食品和低温生物学等领域具有良好的应用前景。但是，目前现有抗冻蛋白产量有限，无法满足应用需求；亟需发掘来源广泛，抗冻机理明确的新来源抗冻蛋白。在课题组前期研究发现南极磷虾蛋白质具有抗冻活性的基础上，以南极磷虾抗冻蛋白为切入点，采用特异性亲和吸附法和色谱法分离纯化南极磷虾抗冻蛋白；利用质谱、光谱、色谱和生物信息学方法相结合解析南极磷虾抗冻蛋白的结构特性。南极磷虾抗冻蛋白的评价方法采用DSC法评价，优化后的南极磷虾抗冻蛋白热滞活性的升降温速率为1.0 ℃/min，样品浓度为1.0 mg/mL，冰晶含量为10 %左右，SDS-PAGE显示其分子量约为76 kDa左右，其热滞活性为1.76 ℃。项目确定了南极磷虾AFPs中氨基酸的种类及比例；在紫外波长范围内主要有两个吸收峰，220 nm附近由肽键引起的较强的紫外吸收峰和280 nm附近由芳香族氨基酸所引起的较弱紫外吸收峰；此外，由红外光谱以及圆二色性光谱分析，可知南极磷虾AFPs具有α-螺旋、β-折叠以及无规卷曲结构，其二级结构大致范围为：α-螺旋的比例24.18-24.96%，β-折叠的比例为31.90-33.17%，β-转角的比例为36.65-38.07%，无规卷曲的比例为5.22-5.85%。目前，正在进行南极磷虾抗冻蛋白的抗冻机理研究。本项目的研究结果有望丰富抗冻蛋白的理论研究，拓展南极磷虾蛋白应用的新领域和促进天然抗冻剂的开发。因此，具有重要的科学意义和应用前景。