自组装多功能纳米硫酸软骨素ABC酶的制备方法研究

Chondroitinase ABC (ChSase ABC) is one of the most widely used chondroitinase which has great potential biological functions. This project is aimed to establish a integration procedure including separation, purification and immobilization. Firstly, ChSase ABC was connected to Elastin polypeptide (ELP) and Silk-like polypeptides by linker, so as to get a flexibly controlled and mechanically strengthful fusion protein and the system of fusion expressing protein. In the separation process, the artificial neural network model was applied to study the peptide self-assembly process and then get the optimum separation conditions, under which the enzyme activity and the yield was in a good balance. By using the self-assembly property, the integration procedure including separation, purification and immobilization was established. Meanwhile, the developed model was used to study the self-assembly process and then reveal the fundamental of the process. The results obtained in this project will be helpful to explore the bio-production technique of the ChSase ABC and get intensively understanding of the ELP self-assembly process, which will be also a benefit to the researchers studying the similar systems.

硫酸软骨素酶ABC（ChSase ABC）为特异性最为广泛的一类硫酸软骨素裂解酶，具有卓越的生物学功能。本项目拟以ChSase ABC为目标酶，经连接肽与类弹性蛋白（Elastin polypeptide，ELP）和类丝蛋白多肽结合，构建可控性好、机械强度高的融合蛋白及可溶表达体系，探讨高效融合表达策略；纯化过程中，建立神经网络模型描述多肽自组装过程，探索最佳条件，使多肽自组装成构象合理及尺寸适当的球体，在保证酶学特性的同时实现酶的快速分离纯化，并同步完成固定化。同时，利用所建模型分析ELP融合蛋白自组装过程中各影响因素的协同作用，揭示其环境应答机理。最终开发出具有自主知识产权的新型、经济的自组装纳米ChSase ABC酶，实现分离纯化、固定化一体化过程集成，并实现自组装过程模型化描述，对提升我国ChSase ABC纯酶生产的技术水平具有重要科学意义，同时为其他类似体系的开发提供科学依据。

硫酸软骨素裂解酶(chondroitinase, ChSase)是一类能将硫酸软骨素、软骨素、透明质酸等糖胺聚糖降解为不饱和二糖及寡糖的裂解酶。本研究主要从以下四个方面对ChSase ABC I进行了系统研究。首先使用融合标签MBP来提高ChSase ABC I的可溶性表达，重组MBP-ChSase ABC I产量可达到3180 IU/L发酵液。其次研究了His标签对ChSase ABC I酶活和性质的影响，结果发现，His-ChSase ABC I的比酶活为201.9 IU/mg蛋白，比ChSase ABC I低两倍，同时His标签降低了ChSase ABC I的催化效率，并对其二级结构产生了影响。再次，为了一步得到高纯化率的ChSase ABC I，研究了ELP对ChSase ABC I表达及纯化的影响，成功构建了pMAL-c2x-(ELP)100-ChSase ABC I，实验结果表明由于ChSase ABC I的分子量过大，没有实现预期，不及MBP和His标签的分离纯化效果。最后，为进一步提高ChSase ABC I表达量，课题组成功开发了一种新的融合标签GAPDH，结果表明，GAPDH-ChSase ABC I的表达水平比ChSase ABC I提高了2.25倍，同时酶活提高了将近3倍。与常用的融合标签MBP对比，GAPDH占有明显的优势。本研究对于ChSase ABC I的工业化应用有一定的指导意义，同时为ChSase ABC I的工业化生产提供一种可能。