深海细菌新型M12家族弹性蛋白酶myroilysin对胶原蛋白的膨胀机制与应用

There are a lot of protease-producing bacteria in deep-sea sediment. Research and development on the bacterial proteases from deep-sea sediment is helpful to find novel proteases with new function and use. Our previous study showed that the M12 novel elastinase myroilysin secreted by the deep-sea bacterium Myroides profundi D25 has strong collagen-swelling ability, indicating that it may be a good modifier of collagen. Based on the previous results, this program will first study the mechanism of myroilysin to swell collagen. The effect of myroilysin on the structures of tendon, fascicle, fibril and molecular of collagen, the unwinding activity of myroilysin to collagen molecular, the degradation activity of myroilysin to the proteoglycans and glycoproteins in collagen will be studied to elucidate the collagen-swelling mechanism of myroilysin. Then, myroilysin will be used to prepare dermal collagen scaffolds by means of the collagen-swelling ability of myroilysin. In vitro and in vivo experiments will be performed to analyze the property of the scaffolds.The potential of myroilysin in collagen scaffold preparation will be evaluated. So this program will explore the use of novel protease form deep-sea bacteria.

深海沉积物中存在着大量产蛋白酶细菌，对深海蛋白酶资源的研究和开发可以发现具有新的功能和用途的蛋白酶。我们前期研究发现深海细菌Myroides profundi D25分泌的M12家族新型弹性蛋白酶myroilysin对胶原蛋白有很强的膨胀作用，但不降解胶原蛋白，预示着该酶是一种很好的胶原蛋白改性剂。本项目将在前期研究的基础上首先研究myroilysin膨胀胶原蛋白的机制，通过研究myroilysin处理对胶原蛋白腱、纤维束、纤丝和胶原分子等不同级别结构的影响和分析myroilysin的解旋酶活性、对胶原蛋白中的糖蛋白和蛋白聚糖等成分的降解活性等，阐明myroilysin膨胀胶原蛋白的机制。然后利用myroilysin能膨胀胶原蛋白的特性制备真皮胶原基质支架，并通过一系列的体外体内实验分析支架的性能，评价myroilysin在胶原基质支架制备中的应用潜力。从而开发深海新型细菌蛋白酶的用途。

Myroilysin是我们筛选的深海产蛋白酶细菌Myroides profundi D25分泌的一个新型弹性蛋白酶。前期研究表明，myroilysin能膨胀胶原蛋白，并且能高效脱除猪皮中的细胞，因此在无细胞真皮基质支架的制备中有应用潜力。本课题在前期研究的基础上，取得一下研究成果。阐明了myroilysin膨胀胶原蛋白的机制。阐明了myroilysin吸附和降解弹性蛋白的机制。对myroilysin进行了发酵优化并建立了中试发酵工艺，大幅提高了myroilysin的产量。以猪皮为材料，利用myroilysin能膨胀胶原蛋白的特性，建立了制备真皮胶原基质支架的技术。支架经矿化制备了猪皮胶原-HAP复合支架材料，用作骨修复的植入材料。体外和动物体内实验表明，胶原-HAP复合支架不仅具有较好细胞生长支持作用，而且具有良好的组织相容性，有成为理想的骨组织工程支架材料的潜力。另外，还探索了利用myroilysin制备皮肤修复支架和眼角膜修复支架的技术。这些研究为新型海洋蛋白酶myroilysin的应用开发奠定了很好的基础。本课题资助发表SCI论文12篇，授权国家发明专利1项。