基于酶促转化和催化氧化的丝素/弹性蛋白组织工程材料构建与调控

Sike fibroin has the property of excellent biocompatibility, it can be potentially used as scaffolds for tissue engineering. In this study, silk fibroins are dissolved in aqueous solutions before enzymatic treatments. The enzymes of serine hydroxymethyltransferase(SHMT) and tyrosine phenol-lyase(TPL) are applied in the treating of soluble silk fibroins, respectively, aimming at converting glycine and serine of fibroins to tyrosine residues. The enzymatic oxidations of silk fibroin are carried out with tyrosinase(TYR) subsequently, which is expected to graft elastin onto fibroin surfaces and obtain a silk fibrion/elastin composite. The mechanisms of the enzyme-catalyzed conversion, as well as the correlations between the enzyme-catalyzed conversion and oxidation, are to be explained according to the analysis results of the changes of fibroin structures and compositions. The efficacies of the production of active groups of dopaquinone and the grafting of amino compounds onto fibroin surface, are to be further improved by optimizing the enzymatic incubating conditions. Considering the oxidized fibroin molecules could react with silk polypeptides and elastin simultaneously, the internal mechanisms of the two competitive reactions are concerned, which undoubtedly could provide advantages to get a more acceptable process for fibroin modification. Furthermore, the structures, morphologies and properties of the regenerated fibroin/elastin membranes based on different processes are to be compared,which might improve the efficacy of the enzymatic modification of fibroin/elastin complex and the quality of the protein membranes as well. The newly established enzymatic method for the production of silk fibroin/elastin scaffolds for tissue engineering could afford us theoretical and scientific basises for the biomolecular structural design and bio-modification of the regenerated protein material.

丝素蛋白具有良好的生物相容性，可应用于组织工程支架材料制备。本研究拟以丝素为原料制备蛋白溶液，通过丝氨酸羟甲基转移酶(SHMT)、β-酪氨酸酶(TPL)分步催化转化，实现丝素中甘氨酸、丝氨酸转化成酪氨酸剩基；结合酪氨酸酶(TYR)催化氧化，实现丝素与弹性蛋白交联，制备丝素/弹性蛋白复合材料。通过丝素蛋白结构与组成变化分析，探明酶促转化的反应机制，研究其与催化氧化反应的相关性；考察丝素蛋白表面多巴醌活性基形成的影响因素，构建丝素蛋白与氨基化合物反应的增效方法；通过丝素/弹性蛋白体系反应产物特性分析，研究丝素自交联、丝素与弹性蛋白交联两种反应的竞争机制，提出调控方法；在丝素/弹性蛋白复合膜材料形态、结构和性能分析基础上，对丝素/弹性蛋白交联反应体系及后续复合蛋白膜成型方法进行优化，构建一种生物酶法丝素/弹性蛋白组织工程材料制备方法，为再生蛋白材料的结构设计与功能化改性提供理论基础和科学依据。

丝素蛋白（Silk fibroin, SF）有良好吸湿性、生物相容性和环境友好性，不仅可用于纺织工业，在生物医用材料领域也有潜在用途。为改善丝素基再生蛋白材料的性能，本项目借助于生物酶法，探究了丝素酶催化氧化与酶促接枝改性的影响机制，制备了包括丝素/弹性蛋白在内的丝素基生物材料。主要研究内容包括四方面，① 丝素蛋白中酪氨酸剩基数的强化方法：分别采用a.构建β-酪氨酸酶（Tyrosine Phenol-lyase, TPL）催化丝素中酪氨酸前体转化、b.以含酪氨酸小分子肽接枝来强化丝素蛋白的反应性，研究表明后者能显著增加酪氨酸酶（Tyrosinase, TYR）对丝素的催化接枝改性效果；② 酶促丝素中酪氨酸剩基形成多巴醌的氧化机理：分别探究了TYR酶催化氧化丝素产生多巴醌活性基的影响机制、辣根过氧化物酶（Horseradish Peroxidase, HRP）催化氧化丝素中酪氨酸产生自由基及引发丝素自交联的反应机理；③ 丝素蛋白自交联和与弹性蛋白（或外源功能大分子）酶促交联的过程调控：通过a.在体系中分步添加多酚类介体（儿茶素），调控TYR催化丝素与弹性蛋白的交联过程，b.通过TNBS选择性封端氨基削弱酶促丝素蛋白自交联，促进TYR催化丝素与外源功能性大分子接枝，实现竞争性酶促反应的过程调控；④ 丝素/弹性蛋白及丝素/功能大分子的生物复合材料构建和性能研究：以TYR催化丝素和弹性蛋白交联，制得的丝素/弹性蛋白复合膜热性能和机械性能较丝素膜改善，其中风干膜弹性较好，纳米纤维支架表现出优良的形貌，NIH/3T3细胞在其支架上的存活率>90%，表现出较好的生物相容性；研究中还借助于HRP体系，催化氧化丝素中酪氨酸残基接枝聚合丙烯酸（AA），制得的SF-g-PAA膜在仿生矿化中获得了较好的羟基磷灰石（HAp）沉积效果，MG-63成骨细胞在其表面黏附和增殖效果较好，拓展了丝素蛋白材料在骨组织工程中的应用。本项目借助酶法实现了基于酪氨酸剩基的丝素蛋白改性和功能化加工，对生物医用材料的设计与构建有指导意义。