基于接枝型亲和色谱的植物蛋白酶高效纯化方法与机理研究

The plant protease is a kind of important enzyme in food industry. However, since the composition of plant is complex and its content of protease is relatively low, it is difficult to purify the plant protease by conventional methods. Currently, due to the lack of highly efficient method for purification of plant protease and its relevant mechanism, the research and application of the plant protease are greatly limited. Thus, it is of great importance to establish a highly efficient method for purification of plant proteases. Based on previous studies on plant proteases and polymer-grafted media by the applicant, in order to achieve highly efficient purification of plant protease, a novel polymer-grafted affinity media is designed by grafting the polymers exhibiting various morphologies to agarose beads. Subsequently, The polymer is functionalized with triazine-based dye ligand and its fragments to obtain the novel polymer-grated affinity beads. Through adsorption-related experiments and computer simulation, the interaction between the affinity ligands and the model plant proteases is studied to describe the adsorption behaviors and reveal the adsorption mechanism. Based on the mechanism obtained, the model plant proteases will be purified in actual situation by regulating the operation conditions to verify the purification efficiency and the adsorption mechanism. The polymer-grated affinity beads will be further used for the purification of other plant proteases to expand their applicability. The present research is helpful not only to illustrate the mechanism of the polymer-grated affinity chromatography and reinforce the theoretical foundation, but also to improve the purification efficiency and prompt the development of plant protease resources. Thus, this research is of great scientific and practical significance.

植物蛋白酶是食品工业中一类重要酶制剂。由于植物组成复杂，蛋白酶含量较低，因此使用常规手段纯化难度较大。目前针对植物蛋白酶的高效纯化方法较少且缺乏机理研究，这使其研究与应用受到极大限制。因此，建立植物蛋白酶的高效纯化方法并揭示其纯化机理具有重要理论意义和应用价值。基于申请人前期对植物蛋白酶及接枝型色谱的相关研究，本项目以具有不同形态的聚合物为接枝聚合物，以三嗪基-染料分子及其片段为亲和配基，设计新型接枝型亲和色谱以实现植物蛋白酶的高效纯化。通过吸附实验和计算机模拟，研究配基与模式植物蛋白酶之间的相互作用，阐明吸附行为，揭示纯化机理。以此为基础，研究模式植物蛋白酶的实际纯化过程，检验纯化机理；研究其它植物蛋白酶的纯化过程，开发新型植物蛋白酶资源。本项目不仅可以阐明接枝型亲和色谱的纯化机理，强化纯化过程的理论研究，而且还将改善植物蛋白酶的纯化效率，推动其资源化利用，因此具有重要的理论和实际意义。

植物蛋白酶在食品工业中应用广泛，市场价值巨大。然而，植物组成复杂且蛋白酶含量较低，这导致难以通过常规手段进行纯化。基于此，本项目以三嗪基-染料分子作为亲和配基，设计新型接枝型亲和色谱展开研究以阐明接枝型染料亲和色谱的纯化机理和建立植物蛋白酶的高效纯化方法。.本项目通过光谱学分析和分子对接实验，比较了三嗪基染料分子Reactive Blue-4（RB-4）和Procion Red MX-5B（PR）对牛血清白蛋白（BSA）和菠萝蛋白酶的吸附作用，结果表明RB-4与蛋白质的结合是自发放热过程且以疏水相互作用为主，其结合位点数约为2，结合常数KA在100-1000之间；对于PR，随着苯环数的减少，其作用模式转变为以静电相互作用为主，结合位点数约为1，结合常数KA在10-100之间。另外，RB-4和PR对菠萝蛋白酶表现出类似的结合特征。.建立了不同接枝型色谱介质的制备方法，其中Dextran、Ficoll和PAMAM的最大接枝量分别可达到16.9、56.8和27.6 mg/mL；表征结果显示接枝后三种介质的形貌特征和粒径分布范围未发生明显变化，比表面积均显著增大，Dextran-和Ficoll-Sepharose 4B的表观孔半径显著减小而PAMAM-Sepharose 4B的表观孔半径变化不大。.通过吸附实验，比较了以RB-4为配基的亲和色谱介质和接枝型亲和色谱介质对BSA和菠萝蛋白酶的等温吸附和吸附动力学过程，结果显示吸附过程是放热反应，符合Langmiur模型，主要作用力为疏水相互作用；在相同条件下，接枝型亲和色谱介质的吸附量显著高于常规亲和色谱介质且吸附速率更快；不同接枝型色谱介质的吸附实验表明接枝聚合物的结构对吸附过程有显著影响，其中球形聚蔗糖分子最有利于吸附。.使用RB-4-Dextran-Sepharose 4B建立了猕猴桃蛋白酶的纯化方法，其回收率可以达到52.3%，纯化倍数4.2倍，比活力达到254.1 U/mg。.本项目的实施不仅明确了三嗪基-染料分子的亲和作用模式，解释了接枝型亲和色谱的作用规律，而且还将积极推动植物蛋白酶的分离、纯化和发掘，具有重要应用价值。