基于磁性纳米载体固定化提高普鲁兰酶耐热性的机理研究

Pullulanase (EC 3.2.1.41), which belongs to the starch debranching enzymes, was the key enzyme in the starch processing industry. However, commercial grade pullulanase as a biocatalyst always presents some drawbacks that limit its popularization in food industry, such as high cost and easy deactivation at higher temperature. In the previous work, we found that the thermostability of oriented immobilized pullulanse was enhanced significantly. However, the thermo-resistant mechanism was unclear. In this project, the relationship between the spatial conformation change of internal disulfide bond and hydrophobic groups and the thermal stability of immobilized pullulanase was studied to explore the thermo-resistant mechanism. To achieve this goal, the binding rules of enzyme to the support and enzyme to substrate were firstly investigated from the perspective of thermodynamics and stoichiometry; the reaction dynamics model of immobilized pullulanase was then established based on the enzyme-subtract and enzyme-substrate-inhibitor dynamics; molecular dynamic simulation was further used to speculate the spatial conformation change of enzyme in carrier-enzyme-substrate system during catalysis, including the distribution of interior hydrophobic groups, disulfide bond, catalytic center and binding sites. These studies would provide some scientific basis for developing techniques to prepare the heat-resistant pullulanase and similar enzymes.

普鲁兰酶属淀粉脱支酶，为淀粉加工业中重要的关键酶制剂，然而由于存在价格昂贵及热稳定性差等缺陷，该酶的工业应用受到了限制，因此提高普鲁兰酶的耐热性并了解其耐热机理具有较大实际应用价值。申请人前期定向固定化能够实现普鲁兰酶耐热性的显著提高，然而其耐热机制却并不清楚。本项目旨在明确酶内部二硫键及疏水基团空间变化和耐热性之间的关联性，探究固定化酶耐热机制。基于此，首先利用热力学及化学计量学，阐明酶-载体及酶-底物结合规律，并结合酶-底物及酶-底物-抑制剂体系的反应动力学，构建固定化酶热-动力学模型；然后通过动态分子模拟，以热-动力学模型为参数，推测固定化酶体系催化过程中酶-底物-载体三者的空间构象变化，包括内部疏水基团、二硫键、催化中心及结合位点的空间排布规律，明确固定化普鲁兰酶耐热机制。项目的顺利实施，以期为高耐热性普鲁兰酶及其他类似酶的制备技术的发展提供理论指导。

普鲁兰酶为淀粉加工业中重要的关键酶制剂，然而由于存在价格昂贵及热稳定性差等缺陷，该酶的工业应用受到了限制。本课题以磁性壳聚糖纳米颗粒为载体，利用生物素-链霉素亲和作用定向固定普鲁兰酶，提高普鲁兰酶稳定性同时保留其较高的酶活。通过活化能常数及失活能常数的计算，建立了酶的耐热预测模型，结果表明热稳定性模型有效预测了酶活力变化情况，证明预测模型能有效获得酶反应器的最佳操作条件；对于固定化普鲁兰酶，其最佳酶反应器温度为50 ℃，此时其半衰期为228.8 h，而自由酶酶反应器最佳反应温度为40 ℃，其半衰期为138.0 h，表明固定化大大提高了普鲁兰酶的热稳定性及使用寿命。采用ITC分析自由酶及固定化酶的热力学结合规律，结果表明，固定化酶的米氏常数Km高于自由酶，虽然固定化酶的最适反应温度没有发生改变，但是酶与底物的亲和常数Kd均要高于自由酶，这说明固定化后，酶和底物的亲和能力变强了，间接反映了在相同的实验条件下固定化酶具有更长的半衰期。采用动态分子模拟探究升温过程对普鲁兰酶分子构象的影响，结果表明在pH 7.0，60 ℃条件下，与其他部分的氨基酸残基相比，普鲁兰酶分子500SER-550ALA范围内的氨基酸残基更不稳定，柔韧性更大，这些氨基酸残基均位于普鲁兰酶结构域A中，说明温度对普鲁兰酶结构域A构象影响更大；随着模拟时间的变化，在480PHE及560ASP残基附近，其二级结构逐渐由Turn转角及bend弯曲变成无规则卷曲，在520SER残基附近，其二级结构逐渐由Alpha螺旋逐渐转变成Turn转角，表明其分子间氢键断裂，二级结构遭到破坏。推测固定化提高酶活耐热性与提高500SER-550ALA范围内的氨基酸残基空间构象的刚性有关。本项目的研究为高耐热性普鲁兰酶的制备技术的发展提供理论指导