融合His-tag的有机磷降解酶的高效表达及一步分离固定化研究

In order to solve the problems of organophosphate degrading enzymes such as the low expression level, low activity, poor stability, etc, the topic of "Study of high-level expression of organophosphate degrading enzyme using histidine as a fusion tag and its separation and immobilization by one-step method" was proposed. Recombinant plasmids and recombinant strains of organophosphate degrading enzyme A (OpdA) with 6×histidine tag at N- or C- terminus will be obtained through site-directed mutagenesis of the coding sequence of amino acid of the OpdA active site, and the transcriptional expression of promoter will be induced by IPTG or self-induction system to solve these problems of low expression and low activity; Then, the metal ion functionalized core-shell structured magnetic mesoporous silica nano-flowers will be synthesized. The separation and purification of OpdA will be realized through the specific affinity of metal ion to histidine-tagged enzyme. In order to improve the stability of OpdA, the separation and immobilization of OpdA by one-step method will be carried out by using the immobilized metal affinity magnetic nanoparticle as a carrier. The catalytic performance and stability of the immobilized enzyme in the degradation of organophosphate compounds will be also investigated. The successful implementation of this project can not only reveal the relationship between the cloning expression of OpdA gene and its conformation and activity, but also reveal the relationship among the preparation, structure, microenvironment and catalytic performance of the immobilized enzyme. The results of this project will provide theoretical basis and technical support for cloning expression, separation, purification, immobilization and degradation of organophosphate compounds.

针对目前产有机磷降解酶(OpdA)的菌株表达量不高、酶活低、稳定性差等问题，本项目提出“融合His-tag的有机磷降解酶的高效表达及一步分离固定化研究”课题。通过活性位点氨基酸编码基因序列定点突变，构建N或C端带His-tag的OpdA重组质粒和重组菌株，采用诱导剂IPTG和自诱导系统诱导启动子转录表达，以解决菌体表达量不高、酶活性低等缺点；制备金属离子修饰的核壳结构磁性介孔氧化硅纳米花，利用所获得的固定化亲和磁性纳米粒子对含His-tag酶的特异亲和性，实现OpdA的分离纯化；为提高酶的稳定性，利用该纳米粒子实现OpdA的一步分离固定化，考察其在有机磷化合物降解中的催化性能和稳定性。本研究的成功实施不仅可揭示基因克隆表达与酶构象、活性之间的联系，还可揭示固定化酶的制备、结构、微环境及催化性能间的关系，将为新型酶的克隆表达、分离纯化和固定化以及有机磷化合物的降解提供理论基础和技术支持。

有机磷化合物以其化学性质稳定、对病害虫高毒性和价格低廉等特点，被广泛应用于农药、杀虫剂和生化武器等领域。然而，伴随含有机磷化合物等产品的大量不合理使用，导致环境中有机磷化合物的残留量严重超标，对环境和人类的健康产生了巨大的威胁。有机磷降解酶催化降解法是一种被广泛研究的绿色、环保、无污染的降解方法，能够实现有机磷化合物的高效降解。.鉴于目前产有机磷降解酶(OpdA)的菌株表达量不高、酶活低、稳定性差、分离纯化过程复杂等问题，项目提出了“融合His-tag的有机磷降解酶的高效表达及一步分离固定化研究”课题。主要研究内容：（1）通过构建N端带His-tag的OpdA重组质粒和重组菌株，采用诱导启动子转录表达，获得表达量高且带His-tag的OpdA；（2）利用酶His-tag上的咪唑基与过渡金属离子间的特异性亲和吸附作用力，制备了金属离子修饰的核壳结构磁性介孔氧化硅纳米花（IMS-MNFs/Ni-NTA）、金属氧化物（NiCo2O4）和蛋黄壳复合型纳米材料（Co/C@SiO2@Ni/C），并以其为吸附介质和载体，实现了OpdA的分离纯化及一步分离固定化，相对于游离酶，所获得的固定化酶具有更好的热稳定性和酸碱耐受性；（3）以甲基对硫磷为模型底物，考察了制备的纳米催化剂的酶学性能和重复使用稳定性，结果表明固定化酶具有较高的催化效率，重复使用多次后甲基对硫磷仍能被有效降解，固定化酶仍能保持良好的催化性能，说明其有良好的循环使用稳定性。本研究的成功实施不仅可揭示酶的外源表达与酶构象、活性之间的联系，还可揭示固定化酶的制备、结构、微环境及催化性能间的关系，将为新型酶的克隆表达、分离纯化和固定化以及有机磷化合物的降解提供理论基础和技术支持。