利用蛋白质工程技术优化双酶催化串联反应合成过氧乙酸工艺及其双酶交联聚集体的制备

As a kind of important chemical raw material, peracetic acid could be synthesized using the traditional chemical technology, however, several technology problems had to be concerned, including product property control and explosion incident control during production. A green synthesis technology for peracetic acid could substitute for the chemical synthesis technology, using a enzymatic cascade reaction catalyzed by glucose oxidase and acyl transferase from Mycobacterium smegmatis (MsAcT), respectively. In this project, the enzymatic cascade reaction for peracetic acid synthesis would be optimized to improve the operation stability and catalytic efficiency using protein engineering technology. First, acyl transferase mutants with high affinity to hydrogen peroxide would be constructed, screened, and then selected so that the total activity ratio between glucose oxidase and acyl transferase (MsAcT) could be decreased in the cascade reaction system, which resulted in the enhancement of the operation stability. Second, MsAcT was site-selectively glycosylated with maleimide-lactoside and then was crosslinked with glucose oxidase in the presence of Concanavalin A, a kind of molecular glues, which resulted in the formation of glucose oxidase-MsAcT cross-linked aggregates. The catalytic efficiency of glucose oxidase-MsAcT cross-linked aggregates could be regulated using the glycosylation site variation of MsAcT. Based on the above research work, three prospective results would be attained. First, the molecular mechanism on the perhydrolysis activity catalyzed by MsAcT would be solved. Second, a novel preparation method for multi-enzymes cross-linked aggregates would be developed, in which the catalytic efficiency of the multi-enzymes cross-linked aggregates could be regulated using the glycosylation site variation. Third, glucose oxidase-MsAcT cross-linked aggregates with intellectual-property rights was developed, which helped to promote the application filed of the enzymatic-synthesized peracetic acid using the cascade reaction catalyzed by glucose oxidase and MsAcT, respectively.

过氧乙酸是一种重要的化工原料，传统的化学合成工艺存在严重的安全隐患及产品质量不稳定等缺点。利用酰基转移酶的过水解活性，建立葡萄糖氧化酶-酰基转移酶催化串联反应合成过氧乙酸的工艺，可解决上述问题。本项目旨在利用蛋白质工程技术，提高该工艺的稳定性和催化效率。具体策略(1)提高酰基转移酶对过氧化氢的亲和力，可降低双酶催化串联反应体系中残留过氧化氢的浓度，增强反应体系的稳定性；(2)在酰基转移酶表面不同位点引入Cys置换突变，并对其进行糖基化修饰。在分子粘合剂介导下，不同位点被糖基化修饰的酰基转移酶分别与葡萄糖氧化酶共固定化形成交联聚集体，筛选出催化效率最高的双酶交联聚集体。基于上述研究结果，可阐明酰基转移酶催化过水解反应的分子机制；建立通过对蛋白质进行位点选择性糖基化修饰，调整酶蛋白在多酶交联聚集体空间中的相对位置的新技术；获得具有自主知识产权的葡萄糖氧化酶/酰基转移酶交联聚集体

过氧乙酸是一种重要的化工原料，传统的化学合成工艺存在严重的安全隐患及产品质量不稳定等缺点。利用酰基转移酶的过水解活性，建立葡萄糖氧化酶-酰基转移酶催化串联反应合成过氧乙酸的工艺，可解决上述问题。本项目旨在利用蛋白质工程技术，提高该工艺的稳定性和催化效率。..本研究项目执行期间，主要完成了下面四个方面的研究工作，分别是：（1）评估并筛选出能与GOD高效偶联催化的酶蛋白；（2）构建并筛选出对过氧化氢亲和力显著提高的MsAcT蛋白突变体，并测定其与GOD偶联后的催化效率；（3）固定化MsAcT及GOD/MsAcT酶制剂的制备；（4）基于MsAcT蛋白质序列，挖掘并评估新型的酰基转移酶。具体实验结果如下：..从四种具有过水解催化活性的酶蛋白，包括Mycobacterium smegmatis 酰基转移酶(MsAcT)，Bacillus subtilis头孢菌素乙酰水解酶 (BsCAH)，Bacillus pumilus 乙酰木聚糖酯酶(BpAXE) 和Thermotoga maritima 乙酰木聚糖酯酶(TmAXE) 中，筛选出可能葡萄糖氧化酶高效偶联的酶蛋白（MsAcT），葡萄糖氧化酶：MsAcT的最优酶活比为50:1。利用蛋白质工程技术，改在MsAcT蛋白活性中心的氨基酸残基，筛选到双突变体MsAcT-S54I/L119Q对过氧化氢的亲和力提高了49.8倍。双突变体MsAcT-S54I/L119Q与葡萄糖氧化酶的最优酶活比降低到10:1，提高了5倍。利用四甲氧基硅烷包埋MsAcT后，固定化酶制剂较游离酶，其动力学选择性 kcat/Km[转酯化反应]/kcat/Km[水解反应]提高了6.88倍。纳米四氧化三铁载体依次用TEOS、APTES和戊二醛修饰后，再与GOD/MsAcT混合酶蛋白共固定化形成交联酶聚集体。结果表明：利用该工艺获得的GOD/MsAcT交联酶聚集体，单位酶活的催化效率提高了2.3倍。除了项目申请书规定的研究内容，课题组进一步拓展了相关研究工作，利用组蛋白重构技术，挖掘到MsAcT蛋白的系列祖先蛋白，并进行了功能验证，祖先蛋白表现出许多新型催化活性，具有潜在的工业化应用价值。