耐高温葡萄糖异构酶在枯草芽孢杆菌中的分泌表达及在高果糖浆合成中的应用基础研究

Glucose isomerase (Gi) which makes the conversion of D-glucose into D-fructose, is an important industrial enzyme for producing high fructose syrup. Aiming at the technology problem of poor thermostability at high temperature among existing commercial Gis and its production using food grade expression system, relative fundamental studies were developed. Firstly, it was necessary to obtain a non reported thermmophilus Gi gene from Genbank, followed by gene synthesis and heterologous expression in Escherichia coli. Furthermore, the thermostability of Gi was improved by molecular modification of homologous modeling, molecular docking and genetic mutation technologies. Then, the gene of orthomutation enzyme was introduced into the Bacillus subtilis expression vector, and the secretory expression was able to be accomplished using the signal peptide of vector. A series of crucial environmental factors which had the greatest influence on the expression were optimized to realize over expression of refractory Gi. Finally, the experiments on the conversion of glucose into high fructose syrup catalyzed by recombinant Gi were conducted in order to obtain the optimal, high-performance and low cost conversion system. The Gi with better thermostability at high temperature, independent intellectual property right and approving to food safety standards, provided a substantial academic foundation for industrial production and technology development of high fructose syrup.

葡萄糖异构酶，可将D-葡萄糖转化为D-果糖，是制得高果糖浆的重要工业酶。本项目针对商业酶高温热稳定性差、无法用符合食品安全的表达系统生产的技术问题开展了基础研究。首先，从Genbank中筛选文献未报道的嗜热葡萄糖异构酶，合成基因，完成在大肠杆菌中的异源表达；运用同源建模、分子对接、基因突变等技术进行分子改造，进一步提高酶的高温热稳定性。其次，将突变基因导入表达载体，构建枯草芽孢杆菌基因工程菌，通过优化影响蛋白表达的关键环境因素，实现耐高温葡萄糖异构酶的高效分泌表达。最后，对重组酶催化葡萄糖生成高果糖浆的过程进行研究，获得转化效率高、反应成本低的最优转化体系。本项目所开发的具有自主知识产权、高温热稳定性优良、符合食品安全标准的葡萄糖异构酶，将为推进高果糖浆的工业化生产技术奠定坚实的理论基础。

葡萄糖异构酶可将D-葡萄糖异构化为D-果糖，获得的高果糖浆作为甜味剂可应用于食品等诸多领域。为了实现一步生物转化法制备HFCS-55，本项目所开展的研究如下：.1) 运用基因挖据技术，从NCBI数据库筛选到未被研究的Thermotoga petrophila GI（TPGI）和Thermoanaerobacter ethanolicus GI（TEGI），选择更加适合的大肠杆菌为表达系统，酶活更高的TEGI为研究对象，详细优化了TEGI重组菌发酵产酶条件，最终其发酵生物量提高200%，酶活力提高12%。.2) 针对酶在非天然工业环境下表现出催化活性低、稳定性差等限制其工业应用的关键催化特性，本研究运用蛋白质工程手段进行酶分子改造，获得突变酶TEGI-W139F-V186T。酶学性质研究发现，突变酶对底物的亲和力和催化效率高于原始酶，突变酶更优的高温热稳定性和更低的最适反应pH表明其更有利于工业应用。.3) 开发聚乙烯亚胺-戊二醛制备新型固定化耐高温葡萄糖异构酶催化剂的工艺，详细优化了交联剂、絮凝剂和载体添加量，以及固定化pH、温度和时间等环境因素，获得最佳固定化条件。该固定化酶于85℃进行10批次异构化反应，可以连续制得F55型高果糖浆，有效降低了生产成本。