蔗糖异构酶催化高纯异麦芽酮糖生产的分子机制研究

Sucrose isomerase catalyzes the production of isomaltulose from sucrose, whereas its product component is changeable due to sucrose isomerase sourced from different microorganisms. Therefore, the product diversity of sucrose isomerase will be studied in this project with the combined rational and irrational protein design strategy. Recombinant sucrose isomerases will be obtained by site-directed mutagenesis combined with homology modeling and ARTP mutagenesis, in which the recombinant protein producing low ratio of glucose and fructose but high ratio of isomaltulose is selected by sugar analysis. Post-translational modification is analyzed using Top-down/Bottom-up technology to examine the relationship between post-translational modification and product specificity of sucrose isomerase. The molecular technology is also applied to study the influence of expressing location on the product diversity. The key control factors influencing product diversity such as key amino acids, expressing location and post-translational modification will be confirmed based on the relationship between mutant sites and product component. The molecular mechanism of sucrose isomerase catalyzing sucrose transformation will be discussed and the engineering strain producing the highly purified isomaltulose will be constructed by molecular recombinant technology.

异麦芽酮糖是由蔗糖异构酶催化蔗糖转化形成的，因蔗糖异构酶来源不同，催化形成的蔗糖异构化产物组成比例也各不相同。本项目针对蔗糖异构酶催化蔗糖转化产物多样性问题，组合运用理性和非理性蛋白质设计策略，采用同源模建定点突变技术和ARTP诱变技术，并结合糖分析手段，筛选获得葡萄糖与果糖比例降低、异麦芽酮糖得率提高的蔗糖异构酶改组蛋白，以确定直接影响蔗糖异构酶催化蔗糖转化产物组成比例的关键氨基酸位点和基序；利用蛋白组学Top-down/Bottom-up技术，分析比较翻译后修饰对蔗糖异构酶催化蔗糖转化产物特异性的贡献和影响；借助分子生物学手段，分析比较蔗糖异构酶表达场所与产物组成比例间的关系。由此，通过实验数据综合分析，以揭示影响蔗糖异构酶产物多样性的关键氨基酸、表达形式和翻译后修饰等关键控制位点，阐明蔗糖异构酶催化蔗糖异构化的分子机制，并通过分子改造，获得高纯异麦芽酮糖生产的重组蔗糖异构酶工程菌。

异麦芽酮糖是由蔗糖异构酶催化蔗糖转化形成的，因蔗糖异构酶来源不同，催化形成的蔗糖异构化产物组成比例也各不相同。本项目针对蔗糖异构酶催化蔗糖转化产物多样性问题，通过关键氨基酸位点分析与动力学模拟，确定了影响蔗糖异构酶催化蔗糖转化产物组成比例的关键氨基酸位点，发现了融合标签种类和融合位置影响蔗糖异构酶的产物组成，为此，构建了高效分泌表达的生产蔗糖异构酶的重组枯草芽孢杆菌工程菌,用于催化蔗糖异构化生产异麦芽酮糖，并采用分子伴侣共表达和理性设计策略提高了重组蔗糖异构酶的可溶性表达水平，利用固体结合肽标签实现了蔗糖异构酶的定向固定化和溶胶-凝胶法固定化酶的制备，为重组蔗糖异构酶的工业化应用奠定了物质基础。