耐热Ｄ-甘露糖异构酶的构建及分子机制研究

D-Mannose, as a natural nutritional and health-beneficial dietary supplement, has been widely used in the food and health care industry. D-Mannose isomerase (EC 5.3.1.7, MIase) is an aldo-keto isomerase that can reversibly catalyze isomerization between D-mannose and D-fructose, which has becoming a promising enzyme for the production of D-mannose, as it operates with lower cost and no secondary pollution. However, the poor thermostability of MIase is serious constraints on industrial application. To address these issues, presently, the thermostability of MIase from B. subtilis WB800 (pMA5-yihS) will be evolved by DNA shuffling, computer aided rational design and ISM, terminal deletion, Short peptide fusion. According to module optimization, the best mutant strain with the highest thermostability will be selected after screening. The key sites and domain related to heat resistance were analyzed by bioinformatics tools. And the mechanism of heat resistance was further explained. The project definitely could enhance the application of the MIase in prepatation of D-mannose, and provide the theoretical basis for enhance thermos resistance mechanism and modification of MIase family.

D-甘露糖作为天然的膳食营养补充剂，已广泛应用于食品医疗保健行业。D-甘露糖异构酶(MIase)，是一种能可逆催化D-果糖转化成D-甘露糖的酮-醛糖异构酶。酶法制备D-甘露糖因具有无污染、低耗能等优点成为D-甘露糖最具前景的生产方法，但同时也因为MIase耐热性差限制了它的工业化应用。为解决这一困境，本项目以前期在B. subtilis WB800中实现高效表达的E. coli来源的MIase酶编码基因yihS为对象，应用DNA shuffling、计算机辅助理性设计结合迭代饱和突变、末端缺失、短肽融合等多种策略对MIase进行分子改造提高其热稳定性，对不同策略的正突变进行模块化组合进一步提升MIase热稳定性，并通过生物信息学手段分析MIase热稳定相关的关键结构域和位点，阐述其耐热机制。本项目将为D-甘露糖酶法制备产业化起到推动作用，为MIase家族酶的耐热机制研究提供重要的理论参考。

D-甘露糖是一种具有重要经济价值的天然生物活性单糖，已被证实有益于增强免疫力，对代谢综合征、糖尿病、肠道疾病、尿路感染等疾病有改善作用，对人类健康意义重大，在食品、保健品、护肤品和家禽畜牧养殖业中应用广泛。本项目以E. coli BL21提取的DNA为模板，通过PCR方法扩增出D-甘露糖异构酶yihS基因进行同源表达，目的蛋白获得较高表达量。该酶在40℃和pH 7.5条件下表现出催化D-果糖的最高活性，转化率为25%左右；通过研究底物浓度对酶活性的影响，得出该酶的活性不受底物浓度的抑制，以D-果糖为底物时，动力学参数Km为123.32 mM，Vmax为113.64 µmol min‒1 mg‒1，催化效率kcat/Km为0.691 s‒1 mM‒1。基于前述实验，通过定点饱和突变定向进化技术结合分子动力学模拟技术，实现了D-MIase的半理性设计与分子改造，提高了酶的耐热稳定性。野生酶经55℃加热处理30 min后，损失了67%的酶活，而突变体R335L、R335K酶活分别下降35%、48%，表现出相对较好的耐热稳定性。通过计算机模拟，发现温度变化对D-MIase位点229Asp~242Phe影响较大，此区域可能与D-MIase热稳定性有较大的相关性。此外，在此基础上尝试制备磁性纳米粒子对酶进行固定化，为其工业应用提供理论依据。本项目进一步丰富了D-甘露糖的生物酶法高效合成的基础理论研究，并为D-甘露糖异构酶的生物合成提供一定的技术支持。