融合蛋白催化D-葡萄糖合成D-阿洛酮糖的研究  

Study on Synthesis of D-Allulose from D-Glucose Catalyzed by Fusion Proteins

作　　者：张家赫 冯敬杰 刘微微 常楚婷 郭庆彬 丁文涛 王昌禄[1,2] ZHANG Jiahe;FENG Jingjie;LIU Weiwei;CHANG Chuting;GUO Qingbin;DING Wentao;WANG Changlu(College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457,China;State Key Laboratory of Food Nutrition and Safety,Tianjin University of Science and Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学食品科学与工程学院,天津300457 [2]天津科技大学省部共建食品营养与安全国家重点实验室,天津300457

出　　处：《食品科学技术学报》2024年第6期113-120,153,共9页Journal of Food Science and Technology

基　　金：国家自然科学基金项目(31501041、32272277);中国博士后科学基金项目(2019M650450)。

摘　　要：D-阿洛酮糖是一种新型功能性代糖,目前主要以价格较高的D-果糖为底物,通过D-阿洛酮糖3-差向异构酶转化获得。寻找低价原料替代D-果糖生产D-阿洛酮糖对其产业化具有重要意义。将葡萄糖异构酶与D-阿洛酮糖3-差向异构酶通过柔性或刚性连接肽,以不同顺序首尾相连,构建4种构型的融合蛋白,从中选择催化效果最好的融合蛋白构型并对此融合蛋白的催化条件进行优化,实现了以价格较低的D-葡萄糖为底物经催化合成D-阿洛酮糖。研究结果表明:融合蛋白AE3G和AS3G同时具备葡萄糖异构酶和D-阿洛酮糖3-差向异构酶活性,能够将D-葡萄糖转化为D-果糖和D-阿洛酮糖。采用刚性连接肽连接的AE3G活性优于使用柔性连接肽连接的AS3G。优化后的AE3G催化条件为65℃,Tris-HCl缓冲液为50 mmol/L,pH值为7.5,Co 2+浓度为0.5 mmol/L,Mn 2+浓度为0.5 mmol/L,湿细胞质量浓度为75 g/L。在此条件下,分别以100 g/L D-葡萄糖和体积分数为5%的F55果葡糖浆为底物,利用融合蛋白催化产生17.2 g/L和12.5 g/L D-阿洛酮糖,反应液中D-葡萄糖、D-果糖和D-阿洛酮糖的质量比为2.5∶2.3∶1.0。研究旨在证明融合蛋白催化D-葡萄糖转化成D-阿洛酮糖的可行性。研究结果表明,D-阿洛酮糖平衡浓度高于文献报道值,希望为低成本生产D-阿洛酮糖提供理论参考。D-allulose is a novel functional sugar substitute primarily produced from the high-cost substrate D-fructose,using D-allulose 3-epimerase to achieve the conversion.Identifying low-cost raw material alternatives to D-fructose for D-allulose production is crucial for its industrialization.In this study,glucose isomerase and D-allulose 3-epimerase were linked with either flexible or rigid peptide linkers in different sequential orientations,constructing four configurations of fusion proteins.The fusion protein configuration,exhibiting the best catalytic performance,was selected and optimized to catalyze D-allulose from the lower-cost substrate D-glucose.The results showed that fusion proteins AE3G and AS3G possessed both glucose isomerase and D-allulose 3-epimerase activities,converting D-glucose to D-fructose and D-allulose.AE3G,connected with a rigid peptide linker,demonstrated higher activity than AS3G,which connected with a flexible linker.The optimized catalytic conditions for AE3G were temperature of 65℃,Tris-HCl buffer of 50 mmol/L,pH of 7.5,Co 2+concentration of 0.5 mmol/L,Mn 2+concentration of 0.5 mmol/L,and wet cell concentration of 75 g/L.Under these conditions,using 100 g/L D-glucose or volume fraction 5%F55 high-fructose corn syrup as substrates,the fusion protein produced 17.2 g/L and 12.5 g/L D-allulose,respectively,with a mass ratio of D-glucose,D-fructose,and D-allulose in the reaction mixture of 2.5∶2.3∶1.0.This study aimed to verify the feasibility of using fusion proteins to catalyze the conversion of D-glucose to D-allulose,and the results indicated that the equilibrium concentration of D-allulose was higher than reported values,providing a theoretical basis for low-cost D-allulose production.

关 键 词：融合蛋白 葡萄糖异构酶 D-阿洛酮糖3-差向异构酶 D-阿洛酮糖 果葡糖浆 D-葡萄糖 

分 类 号：TS201.2[轻工技术与工程—食品科学]