Li Lei, Zhang Qianqian, Wang Tong, Qi Hongbin, Wei Meijing, Lu Fuping, Guan Lijun, Mao Shuhong, Qin Hui-Min
Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, P. R. China.
Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P. R. China.
J Agric Food Chem. 2022 Dec 28;70(51):16298-16306. doi: 10.1021/acs.jafc.2c07153. Epub 2022 Dec 14.
d-Allulose, a rare sugar and functional sweetener, can be biosynthesized by d-allulose 3-isomerase (DAE). However, most of the reported DAEs exhibit poor resistance under acidic conditions, which severely limited their application. Here, surface charge engineering and random mutagenesis were used to construct a mutant library of CcDAE from H10, combined with high-throughput screening to identify mutants with high activity and resistance under acidic conditions. The mutant M3 (I114R/K123E/H209R) exhibited high activity (3.36-fold of wild-type) and acid resistance (10.6-fold of wild-type) at pH 4.5. The structure-function relationship was further analyzed by molecular dynamics (MD) simulations, which indicated that M3 had a higher number of hydrogen bonds and negative surface charges than the wild type. A multienzyme cascade system including M3 was used to convert high-calorie sugars in acidic juices, and functional juices containing 7.8-15.4 g/L d-allulose were obtained. Our study broadens the manufacture of functional foods containing d-allulose.
阿洛酮糖是一种稀有糖和功能性甜味剂，可通过阿洛酮糖3-异构酶（DAE）进行生物合成。然而，大多数已报道的DAE在酸性条件下表现出较差的耐受性，这严重限制了它们的应用。在此，通过表面电荷工程和随机诱变构建了来自H10的CcDAE突变体文库，并结合高通量筛选来鉴定在酸性条件下具有高活性和耐受性的突变体。突变体M3（I114R/K123E/H209R）在pH 4.5时表现出高活性（野生型的3.36倍）和耐酸性（野生型的10.6倍）。通过分子动力学（MD）模拟进一步分析了结构-功能关系，结果表明M3比野生型具有更多的氢键和负表面电荷。使用包含M3的多酶级联系统来转化酸性果汁中的高热量糖，并获得了含有7.8-15.4 g/L阿洛酮糖的功能性果汁。我们的研究拓宽了含阿洛酮糖功能性食品的制造途径。
