State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu Province, China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu Province, China.
J Agric Food Chem. 2022 Sep 28;70(38):12128-12134. doi: 10.1021/acs.jafc.2c04800. Epub 2022 Sep 13.
As a natural sweetener with low calories and various physiological activities, d-allulose has drawn worldwide attention. Currently, d-allulose 3-epimerase (DAEase) is mainly used to catalyze the epimerization of d-fructose to d-allulose. Therefore, it is quite necessary to enhance the food-grade expression of DAEase to meet the surging market demand for d-allulose. In this study, initially, the promising variant H207L/D281G/C289R of H10 DAEase (CcDAEase) was generated by protein engineering, the specific activity and the of which were 2.24-fold and 13.45-fold those of the CcDAEase wild type at 60 °C, respectively. After that, P was determined as the optimal promoter for the recombinant expression of CcDAEase in , and catabolite-responsive element (CRE) box engineering was further performed to eliminate the carbon catabolite repression (CCR) effect. Lastly, high-density fermentation was carried out and the final activity peaked at 4971.5 U mL, which is the highest expression level and could effectively promote the industrial production of DAEase. This research provides a theoretical basis and technical support for the molecular modification of DAEase and its efficient fermentation preparation.
作为一种低热量、具有多种生理活性的天然甜味剂，d-阿洛酮糖已引起全球关注。目前，d-阿洛酮糖 3-差向异构酶（DAEase）主要用于催化 d-果糖向 d-阿洛酮糖的差向异构化。因此，提高 DAEase 的食品级表达量以满足 d-阿洛酮糖不断增长的市场需求是非常必要的。在本研究中，首先通过蛋白质工程生成了 H10 DAEase（CcDAEase）有前途的 H207L/D281G/C289R 变体，其在 60°C 时的比活性和温度系数分别是 CcDAEase 野生型的 2.24 倍和 13.45 倍。之后，确定 P 是 CcDAEase 在 中重组表达的最佳启动子，并进一步进行了分解代谢物响应元件（CRE）盒工程以消除碳分解代谢物阻遏（CCR）效应。最后，进行了高密度发酵，最终酶活达到 4971.5 U·mL，这是最高的表达水平，可以有效促进 DAEase 的工业生产。本研究为 DAEase 的分子修饰及其高效发酵制备提供了理论依据和技术支持。
