Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu, China.
Biotechnol J. 2024 Aug;19(8):e2400280. doi: 10.1002/biot.202400280.
D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants.
The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEase variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle.
All these results indicated that the DAEase variant would be a potential candidate for the industrial production of D-allulose.
D-阿洛酮糖是一种最知名的稀有糖，广泛应用于食品、化妆品和制药行业。D-阿洛酮糖的最常见生产方法是 D-阿洛酮糖 3-差向异构酶（DAEase）催化的 D-果糖转化。为了解决野生型 DAEase 普遍存在的催化效率低和热稳定性差的问题，本研究采用 D-阿洛酮糖生物传感器开发了一种高通量筛选 DAEase 变体的便捷高效方法。
通过半理性分子修饰，同时提高了来自 Caballeronia insecticola 的 DAEase 的催化活性和热稳定性。与野生型酶相比，DAEase 变体的催化活性提高了 14.7%，在 65°C 时的半衰期（t）值从 1.60 增加到 27.56 h，提高了 17.23 倍。令我们高兴的是，表达这种 DAEase 变体的枯草芽孢杆菌 WB800 全细胞可将 500 g/L 的 D-果糖在 1 h 内转化为 33.6%的 D-阿洛酮糖。此外，还评估了细胞固定化的实用性，从第二次到第七次循环，固定化细胞的相对活性保持在 80%以上。
所有这些结果表明，该 DAEase 变体将是 D-阿洛酮糖工业生产的潜在候选者。
