The rare sugar d-allulose is an attractive sucrose substitute due to its sweetness and ultra-low caloric value. It can be produced from D-fructose using d-allulose 3-epimerase (DAE) as the biocatalyst. However, most of the reported DAEs show low catalytic efficiency and poor thermostability, which limited their further use in food industrial. Here, a putative d-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense (HcDAE) was characterized, showing optimal activity at pH 8.0 and 70 °C in the presence of Mg²⁺. Saturation mutagenesis of Y7, C66, and I108, the putative residues responsible for substrate recognition at the O-4, -5, and -6 atoms of D-fructose was performed, and it yielded the triple mutant Y7H/C66L/I108A with improved activity toward D-fructose (345 % of wild-type enzyme). The combined mutant Y7H/C66L/I108A/R156C/K260C exhibited a half-half (t_1/2) of 5.2 h at 70 °C and an increase of the T_m value by 6.5 °C due to the introduction of disulfide bridges between intersubunit with increased interface interactions. The results indicate that mutants could be used as industrial biocatalysts for d-allulose production.

稀有糖d-阿洛酮糖因其甜度和超低热值而成为有吸引力的蔗糖替代品。它可以使用d-阿洛酮糖3-差向异构酶 (DAE) 作为生物催化剂从 D-果糖生产。然而，大多数报道的DAEs催化效率低，热稳定性差，限制了它们在食品工业中的进一步应用。在这里，对来自刚果嗜盐菌(HcDAE)嗜热生物的推定d-阿洛酮糖 3-差向异构酶进行了表征，在存在 Mg ²⁺的情况下，在 pH 8.0 和 70 °C 下显示出最佳活性. 对 D-果糖的 O-4、-5 和 -6 原子处负责底物识别的推定残基 Y7、C66 和 I108 进行饱和诱变，产生了三重突变体 Y7H/C66L/I108A对 D-果糖的活性（野生型酶的 345%）。组合突变体 Y7H/C66L/I108A/R156C/K260C在 70 °C 下表现出5.2 小时的一半 ( t _1/2 ) 并且由于在两者之间引入二硫键，T _m值增加了 6.5 °C。具有增加的界面相互作用的亚基间。结果表明突变体可用作生产d-阿洛酮糖的工业生物催化剂。