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Improving the enzyme property of D-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense through rational design
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2021-06-12 , DOI: 10.1016/j.enzmictec.2021.109850
Zhangliang Zhu 1 , Lei Li 1 , Wei Zhang 1 , Chao Li 1 , Shuhong Mao 1 , Fuping Lu 1 , Hui-Min Qin 1
Affiliation  

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 (t1/2) of 5.2 h at 70 °C and an increase of the Tm 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.



中文翻译:

通过合理设计改善嗜热刚果嗜盐菌D-阿洛酮糖3-差向异构酶的酶学特性

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。

更新日期:2021-06-23
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