甜蜜
催化效率
化学
果糖
酶
重组DNA
催化作用
突变
生物化学
细菌
糖
食品科学
立体化学
生物
突变
基因
遗传学
作者
Liming Guan,Ling Zhu,Kunlun Wang,Yang Gao,Jialei Li,Song Yan,Xindi Zhang,Nina Ji,Jia Fan,Ye Zhou,Xinmiao Yao,Бо Ли
标识
DOI:10.3389/fbioe.2024.1365814
摘要
D-Allulose has become a promising alternative sweetener due to its unique properties of low caloric content, moderate sweetness, and physiological effects. D-Allulose 3-epimerase (DAEase) is a promising enzyme for D-Allulose production. However, the low catalytic efficiency limited its large-scale industrial applications. To obtain a more effective biocatalyst, a putative DAEase from Christensenellaceae bacterium (CbDAE) was identified and characterized. The recombinant CbDAE exhibited optimum activity at pH 7.5°C and 55°C, retaining more than 60% relative activity from 40°C to 70°C, and the catalytic activity could be significantly increased by Co 2+ supplementation. These enzymatic properties of purified CbDAE were compared with other DAEases. CbDAE was also found to possess desirable thermal stability at 55°C with a half-life of 12.4 h. CbDAE performed the highest relative activity towards D-allulose and strong affinity for D-fructose but relatively low catalytic efficiency towards D-fructose. Based on the structure-guided design, the best double-mutation variant G36N/W112E was obtained which reached up to 4.21-fold enhancement of catalytic activity compared with wild-type (WT) CbDAE. The catalytic production of G36N/W112E with 500 g/L D-fructose was at a medium to a higher level among the DAEases in 3.5 h, reducing 40% catalytic reaction time compared to the WT CbDAE. In addition, the G36N/W112E variant was also applied in honey and apple juice for D-allulose conversion. Our research offers an extra biocatalyst for D-allulose production, and the comprehensive report of this enzyme makes it potentially interesting for industrial applications and will aid the development of industrial biocatalysts for D-allulose.
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