Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution  

作　　者：Shuhan Liu Yifan Wang Demin Kong Jing Wu Zhanzhi Liu 

机构地区：[1]State Key Laboratory of Food Science and Technology,Jiangnan University,1800 Lihu Avenue,Wuxi 214122,Jiangsu Province,China [2]School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education,Jiangnan University,1800 Lihu Avenue,Wuxi 214122,Jiangsu Province,China [3]International Joint Laboratory On Food Safety,Jiangnan University,1800 Lihu Avenue,Wuxi 214122,Jiangsu Province,China

出　　处：《Systems Microbiology and Biomanufacturing》2022年第4期685-694,共10页系统微生物学与生物制造（英文）

基　　金：The authors are grateful for the financial support from the National Natural Science Foundation of China(NSFC)(32101884);Natural Science Foundation of Jiangsu Province(BK20190586).

摘　　要：d-allulose,the epimer at C-3 position of d-fructose,is a low-calorie functional rare sugar,which is regarded as one of the most potential sweeteners.At present,the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase(DAE).However,industrial applications of DAE are still limited by its poor thermostability.Herein,directed evolution was applied to improve the thermostability of dAE from Clostridium cellulolyticum H10(CcDAE).Two optimal mutants D281G and C289R,exhibiting 13.80-fold and 13.88-fold t_(1/2 )values as that of wild type at 65℃,respectively,were obtained.To further enhance the thermostability,the triple mutant A107P/D281G/C289R was constructed after combina-tion of mutants D281G,C289R,and previously identified thermostability-enhanced mutant A107P.The T_(m) and optimal temperature of triple mutant were increased by 14.39℃and 5℃,respectively,compared to the wild type,meanwhile,the half-life of triple mutant was 58.85-fold as that of wild type at 65℃.Furthermore,the conversion rate of triple mutant was increased from 24.76%of wild type to 27.53%using 300 g/L d-fructose as substrate at 70℃.The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.

关 键 词：D-allulose 3-epimerase Directed evolution Ultra-high throughput screening Thermostability 

分 类 号：O62[理学—有机化学]