期刊
BIOPROCESS AND BIOSYSTEMS ENGINEERING
卷 46, 期 9, 页码 1279-1291出版社
SPRINGER
DOI: 10.1007/s00449-023-02896-z
关键词
Cellobiose 2-epimerase; Lactulose; Thermostability; Catalysis; Computer-aided design
A non-characterized cellobiose 2-epimerase (CE) from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database, and a smart mutation library was constructed to obtain a positive mutant D226G with improved thermostability and catalytic efficiency. Molecular modeling revealed that the improvement of D226G was attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket, and the increment of substrate-binding capability. The yield of lactulose synthesis catalyzed by D226G reached 45.5%, higher than that achieved with CmCE. This study provides insight into the design of efficient CEs for biosynthesis of lactulose.
Cellobiose 2-epimerase (CE) is ideally suited to synthesize lactulose from lactose, but the poor thermostability and catalytic efficiency restrict enzymatic application. Herein, a non-characterized CE originating from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database. Then, a smart mutation library was constructed based on FoldX ??G calcu-lation and modeling structure analysis, from which a positive mutant D226G located within the a8/a9 loop exhibited longer half-lives at 65-75 degrees C as well as lower Km and higher k(cat)/K-m values compared with CmCE. Molecular modeling demon-strated that the improvement of D226G was largely attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket and the increment of substrate-binding capability. Finally, the yield of synthesizing lactulose catalyzed by D226G reached 45.5%, higher than the 35.9% achieved with CmCE. The disclosed effect of the flexible loop on enzymatic stability and catalysis provides insight to redesign efficient CEs to biosynthesize lactulose.
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