Back to Search
Start Over
Surface engineering of cyclodextrin glycosyltransferase reveals structural compactness and rigidity responsible for enhanced organic solvents resistance.
- Source :
-
Molecular Catalysis . Nov2023, Vol. 550, pN.PAG-N.PAG. 1p. - Publication Year :
- 2023
-
Abstract
- • Semi-rational engineering on surface residues of CGTase for enhanced OSs resistance. • A variant G539I/R146F/D147N with enhanced OSs resistance and thermostability was obtained. • Strengthened structural compactness and rigidity of CGTase is conducive to its OSs resistance. Cyclodextrin glycosyltransferase (CGTase) is a preferable biocatalyst for production of cyclodextrin and phenols glycosylated derivatives due to its cost-effective donors and broad range of acceptors. Organic solvents (OSs) are often beneficial for solubilizing hydrophobic substrates and product specificity, but often harmful towards CGTases. Herein, using Paenibacillus macerans CGTase (Pm CGTase) as a template and DMSO as a screening solvent, semi-rational engineering was performed at 18 surface residues by a developed high throughput screening method. Using site saturation mutagenesis and iterative saturation mutagenesis, 5 OSs-resistant variants (G539I, G539V, G539I/R146F, G539V/R146A, G539I/R146F/D147N) were obtained. Compared with WT, the best variant G539I/R146F/D147N showed a 79.3 % enhanced residual activity at 30 % DMSO and a 60 % increased t 1/2 value at both 40 and 50 °C. In addition, a decreased K m value and an increased k cat value led to 41.9 % higher catalytic efficiency of G539I/R146F/D147N than WT in 15 % DMSO. This variant was also applied to glycosylation of sophoricoside in 20 % DMSO, leading to 26.2 % increased conversion. Molecular dynamics demonstrates that its enhanced OSs resistance may be attributed to the strengthened structural compactness and rigidity. Our results provide guidance for engineering OSs resistance of Pm CGTase, which would further improve its application potential. [Display omitted] [ABSTRACT FROM AUTHOR]
- Subjects :
- *CYCLODEXTRINS
*GLYCOSYLTRANSFERASES
*ORGANIC solvents
*PAENIBACILLUS
*PHENOLS
Subjects
Details
- Language :
- English
- ISSN :
- 24688231
- Volume :
- 550
- Database :
- Academic Search Index
- Journal :
- Molecular Catalysis
- Publication Type :
- Academic Journal
- Accession number :
- 173176703
- Full Text :
- https://doi.org/10.1016/j.mcat.2023.113613