Surface engineering of cyclodextrin glycosyltransferase reveals structural compactness and rigidity responsible for enhanced organic solvents resistance

被引:3
作者
Han, Ruizhi [1 ,2 ]
Jiang, Yulin [1 ]
Liu, Siyan [1 ]
Ji, Yu [2 ]
Schwaneberg, Ulrich [2 ,3 ]
Ni, Ye [1 ]
机构
[1] Jiangnan Univ, Sch Biotechnol, Key Lab Ind Biotechnol, Wuxi 214122, Peoples R China
[2] Rhein Westfal TH Aachen, Inst Biotechnol, D-52074 Aachen, Germany
[3] DWI Leibniz Inst Interact Mat, D-52074 Aachen, Germany
基金
中国国家自然科学基金;
关键词
Cyclodextrin glycosyltransferase; Semi-rational design; Organic solvent resistance; Surface engineering; MD simulation; ENZYMATIC PRODUCTION; BETA-CYCLODEXTRIN; GLUCANOTRANSFERASE; TRANSGLYCOSYLATION; LIPASE; STEVIOSIDE; EFFICIENCY; TOLERANCE; NARINGIN; IMPROVE;
D O I
10.1016/j.mcat.2023.113613
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
摘要
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 (PmCGTase) 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 t1/2 value at both 40 and 50 degrees C. In addition, a decreased Km value and an increased kcat 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 PmCGTase, which would further improve its application potential.
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页数:10
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