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Efficient Synthesis of Rare Disaccharides by Engineered β-Glucosidase Based on Hydropathy Index

preprint
submitted on 05.03.2020 and posted on 18.03.2020 by Kangle Niu, Zhengyao Liu, Yuhui Feng, Tianlong Gao, Zhenzhen Wang, Piaopiao Zhang, Zhiqiang Du, Daming Gao, Xu Fang

Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis, especially rare disaccharides, is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1bI177S, TrCel1bI177S/I174S, and TrCel1bI177S/I174S/W173H, and one negative variant TrCel1bN240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1bI177S/I174S/W173H was accelerated and then the maximum total production (195.8 mg/ml/mg enzyme) of the synthesized disaccharides was increased 3.5-fold compared to that of wildtype. On the contrary, TrCel1bN240I lost reverse hydrolysis activity. The results demonstrate that the average hydropathy index of the key amino acid residues in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

Funding

National Key R&D Program of China (No. 2018YFA090010)

Key Technologies R&D Program of Shandong Province (No. 2018GSF121021)

the 111 Project (No. B16030)

the State Key Laboratory of Microbial Technology Open Projects Fund (No.31570040)

the National Natural Science Foundation of China (No.31870785)

History

Email Address of Submitting Author

fangxu@sdu.edu.cn

Institution

Shandong University

Country

China

ORCID For Submitting Author

0000-0002-9196-5697

Declaration of Conflict of Interest

Part of the study was authorized by the patent CN 104232606 B.

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