A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

Abstract

Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis 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 TrCel1b^I177S, TrCel1b^I177S/I174S, and TrCel1b^{I177S/I174S/W173H}, and one negative variant TrCel1b^N240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1b^{I177S/I174S/W173H} was accelerated and then the maximum total production (195.8 mg/mL/mg enzyme) of the synthesized disaccharides was increased by 3.5-fold compared to that of wild type. On the contrary, TrCel1b^N240I 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.

Keywords

β-Glucosidase / Hydropathy index / Disaccharide synthesis / Reverse hydrolysis reaction / Site-directed mutagenesis

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Funding

National Key R&D Program of China(2018YFA090010)

Key Technology Research and Development Program of Shandong(2018GSF121021)

the 111 Project(B16030)

the State Key Laboratory of Microbial Technology Open Projects Fund(31870785)

National Natural Science Foundation of China(31570040)