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.

Recent Advances in β-Glucosidase Sequence and Structure Engineering: A Brief Review

“…Rational design (Hydropathy index for enzyme activity) Site-directed mutation ---Transglycosylation: ↑disaccharides productivity by 3.5-fold. [28] Bacillus sp. D1 (BglD1) Semi-rational design Site-directed mutagenesis ---Transglycosylation: ↑ GOS productivity by 11.5%.…”

“…As shown in Table 4, the rational design of targeted BGL catalytic tunneling of subsite residues provides methodological strategies: (1) reducing the binding in glycone (−) subsites; (2) increasing the affinity in aglycone (+) subsites; and (3) disrupting the binding of catalytic water: mainly by removing the hydrogen-bonding interactions with the catalytic water and the retention of nucleophilic water molecules at key amino acid residues, or enhancing the hydrophobicity at the active site entry or acceptor subsite [95]. For example, the Hydropathy Index For Enzyme Activity (HIFEA) strategy to reduce the hydrophilic index of BGL amino acid residues has been used for the rational design of oligosaccharide synthesis [28]. ↑ Disaccharides productivity by 3.5-fold.…”

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

“…In the original publication of the article (Niu et al 2020 ), the statement in the Funding information section was incorrectly given as ‘the National Key R&D Program of China (No. 2018YFA090010)’ and should have read ‘the National Key R&D Program of China (No.…”

Hybrid Computationally Guided Strategies for Engineering a Laminaribiose Phosphorylase to Facilitate the Industrial Production of Laminaribiose In Vitro

“…The biosynthesis of laminaribiose from glucose by β-glucosidase or from glucose and glucose 1-phosphate (G1P) by laminaribiose phosphorylase (LBP, EC 2.4.1.30) has recently received increasing attention due to the readily accessible substrate, mild reaction conditions, environment-friendly process, and excellent regio- and stereoselectivity . The synthesis of laminaribiose by glycosidases has several benefits that embody simplicity, accessibility, and easy operations, although the broad substrate specificity of β-glucosidase synthesizes three main products (laminaribiose, sophorose, and cellobiose). , In contrast, LBP, which catalyzes ATP-independent phosphorolysis reactions with strict regioselectivity, is the ideal enzyme for the transformation of readily accessible saccharides to laminaribiose. − Many studies have investigated the use of sucrose or maltodextrin as a provider of G1P catalyzed by sucrose phosphorylase (SP) or α-glucan phosphorylase (αGP) . Muller et al reported the production of laminaribiose from sucrose and glucose catalyzed by SP and LBP, in which the reaction was carried out at 35 °C with a final yield of 55%.…”

“…13 The synthesis of laminaribiose by glycosidases has several benefits that embody simplicity, accessibility, and easy operations, although the broad substrate specificity of β- glucosidase synthesizes three main products (laminaribiose, sophorose, and cellobiose). 14,15 In contrast, LBP, which catalyzes ATP-independent phosphorolysis reactions with strict regioselectivity, is the ideal enzyme for the transformation of readily accessible saccharides to laminaribiose. 16−18 Many studies have investigated the use of sucrose or maltodextrin as a provider of G1P catalyzed by sucrose phosphorylase (SP) or α-glucan phosphorylase (αGP).…”