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.

中文翻译：

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β-葡萄糖苷酶从葡萄糖高效合成二糖的新策略

寡糖具有重要的治疗应用。寡糖合成的有用途径是β-葡糖苷酶逆水解。但是，二糖从单糖的低转化效率限制了其大规模生产，因为平衡偏向于水解方向。基于对接结果的分析，我们假设催化位点中关键氨基酸残基的亲水性指数与二糖合成密切相关，位于催化位点中的更多亲水性残基会增强逆水解活性。在这项研究中，阳性变体Tr Cel1b ^I177S，Tr Cel1b ^{I177S / I174S}和Tr Cel1b根据酶活性亲水指数（HIFEA）策略设计了^{I177S / I174S / W173H}和一种阴性变体Tr Cel1b ^N240I。加速了Tr Cel1b ^{I177S / I174S /} W173H的^逆水解作用，与野生型相比，合成二糖的最大总产量（195.8 mg / mL / mg酶）增加了3.5倍。相反，Tr Cel1b ^N240I失去了^逆水解活性。结果表明，Tr催化位点关键氨基酸残基的平均亲水指数Cel1b是合成Laminaribiose，槐糖和纤维二糖的重要因素。HIFEA策略为合理设计用于寡糖合成的β-葡萄糖苷酶提供了新的视角。