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<p>The consumption of a high-sugar diet has been widely associated with various health complications. Due to its low caloric content, favorable safety profile, and high sweetness, rubusoside has been recognized as a promising alternative to sucrose and synthetic sweeteners. In this study, we report a β-glucosidase from <i>Bacteroides thetaiotaomicron</i> (BtBGL), which specifically hydrolyzes the β-1,2-glucosidic bond in the sophorosyl disaccharide at the C-13 hydroxyl group of stevioside to produce rubusoside. The M98V/Y718F mutant of BtBGL was obtained through consensus design and site-directed mutagenesis. Compared to the wild-type BtBGL, the optimal temperature of the M98V/Y718F mutant increased from 45&#xa0;°C to 50&#xa0;°C, demonstrating improved thermostability at this temperature. Additionally, the optimal pH decreased from 6.0 to 5.5, while enzyme activity at pH 5.5 increased from 61.17 U/mg (at pH 6.0) to 83.34 U/mg. This shift in optimal pH remains consistent when stevioside is used as the substrate. The increased surface charge, hydrophobicity, and hydrogen bonding in this double-point mutant may contribute to its enhanced activity at the altered pH and improvement in thermal stability. Stevioside (200 g/L) can be completely converted by the M98V/Y718F mutant within 1&#xa0;h, and the highest rubusoside yields of 80% and 81% were achieved at 5.5 and 6.0, respectively, after 2 h. This study provides a reference for the engineering of biocatalysts used in rubusoside production.</p> Graphical Abstract <p></p>

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An Engineered β-glucosidase from Bacteroides Thetaiotaomicron for Production of Rubusoside from Stevioside

  • 期刊论文

  • 发表日期：

    2026年4月1日

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  • Yuan Liao,
  • Kai Chen,
  • Hafza Altaf,
  • Jiajie Ding,
  • Liping Zhu,
  • Honghua Jia,
  • Yan Li

摘要

The consumption of a high-sugar diet has been widely associated with various health complications. Due to its low caloric content, favorable safety profile, and high sweetness, rubusoside has been recognized as a promising alternative to sucrose and synthetic sweeteners. In this study, we report a β-glucosidase from Bacteroides thetaiotaomicron (BtBGL), which specifically hydrolyzes the β-1,2-glucosidic bond in the sophorosyl disaccharide at the C-13 hydroxyl group of stevioside to produce rubusoside. The M98V/Y718F mutant of BtBGL was obtained through consensus design and site-directed mutagenesis. Compared to the wild-type BtBGL, the optimal temperature of the M98V/Y718F mutant increased from 45 °C to 50 °C, demonstrating improved thermostability at this temperature. Additionally, the optimal pH decreased from 6.0 to 5.5, while enzyme activity at pH 5.5 increased from 61.17 U/mg (at pH 6.0) to 83.34 U/mg. This shift in optimal pH remains consistent when stevioside is used as the substrate. The increased surface charge, hydrophobicity, and hydrogen bonding in this double-point mutant may contribute to its enhanced activity at the altered pH and improvement in thermal stability. Stevioside (200 g/L) can be completely converted by the M98V/Y718F mutant within 1 h, and the highest rubusoside yields of 80% and 81% were achieved at 5.5 and 6.0, respectively, after 2 h. This study provides a reference for the engineering of biocatalysts used in rubusoside production.

Graphical Abstract