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<p>2-<i>O</i>-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid widely used in food, cosmetic, and pharmaceutical industries; however, its enzymatic production remains limited by high enzyme consumption and low catalytic efficiency. In this study, a marine-derived cyclodextrin glycosyltransferase (CGTase) was engineered using a semi-rational design strategy to enhance AA-2G synthesis with maltodextrin as a cost-effective glycosyl donor. Based on sequence alignment, two residues were selected for saturation mutagenesis, and beneficial variants (Y260F and A236P) were identified from mutant libraries through activity-based screening. Subsequently, the double mutant Y260F/A236P was constructed and characterized. Compared with the wild-type enzyme, the double mutant achieved an AA-2G concentration of 30.2&#xa0;g/L, corresponding to an 11.8% increase in yield. Kinetic analysis revealed a 20.8% decrease in <i>K</i><sub>m</sub> and a 1.48-fold increase in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> toward L-ascorbic acid. Under optimized conditions, the engineered system achieved high AA-2G yield with significantly reduced enzyme loading (120 U/g substrate), and the double mutant enabled rapid conversion, reaching high yield within 12&#xa0;h. In addition, A236 was identified as a novel mutagenesis site in CGTases. These results provide an efficient and cost-effective strategy for AA-2G production and offer new insights into enzyme engineering.</p>

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Engineering of a marine cyclodextrin glucosyltransferase for efficient synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid using maltodextrin

  • 期刊论文

  • 发表日期：

    2026年6月22日

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  • Wei Wang,
  • Yang Zheng,
  • Jingjing Sun,
  • Chengcheng Jiang,
  • Cong Lin,
  • Jianhua Hao

摘要

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid widely used in food, cosmetic, and pharmaceutical industries; however, its enzymatic production remains limited by high enzyme consumption and low catalytic efficiency. In this study, a marine-derived cyclodextrin glycosyltransferase (CGTase) was engineered using a semi-rational design strategy to enhance AA-2G synthesis with maltodextrin as a cost-effective glycosyl donor. Based on sequence alignment, two residues were selected for saturation mutagenesis, and beneficial variants (Y260F and A236P) were identified from mutant libraries through activity-based screening. Subsequently, the double mutant Y260F/A236P was constructed and characterized. Compared with the wild-type enzyme, the double mutant achieved an AA-2G concentration of 30.2 g/L, corresponding to an 11.8% increase in yield. Kinetic analysis revealed a 20.8% decrease in Km and a 1.48-fold increase in kcat/Km toward L-ascorbic acid. Under optimized conditions, the engineered system achieved high AA-2G yield with significantly reduced enzyme loading (120 U/g substrate), and the double mutant enabled rapid conversion, reaching high yield within 12 h. In addition, A236 was identified as a novel mutagenesis site in CGTases. These results provide an efficient and cost-effective strategy for AA-2G production and offer new insights into enzyme engineering.