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Rational design of α-glucosidase for the synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid
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  • Shaoyan Zheng,
  • Weijie Zhou,
  • Xiangna Lin,
  • Feifei Li,
  • Chunfang Xie,
  • Daling Liu,
Shaoyan Zheng
Jinan University
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Weijie Zhou
Jinan University
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Xiangna Lin
Jinan University
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Feifei Li
Jinan University
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Chunfang Xie
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Daling Liu
Jinan University
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α-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) from L-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G. Main Methods and Major Results This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild type, Isothermal Titration Calorimetry(ITC) results showed that the equilibrium dissociation constant (KD) of the mutant for AA-2G was doubled; the Michaelis constant (Km) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%. Conclusions and Implications Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.
14 Mar 2023Submitted to Biotechnology Journal
14 Mar 2023Submission Checks Completed
14 Mar 2023Assigned to Editor
17 Mar 2023Reviewer(s) Assigned
16 Apr 2023Review(s) Completed, Editorial Evaluation Pending
21 Apr 2023Editorial Decision: Revise Major
04 May 20231st Revision Received
04 May 2023Submission Checks Completed
04 May 2023Assigned to Editor
04 May 2023Reviewer(s) Assigned
01 Jun 2023Review(s) Completed, Editorial Evaluation Pending
02 Jun 2023Editorial Decision: Accept