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<p>Chondroitinase ABC (ChSase ABC), which can digest chondroitin sulfate (CS), has been utilized for the analysis of CS glycan structure and the preparation of low-molecular-weight chondroitin sulfate. However, the catalytic activity of most ChSase ABCs remains insufficient for industrial or large-scale applications. Previous structure studies of ChSase ABCs indicate that the N-terminal domain is typically involved in substrate recognition and binding. Furthermore, the N-terminal domain of the ChSase ABC from <i>Bacteroides thetaiotaomicron</i> (<i>Bt</i>ChSase ABC) also exhibits similarity with the carbohydrate-binding module of other glycosidases. In the present work, the N-terminal of <i>Bt</i>ChSase ABC was chosen for engineering. Through multiple sequence alignment, 9 non-conserved residues were selected as candidate mutation sites, and a mutant library containing 20 variants was constructed by single-point and combinatorial mutations. Among them, the catalytic activity of 13 mutants showed varying degrees of improvement. The mutant L137V showed 79.29% and 84.49% increases in specific activity and <i>V</i><sub>max</sub>, respectively, compared to the wild type. SAX-HPLC analysis of enzymatic hydrolysates confirmed that the mutation retained the enzyme’s substrate recognition and cleavage mechanism. Kinetic parameters revealed that the enhanced catalytic activity of the mutants was mainly attributed to an increased turnover number (<i>k</i><sub>cat</sub>), indicating that the rate-limiting step of catalysis was effectively accelerated.</p>

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Semi-rational design of the N-terminal of Chondroitinase ABC to improve the catalytic activity

  • 期刊论文

  • 发表日期：

    2026年4月18日

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  • Tong Huan,
  • Jing-Yi Zhang,
  • Jia-Ying Huang,
  • Wen-Jie Si,
  • Yue Wu,
  • Ye-Wang Zhang

摘要

Chondroitinase ABC (ChSase ABC), which can digest chondroitin sulfate (CS), has been utilized for the analysis of CS glycan structure and the preparation of low-molecular-weight chondroitin sulfate. However, the catalytic activity of most ChSase ABCs remains insufficient for industrial or large-scale applications. Previous structure studies of ChSase ABCs indicate that the N-terminal domain is typically involved in substrate recognition and binding. Furthermore, the N-terminal domain of the ChSase ABC from Bacteroides thetaiotaomicron (BtChSase ABC) also exhibits similarity with the carbohydrate-binding module of other glycosidases. In the present work, the N-terminal of BtChSase ABC was chosen for engineering. Through multiple sequence alignment, 9 non-conserved residues were selected as candidate mutation sites, and a mutant library containing 20 variants was constructed by single-point and combinatorial mutations. Among them, the catalytic activity of 13 mutants showed varying degrees of improvement. The mutant L137V showed 79.29% and 84.49% increases in specific activity and Vmax, respectively, compared to the wild type. SAX-HPLC analysis of enzymatic hydrolysates confirmed that the mutation retained the enzyme’s substrate recognition and cleavage mechanism. Kinetic parameters revealed that the enhanced catalytic activity of the mutants was mainly attributed to an increased turnover number (kcat), indicating that the rate-limiting step of catalysis was effectively accelerated.