Engineering of Substrate-Binding Domain to Improve Catalytic Activity of Chondroitin B Lyase with Semi-Rational Design

Abstract

Dermatan sulfate and chondroitin sulfate are dietary supplements that can be utilized as prophylactics against thrombus formation. Low-molecular-weight dermatan sulfate (LMWDS) is particularly advantageous due to its high absorbability. The enzymatic synthesis of low-molecular-weight dermatan sulfates (LMWDSs) using chondroitin B lyase is a sustainable and environmentally friendly approach to manufacturing. However, the industrial application of chondroitin B lyases is severely hampered by their low catalytic activity. To improve the activity, a semi-rational design strategy of engineering the substrate-binding domain of chondroitin B lyase was performed based on the structure. The binding domain was subjected to screening of critical residues for modification using multiple sequence alignments and molecular docking. A total of thirteen single-point mutants were constructed and analyzed to assess their catalytic characteristics. Out of these, S90T, N103C, H134Y, and R159K exhibited noteworthy enhancements in activity. This study also examined combinatorial mutagenesis and found that the mutant H134Y/R159K exhibited a substantially enhanced catalytic activity of 1266.74 U/mg, which was 3.21-fold that of the wild-type one. Molecular docking revealed that the enhanced activity of the mutant could be attributed to the formation of new hydrogen bonds and hydrophobic interactions with the substrate as well as neighbor residues. The highly active mutant would benefit the utilization of chondroitin B lyase in pharmaceuticals and functional foods.