Combination of engineering the substrate and Ca²⁺ binding domains of heparinase I to improve the catalytic activity

Abstract

Heparinase I (EC 4.2.2.7), is an enzyme that cleaves heparin, showing great potential for eco-friendly production of low molecular weight heparin (LMWH). However, owing to its poor catalytic activity and thermal stability, the industrial application of heparinase I has been severely hindered. To improve the catalytic activity, we proposed to engineer both the substrate and Ca²⁺ binding domains of heparinase I. Several heparinases I from different organisms were selected for multiple sequence alignment and molecular docking to screen the key residues in the binding domain. Nine single-point mutations were selected to enhance the catalytic activity of heparinase I. Among them, T250D was the most highly active one, whereas mutations around Ca²⁺ binding domain yielded two active mutants. Mutant D152S/R244K/T250D with significantly increased catalytic activity was obtained by combined mutation. The catalytic efficiency of the mutant was 118,875.8 min⁻¹·µM⁻¹, which was improved 5.26 times. Molecular modeling revealed that the improved activity and stability of the mutants were probably attributed to the formation of new hydrogen bonds. The highly active mutant had great potential applications in industry and the strategy could be used to improve the performance of other enzymes.

Graphical Abstract

Highlights

• Improved catalytic activity of heparinase I by engineering the binding domains of substrate and Ca²⁺.

• The mutant D152S/R244K/T250D showed the highest catalytic performance.

• The increased hydrogen bonds attribute to the increased activity.

Keywords:

• Binding domain
• catalytic activity
• heparinase I
• hydrogen bonds
• molecular docking

Additional information

Funding

This work was supported by Natural Science Foundation of Guangxi Province (No. 2021GXNSFAA196005) and National Natural Science Foundation (No. 22068001).