Engineering the heparin-binding pocket to enhance the catalytic efficiency of a thermostable heparinase III from Bacteroides thetaiotaomicron

•A thermostable heparinase III was characterized and engineered.•Asp321 and Glu105 were identified as essential residues for catalytic efficiency.•S264 F mutation significantly increased thermostability.•The thermostable variant E105R/S264 F showed high catalytic efficiency.•E105R/S264 F variant has potential for producing low-molecular-weight heparin.