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

Abstract

Heparinase has attracted much attention because of its applications in pharmaceutical industry. Herein, the heparinases III from Flavobacterium heparinum (FhepIII) and Bacteroides thetaiotaomicron (BhepIII) were firstly comparatively characterized. BhepIII showed higher catalytic activity and thermostability toward heparin comparing to FhepIII. To further upgraded BhepIII, a protein engineering approach based on B-factor was performed. By site-saturated mutagenesis of the flexible residues within an 8 Å radius around the catalytic residue, Asp321 and Ser264 were identified as essential residues for catalytic efficiency and thermostability, respectively. D321Q mutation enhanced catalytic efficiency (kcat/Km) with a 68.4% increase by increasing the surface potential while S264 F mutation increased thermostability with a half-time at 50℃ (t1/250℃) of 3.8 h versus 2.7 h of the wild-type by increasing rigidity and interactions within the active pocket. Double mutation of S264 F and D321Q resulted in a 245% increase in kcat/Km but with a decreased t1/250℃ (2.0 h). E105R mutation that generated a 348% increase in kcat/Km was further identified by electric potential engineering of the pocket tunnel. Eventually, the variant E105R/S264 F that showed a 418% increase in kcat/Km without compromise of thermostability was constructed. The engineered E105R/S264 F has a great potential for the commercial production of low molecular weight heparin in the future.