Immobilized high-performance heparin lyase III for efficient preparation of low molecular weight heparin

Heparin lyase III has garnered widespread attention due to its high specificity and minimal loss of anticoagulant activity during the preparation of low molecular weight heparin (LMWH), a crucial anticoagulant drug in clinical practice. However, low expression levels and complex preparation processes limit its practical application. To address these challenges, high-performance Bacteroides thetaiotaomicron heparin lyase III (Bhep III) variants were engineered and immobilized for LMWH preparation. First, we enhanced enzyme expression by adding a solubility-enhancing tag and optimizing the N-terminal coding sequence, which resulted in a Bhep III activity level of 2.9 × 10³ U/L with 8-fold increase. After evolution guided the design of rational mutations, the variant Bhep III K85A/Q95F/S471T generated higher activity (5.4 × 10⁴ U/L in 5-L fermenter), which is, to our knowledge, the highest reported to date in the literature, being 1.7-fold that of the wild type and demonstrating 2-fold increase of the thermal stability. By screening and optimizing the C-terminal self-assembling tag, we successfully immobilized Bhep III, further increasing its thermal stability by 12-fold, and allowing for the multi-batch preparation of LMWH with simple centrifugation. The immobilized heparin lyase III demonstrated sufficient reusability in enzymatic reactions, facilitating efficient industrial-scale production of LMWH.