An engineered platform based on chitin-affinity immobilization for producing low molecular weight heparin

Abstract

Using chitin-affinity interaction between triple-functional heparinase I (Hep I) and chitin, an engineered platform was prepared to produce controllable low molecular weight heparin (LMWH). Chitin microspheres with well-defined nanofibrils were fabricated through a "bottom up" pathway. An enhanced soluble protein, ChBD-SUMO-Hep I (CSH-I), was expressed in 3L batch fermentation with a high bioactivity of 2.5×10³ IU/L. Chitin binding domain (ChBD) can specifically bind to chitin in noncovalent way, which leads to the immobilization and purification of enzyme in a single step. The immobilized CSH-I was preferred over its free counterpart due to its higher tolerance to heat and pH, as well as improved shelf-life. The restraint enzyme could be reused up to 8 times to achieve a conversion yield exceeding 90%. By using the bioinspired conjugates, the qualified LMWH fractions were obtained by monitoring the degradation process with an absorbance range of 44.5-68.3 at 232nm.

Keywords: Chitin; Chitin binding domain; Heparin; Heparinase I; Immobilization; Recombinant protein.