Identification and characterization of a novel heparinase PCHepII from marine bacterium Puteibacter caeruleilacunae

Abstract

Abstract Heparin (HP) and heparan sulfate (HS) are multifunctional polysaccharides widely used in clinical therapy. Heparinases (Hepases) are enzymes that specifically catalyse HP and HS degradation, and they are valuable tools for studying the structure and function of these polysaccharides and for preparing low molecular weight heparins. In this study, by searching the NCBI database, a novel enzyme named PCHepII was discovered in the genome of the marine bacterium Puteibacter caeruleilacuae. Heterologously expressed PCHepII in Escherichia coli (BL21) has high expression levels and good solubility, active in sodium phosphate buffer (pH 7.0) at 20°C. PCHepII exhibits an enzyme activity of 254 mU/mg towards HP and shows weak degradation capacity for HS. More importantly, PCHepII prefers to catalyse the high-sulfated regions of HP and HS rather than the low-sulfated regions. Although PCHepII functions primarily as an endolytic Hepase, it mainly generates disaccharide products during the degradation of HP substrates over time. Investigations reveal that PCHepII exhibits a preference for catalysing the degradation of small substrates, especially HP tetrasaccharides. The catalytic sites of PCHepII include the residues His199, Tyr254, and His403, which play crucial roles in the catalytic process. The study and characterization of PCHepII can potentially benefit research and applications involving HP/HS, making it a promising enzyme.