Heparinase III with High Activity and Stability: Heterologous Expression, Biochemical Characterization, and Application in Depolymerization of Heparin

A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg^-1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like →4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 → 4)ΔUA/ΔUA2S[1 → and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.

enzymatic depolymerization; heparin; heparinase; low molecular weight heparin.