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

A novel heparinase III from Pedobacter schmidteae ( Ps Hep-III) with high activity and good stability was successfully cloned, expressed, and characterized. Ps Hep-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. Ps Hep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. Ps Hep-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 Ps Hep-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 Ps Hep-III for characterizing heparin and producing low-molecular-weight heparin effectively.