Cloning, expression, and characterization of a heparinase III coupled with heparinase I for enzymatic depolymerization of heparin.

Abstract

A heparinase III (NsHep-III) from Niabella sp. was identified, cloned, and expressed as soluble form in E. coli BL21 (DE3). With heparin as substrate, the maximum activity of NsHep-III of 90.0 U·mg^- 1 was achieved at pH 7.1 Tris-HCl (containing 15 mM Mg²⁺) and 30^oC. The half-life of NsHep-III was determined to be 5 h at 30^oC. The interactions between NsHep-III and substrates were studied by molecular docking. The combination of NsHep-III and a heparinase I from Bacteroides eggthii (BeHep-I) was employed to cleave heparin. Analysis of the enzymatic products of NsHep-III by SAX-HPLC showed seven different modified disaccharides, indicating that NsHep-III has a wide range of substrate specificity. The results of GPC analysis demonstrated that the average molecular weight of the product of heparin cleavage by the combination of NsHep-III/BeHep-I was reduced to 3969 Da, which accounted for 90% of all the components, and complied with the requirements of the European Pharmacopoeia. NsHep-III has notable activity and efficiency in cleaving heparin, which is potentially useful for the industrial production of low molecular weight heparin.