Expression Purification And Characterization Of Heparanase From Flavobacterium Heparinum In Escherichia Coli

Heparinases are the enzymes that specifically cleave heparin, the only one among three heparanases derived from Flavobacterium heparinum which can cleavage the glycosidic bond of heparin. In this study, the recombinant heparinase I was expressed in E.coli, induced at 15℃, about 90%of the fusion proteins were soluble. Recombinant HepI was heat-labile, crude enzyme solution after GST-tag purification was achieved and the purified enzyme had a specific activity of 70.35IU/mg protein. This represented 82.2%recovery of the total activity of heparinase I (HepI). The purified enzyme the optimum temperature 35℃, the optimum pH 7.0, NaCl 200 mM, CaCl2 1mM in the highest activity. GST-HepI is very sensitive of temperature; temperature is above 40℃ for most of the enzymes are inactivated; in the 4℃, 16h after the loss of enzyme activity of up to about 70%, the enzyme stability.The experiment found that NaCl and CaCl2 have very strong activation and protection of enzymes, in the assay buffer 1mM CaCl2 and 200mM NaCl can increase the activity of enzyme 1.5 times. The SAX-HPLC is also proved that the recombinant GST-HepI cleavage of heparin products and type of Flavobacterium heparinase cleavage product of source of basically the same, suggesting that the GST label on the enzyme substrate specificity has no effect.In addition this paper established a polyacrylamide gel electrophoresis analysis of heparin cracking degree detecting method, the heparin and its degradation degree for rapid identification of the traditional HPLC detection technique, and compared this method is simple and rapid, for the identification of low molecular weight heparin laid the foundation. The electrophoresis results showed that recombinant heparinase I degradation of heparin effect is obvious, can be used for the preparation of low molecular weight heparin.This topic further conducted on heparin enzyme cutting or site-directed mutagenesis to study on heparin enzyme activity related to functional area, heparin enzyme of structure and function.