Study On Strain Screening And Part Enzymatic Properties Of A Novel Heparinase-producing Strain

A novel heparinase-producing strain was screened and isolated from siol samples. The strain was identified as Bacillus subtilis, by testing physiological-biochemical and morphologic characteristics. We name it Lx-10. The heparinase from Lx-10 was a kind of inducible, non-extracellular enzyme.By treatment with both ultraviolet rays and LiCl, a mutant strain B.S.Lx-19 with high yield of heparinase and stability was obtained, whose enzyme activity increased by 40% over strain Lx-10.Fermentation conditions for enzyme production have been established: The optimum condition are carbonic resource as maltose; nitric resource as soya peptone;initial pH for 6.5;inocution for 10%;seeds age for 12h. Compositition of the medium(%) is maltose 1.2, soya peptone 1.2, heparin 0.1, K2HPO40.25, NaH2PO4 0.25, MgSO2'7H2O 0.1, pH 6.5. After fermentation in 2 liter fermentor at 37? with an aeration rate of 1:0.5-1:1 for one day. The average heparinase enzyme activity was 724U/L culture broth.The B.S.Lx-19 heparinase was partial purified from the crude extract. 30%~60% ammonium sulfate fractionation of the crude extract was carried out at 4 ?. The optimum temperature and pH for it were 37? and 6.5. It was stable up to 40? in the pH range from 5 to 8. In addition, the heparinase is stimulated in the presence of Ca2+ and Mn2+, but is inhibited by Cu2+ and Fe3+. Neither PMSF nor EDTA significantly affected enzyme activity. By infrared spectrum and protonresonance spectrum analysis, we found smaller polysaccharides, unsaturated bond and reducing sugar, which are specific products by heparinase.