Heparinases obtained from sphingobacterium daejeonense, preparation therefor and application thereof

A method for obtaining heparinases SDhep I and SDhep II from Sphingobacterium daejeonense bacterium, comprising the following steps:

• (1) the Sphingobacterium daejeonense is inoculated on the slant medium;
• (2) the strain obtained in step (1) is inoculated into the seed culture medium and cultured for 1-2 days, inoculated into a secondary liquid seed medium and cultured for 1-2 days, and then inoculated into 2 L fermentation medium and cultured for 1-5 days, thereafter cells are collected;
• (3) the cells obtained in step (2) are suspended in Tris-HCl buffer, disrupted by a high pressure homogenizer, then centrifuged for a supernatant and subjected to ammonium sulfate precipitation in an ice bath to collect a precipitated component with 35%-85% saturation, and then the precipitate was dissolved in Tris-HCl buffer and dialyzed to obtain an enzyme solution;
• (4) the enzyme solution obtained in step (3) is loaded onto a Q column pre-equilibrated with a Tris-HCl buffer, then equilibrated with the same buffer and eluted with a linear gradient of a 0-0.5 M NaCl in the same buffer;
• wherein the fraction with enzyme activity collected from the effluent and equilibrate solution is heparinase SDhep I, which is not bound to the Q column;
• and wherein the fraction with heparinase activity collected from the eluate is heparinase SDhep II, which is bound to the Q column, then the fraction with heparinase activity collected from the eluate is combined and dialyzed;
• (5) the SDhep I fraction obtained in step (4) is loaded onto the CS column pre-equilibrated with a Tris-HCl buffer, then equilibrated with the same buffer and then eluted with a linear gradient of 0-0.5 M NaCl in the same buffer;
• the fractions with heparinase activity are collected, dialyzed and loaded onto the SP column pre-equilibrated with Tris-HCl buffer, then equilibrated with the same buffer and then equilibrated with Tris-HCl buffer with a linear gradient of 0-0.5 M NaCl;
• the fractions are collected and concentrated to 0.2 mL by an ultrafiltration centrifuge tube intercepting molecular weight of 30 kD;
• the concentrated enzyme is loaded onto a Sephadex G-100 column pre-equilibrated with a Tris-HCl buffer and then eluted with the same buffer;
• the fractions with activity are collected, combined and concentrated to obtain purified SDhep I;
• (6) SDhep II obtained in step (4) is loaded on a CS column pre-equilibrated with Tris-HCl buffer, then equilibrated with the same buffer, and then eluted with a linear gradient 0.1-0.6 M NaCl in the same buffer;
• the fractions with heparinase activity in eluent solution are tested, collected, combined and dialyzed;
• the enzyme solution is loaded onto a Q-column pre-equilibrated with Tris-HCl buffer, and eluted with a linear gradient of 0.08 M NaCl in the same buffer;
• the fractions are tested, collected and loaded onto a CS column pre-equilibrated with a Tris-HCl buffer, then equilibrated with the same buffer and then eluted with a linear gradient of 0-1 M NaCl in the same buffer;
• the fractions with activity are collected, combined, and concentrated to obtain purified SDhep II.