Purification of Heparinase I, II and III from Flavobacterium heparinum

Abstract

A purified heparinase I, II and III free of lyase activity and each having a molecular weight of 42,800 84,100, 70,800, respectively, are produced by culturing Flavobacterium heparinum. The kinetic properties of the heparinases have been determined as well as the conditions to optimize their activity and stability.

1. An isolated heparinase II from Flavobacterium heparinum free of lyase activity other than heparinase II activity, having a molecular weight of 84,100, cleaving heparin and heparan sulfate and having a pH optimum  pI of 8.9-9.1. 
     
     
       2. An isolated heparinase III from Flavobacterium heparinum free of lyase activity other than heparinase III activity, having a molecular weight of 70,800, cleaving heparan sulfate, and having a pH optimum  pI of 9.9-10.1. 
     
     
       3. The heparinase III of  claim 2  which does not cleave heparin sulfate. 
     
     
       4. The heparinase III of  claim 2  with albumin. 
     
     
       5. A method for purifying heparinase I, II, and III from a biologically pure culture of Flavobacterium heparinum comprising the steps of
 lysing Flavobacterium heparinum cells in a biologically pure culture of Flavobacterium heparinum,  
 removing cell debri  debris and nucleic acids from the cell lysate,  
 absorption of heparinase I, II, and III to hydroxyapatite,  
 absorption of non-heparinase I, II, and III proteins to QAE-resin,  
 recovery of the heparinase I, II, and III not bound to the QAE-resin,  
 separation of heparinase I, II, and III by HPLC on a hydroxylapatite column,  
 recovery of the heparinase I, II, and III separated on the hydroxylapatite column,  
 purification of the separated heparinases by cation exchange FPLC,  
 recovery of the heparinase I, II, and III separated by cation exchange FPLC,  
 purification of the separated heparinases by gel permeation HPLC, and  
 recovering the heparinases I, II, and III separated by gel permeation HPLC.  
 
     
     
       6. The method of  claim 5  wherein the nucleic acids are removed by precipitation with protamine. 
     
     
       7. The method of  claim 6  wherein the heparinases are separated on the hydroxylapatite column by elution with a salt gradient. 
     
     
       8. The method of  claim 6  wherein the heparinases are eluted from the cation exchange column by a gradient of increasing salt concentration. 
     
     
       9. The isolated heparinase II of  claim 1  having a pH optimum of  6 . 9  on heparan sulfate as a substrate. 
     
     
       10. The isolated heparinase III of any of claims  2 - 4  having a pH optimum of  7 . 6  on heparan sulfate as a substrate.