Heparinase enzyme production

We propose a new approach that would allow the full heparinization of the extracorporeal device, yet could enable, on-demand, elimination of heparin in the patient s bloodstream. This approach consists of a blood filter containing immobilized heparinase, which could be placed at the effluent of any extracorporeal device (Figure 1). Such a filter could theoretically be used to eliminate heparin after it had served its purpose in the extracorporeal device and before it returned to the patient. In this chapter we discuss our efforts to develop such a filter. Our work has focused on several areas (1) enzyme production (2) enzyme purification (3) characterization of heparinase (4) immobilization of heparinase and (5) in vitro testing of immobilized heparinase. 

Although the size distribution of fragments from heparinase (and hep-aranase) digests reflects the relative content of regular sequences 5 in different heparin preparations and fractions, these sequences may be quantitated only when the enzyme efficiency is high, and products are... 

While our studies on heparinase production and purification have been encouraging, less success has been achieved in the immobilization procedures (Table II). Studies are in progress to understand better the important parameters in immobilization procedures and in establishing new supports. Initial results indicate that a noncharged support with a high surface area is best (Table II). Additionally, our preliminary evidence is that high levels of heparinase (> 1 mg/mL) and the presence of substrate in the immobilization reaction enhance the recovery of immobilized enzyme activity. 

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