The release of heparinase from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock.
Heparinase was released from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock procedure. The procedure involves resuspending exponentially growing cells consecutively into (1) 40% sucrose, (2) 10 mM sodium phosphate, 2 mM magnesium chloride, pH 7, and (3) 10 mM sodium phosphate, 300 mM sodium chloride, 2 mM magnesium chloride, pH 7. Typically, 50-75% of the total heparinase activity is recovered by this procedure with an observed 7-15-fold increase in purity. The majority of heparinase activity is released in the final step of the procedure allowing for resolution from cytoplasmic and nonspecific periplasmic material. F. heparinum cells can be stored in 40% sucrose at 4 degrees C for up to one week without significant losses in recovery yields.
Zimmermann
J J
JJ
IBEX Technologies, Montreal, Quebec.
Oddie
K
K
Langer
R
R
Cooney
C L
CL
D001709:Biotechnology; D002462:Cell Membrane / Q000201:enzymology; D004492:Edetic Acid; D005417:Flavobacterium / Q000201:enzymology* / Q000254:growth & development; D019763:Heparin Lyase; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D013395:Sucrose
D013395:Sucrose; D004492:Edetic Acid; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase; D008274:Magnesium