Journal of Lipid Research, Vol 20, 519-524, Copyright © 1979 by Lipid Research, Inc.
Purification of phospholipase C from Bacillus cereus by hydrophobic chromatography on palmitoyl cellulose
S Imamura and Y Horiuti
Phospholipase C (phosphatidylcholine choline-phosphohydrolase, EC 3.1.4.E)
from Bacillus cereus (IAM-1208) was adsorbed to palmitoyl cellulose from a
crude enzyme solution at pH 5--9. The adsorption was not influenced by
ionic strength up to 2 M NaCl. The adsorbed enzyme was eluted almost
completely by washing the cellulose with a suitable detergent, such as
Triton X-100, Adekatol SO-120, Cation DT-205, or sodium deoxycholate. The
enzyme was then purified by column chromatography on a palmitoylated
textile (palmitoylated gauze) with an overall recovery of 91% and a
467-fold increase in specific activity over that of enzyme in the crude
culture supernatant. Subsequent fractionation with acetone and
chromatography on a Sephadex G-75 column separated two nearly homogeneous
phospholipase C's. The enzyme adsorbed on palmitoyl cellulose was active,
although its activity was about one- fourth that of free phospholipase C.
Therefore, the enzyme appeared to be adsorbed to the cellulose through a
hydrophobic site that was distinct from the catalytic site on the enzyme
molecule.
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