new phospholipids was demonstrated by preparing corresponding compounds where
the choline residue was exchanged by glucose (68 %), galactose (75 %), deoxyade-
nosine (75 %), thymine (70 %), etc. on the gram scale.
Lambrecht and Ulbrich-Hofmann (1992) used the fact that PLD is bound prefer-
entially by adsorption to carriers with a hydrophobic surface, and that the binding
efficiency is markedly increased in presence of Ca2+ions for the purification of PLD
from white cabbage leaves. The authors compared these findings with the Ca2+re-
quirement of the enzyme necessary to develop catalytic activity towards phospho-
lipid micelles. From the solution of a crude protein mixture containing 50 mM CaCl 2 ,
PLD was adsorbed onto octyl-Sepharose CL-4B (Pharmacia) and could be eluted
selectively through the removal of Ca2+ions with EDTA. The yield of PLD was
85–90 %, and the purification factor 103. These results were used by the same
authors for an adsorptive immobilization of PLD from white cabbage to different
carrier materials with long-chain anchor groups (octadecyl, octyl, etc.) mediated
by calcium ions. The catalytic activity of the adsorbed PLD turned out to be depen-
dent on both the Ca2+concentration during the binding process and the nature of the
carrier. To obtain maximum activity, 10 mM CaCl 2 was required in the case of oc-
tadecyl-Si 40 Rp 18 (Serva), but 40 mM CaCl 2 for octyl-Sepharose CL-4B. Other
findings of this investigation were that the storage stability increased markedly (PLD
adsorbed to octyl-Sepharose lost 30 % activity within 14 days when stored at 8 8 Cin
an acetate buffer, pH 7, whereas the native enzyme was completely inactive after 4
13.4 Immobilization of phospholipases 283
Figure 9. A schematic illustration of a lipid-coated PLD and transphosphatidylation of egg PC with
alcohol in benzene solution in the presence of aqueous buffer (Okahata et al., 1995).