13.4.2 Immobilization of phospholipase D
Possible applications of immobilized PLD are the synthesis of new phospholipids via
transphosphatidylation, the analytical area, and the processing of crude phospholipid
mixtures by enrichment of their PA content (see Table 1), which improves the emul-
sifying properties and the temperature stability. However, as yet the use of enzyma-
tically modified food additives in Europe is restricted to hydrolyzed products due to
the European food regulations (E 322).
The first extensive study of transphosphatidylation reactions catalyzed by immo-
bilized PLD from white cabbage was published by Tobback et al. (1988). They in-
vestigated many different carrier materials with respect to their suitability for the
attachment of PLD by maintaining the enzymatic activity, including Celite, alumina
oxide, active carbon (adsorptive binding of PLD), octyl-Sepharose, porous glass
(covalent binding of PLD), DEAE-Sephadex (ionic binding of PLD), Ca2+-algi-
nate, and polyvinylalcohol (entrapment of PLD). The quality of the biocatalysts
obtained was tested by their ability to convert dioleoyl-PC into dioleoyl-PE. The
best results were obtained with PLD bound to porous glass and to DEAE-Sephadex
in reaction mixtures containing diethylether orn-propylacetate.
Reuter (1997) immobilized PLD from white cabbage on SiO 2 (Cab-osil M5) to test
the usability of the products obtained for the enrichment of PA in phospholipids and
phospholipid mixtures. For this purpose, the SiO 2 carrier was first derivatized with 3-
aminopropyltriethoxysilane or with 3-glycidoxypropyltrimethoxysilane. PLD was
either bound directly or by additional bifunctional spacers such as glutaralde-
hyde, dimethyladipimidate dihydrochloride (DMAD), decanoic acid dichloride
(DAD) or by an azo linkage, respectively. The standard reaction solution used
13.4 Immobilization of phospholipases 281
Figure 8. Residual activity and binding rate for phospholipase D immobilized to SiO 2 (Cab-osilÒM-5)
via different (bifunctional) spacers: 3--aminopropyltriethoxysilane (amino-s), (3-glycidoxypro-
pyl)trimethoxysilane (epoxy-s), amino-s and glutaraldehyde (GDA), amino-s and decanoic acid dichlo-
ride (DAD), amino-s and dimethyladipimidate dihydrochloride (DMAD), amino-s and epoxy-s con-
taining an additional azo linkage (am-s-N¼N-, ep-s-N¼N-).