of phospholipases as such. Here, the influence of experimental parameters (described
in Sections 13.3.1–13.3.3) on the properties of the carrier-bound enzymes and their
optimization with respect to high residual catalytic activity are investigated. From
the different immobilization methods described in Section 13.3.1, it was clear that
adsorption and covalent attachment were mainly used, and that the carrier materials
most frequently applied were porous glass and synthetic organic polymers. Until
now, immobilized phospholipases have not been used in industrial processes, as
for example the conversion of lecithin to lysolecithin in the presence of PLA 2 ,
nor in connection with the phospholipase-catalyzed synthesis of new phospholipids
on a preparative scale. By far the most investigations in this field have been carried
out with the immobilization of PLA 2.
13.4.1 Immobilization of phospholipase A 2
Fundamental research on this topic has been carried out by Dennis and co-workers.
In an early publication, they described the immobilization of a cobra venom PLA 2 on
porous glass beads by diazo coupling (Adamich et al., 1978). The products obtained
had residual activities of just 1 %. A few years later, Lombardo and Dennis (1985;
1986) reported on studies carried out with PLA 2 from the same source (Indian cobra;
Naja naja naja), but attached covalently to an agarose gel via tresyl chloride. The
protein binding yield was up to 87 %. For test reactions, substrates such as dipal-
mitoyl-PC, diheptanoyl-PC and the corresponding PE-derivatives were used. A se-
lection of data from these publications is listed in Table 3; these show that the activity
of immobilized PLA 2 towards dipalmitoyl-PC/Triton X-100 micelles again is just 1–
2 % of the specific activity of the soluble (native) PLA 2. The exchange of dipalmi-
toyl-PC by dipalmitoyl-PE leads to significantly lower activities, but a reduced ac-
tivity difference between the immobilized and the native species. Transition from the
monomeric to the micellar state of diheptanoyl-PC causes a 40-fold increase in ac-
tivity when the reaction is catalyzed by soluble PLA 2. In the presence of immobilized
PLA 2 , the reaction rate is merely enhanced by a factor of 3.5. Triton X-100 does not
274 13 Preparation and Application of Immobilized Phospholipases
Table 3.Activities of soluble and immobilized PLA 2. (Data from Lombardo and Dennis, 1986).
Substrate Triton X-100 Specific activities (llmol/(ming))
soluble PLA 2 immobilized
PLA 2(%)Dipalmitoyl-PC (5 mM) 20 mM 1120 19 1.70
Dipalmitoyl-PE (5 mM) 20 mM 13 5.3 40.8
Diheptanoyl-PC (0.8 mM) – 86 77 87.5
Diheptanoyl-PC (3.2 mM) – 3400 280 8.24
Diheptanoyl-PE (0.2 mM) – 81 25 30.9
Diheptanoyl-PE (0.2 mM) 2 mM 71 25 32.5