an indication for the adsorption of PLA 2 in its native state, and for the absence of
diffusion limitation.
The activities of native and immobilized PLA 2 as a function of temperature are
compared in Figure 6 (Remus and Grunwald, 1995). In this case, the enzyme was
bound chemically to VA-Epoxy Biosynth (Riedel-de Hae ̈n), a polymeric carrier the
surface of which is preactivated by epoxy groups (see Table 2), which means that the
enzyme should be bound predominantly bye-amino residues of PLA 2. The results
also showed that this type of binding is unfavorable. The activity retention was 4 % at
room temperature, but reached the activity of soluble PLA 2 for 25 8 Cat70 8 C due to a
markedly enhanced temperature stability of the biocatalyst caused by its attachment
to the carrier material which partly compensated for the strong activity loss. The high
activation energy measured for the PC-hydrolysis in presence of PLA 2 immobilized
via the epoxy-groups of the carrier proved that diffusion limitation could not be the
reason for the low activities. Furthermore, the pH-profiles for both, the activity of the
native as well as that of the immobilized PLA 2 were similar, which showed that
partitioning effects could be neglected (see Section 13.3.3).
The amino acids that may be involved principally in the binding of PLA 2 from
porcine pancreas via functional groups or spacers such as epoxy, glutaraldehyde, are
four arginine, nine lysine, and three histidine residues, and theN-terminal alanine.
Some of these amino acids are important for substrate binding (Lys62, Lys56), either
as part of the active site (Arg43, His48) or for the interfacial recognition site. Avoid-
ing enzyme binding through these amino acid residues is most likely the reason for
the successful immobilization experiments of Ferreira et al. (1993), though the ami-
no acid sequence of the cobra venom PLA 2 is not identical with that of porcine
pancreatic PLA 2. Our results for the immobilization of this enzyme (porcine pan-
creatic PLA 2 ) are shown in Figure 7A and B. We used macroporous organofunc-
tional polysiloxanes of the Deloxan-type (Degussa-Hu ̈ls), with a specific surface
276 13 Preparation and Application of Immobilized Phospholipases
Figure 6. The temperature course of the activity of native (^) PLA 2 and PLA 2 covalently attached to
VA-Epoxy Biosynth (Riedel-de Hae ̈n) via an epoxy spacer (&). The immobilized enzyme exhibits a
markedly increased temperature stability.