Front Matter

for the determination of PLD-activity contained 0.625 mM PC, 15 mM SDS, 0.3 M

sodium acetate buffer (pH 5.1), and 40 mM CaCl 2. A comparison of some of the

different biocatalysts with respect to their residual activity and protein binding yield

is shown in Figure 8. The highest activities were retained when PLD was attached to

SiO 2 functionalized with 3-aminopropyltriethoxy silane. Although the type of bind-

ing had to be an ionic or adsorptive one the release of PLD was not observed. The

other synthesis variants exhibit only low activities under these reaction conditions;

however, those catalysts prepared with long-chain spacers such as DMAD and DAD

showed residual activities above 100 % with increasing substrate concentrations. An

explanation for this finding cannot yet be given, apart from the fact that it seems

reasonable to suppose that binding to the carrier surface via a longer-chain favors

flexibility of the enzyme structure as well as accessibility of its active site for the

micelles. This is also confirmed in immobilization experiments performed by Reuter

(1997) with the Fractogel EMD Azlacton carrier (Merck KGaA), which led to highly

satisfactory results concerning the residual activity of PLD. Fractogels (Table 2) are

synthetic, methacrylate-based, beaded polymers. Long, linear, polymer chains – so-

called ‘tentacles’ – are bound covalently to the matrix, bearing functional ligands for

an attachment of biomolecules with minimized steric hindrance. Furthermore, im-

mobilized PLD with high efficacy resulted from covalent binding to carriers such as

Deloxan DAP III (with GDA as spacer), and from adsorptive fixation to the hydro-

phobic surface of Deloxan HAP. However, it must be mentioned that the two quality

criteria, ‘storage stability’ and ‘operational stability’ were not fulfilled sufficiently,

which is probably due to the well-known poor stability of PLD from white cabbage.

For example, the catalysts obtained with the tentacle polymer or with functionalized

SiO 2 , where PLD was bound via decanoic acid dichloride could be used repeatedly

on five occasions without activity loss. Thereafter, the activity decreased continu-

ously. Nevertheless, it should be possible to employ these immobilization techniques

and carrier materials for the synthesis of highly active PLD preparations with long-

term stability using more stable PLDs from microbial sources that are currently

available.

A special type of PLD-immobilization was presented by Okahata et al. (1995),

who prepared lipid-coated PLD by mixing an aqueous PLD-solution with dialkyl

amphiphiles dissolved in acetone (Figure 9). The precipitate obtained, which had

a protein content of about 7 % after lyophilization, was used for the transphospha-

tidylation of egg PC with, for example,n-butanol. The reactions were performed in

biphasic systems consisting of benzene and an acetate buffer. Because of its lipid-

coating, the enzyme – together with the substrate – was soluble in the organic phase,

whereas the choline moiety was released into the aqueous phase. The ratio of reac-

tion rates of lipid-coated PLD fromStreptomycessp. and native PLD was found to be

about 300. Among the lipids used for PLD-coating, anionic lipid molecules turned

out to be most suitable with respect to the residual activity. As the actual reaction is

carried out in a homogeneous phase, its kinetics can be described by Michaelis –

Menten mechanisms over the entire concentration range of the two substrates PC

and the acceptor alcohols. From an analysis of the kinetic data the authors concluded

that PC is bound first to the enzyme before this intermediate reacts with the nucleo-

phile. Furthermore, it could be shown that the substrate specificity was not affected

by lipid coating. The general applicability of lipid-coated PLD for the synthesis of

282 13 Preparation and Application of Immobilized Phospholipases