Front Matter

378 C, whereas the degree of PE- and PC- hydrolysis was about 50 % after 60 h under

the same conditions.

Since the early 1980s many reports have been published about the catalytic ac-

tivity of hydrolytic enzymes in (nearly) water-free organic solvents of low polarity.

Pernas et al. (1990) showed that extracellular PLA 2 (fromNaja najavenom, bee

venom, porcine and bovine pancreas andStreptomyces violaceoruber) is capable

of catalyzing the esterification of lyso-PC with [^3 H]-oleic acid. They made use

of the findings of Zaks and Klibanov (1988) that enzymes incubated in a buffer

solution at a pH value necessary for optimum activity (in this case a Tris/HCl-buffer

of pH 9 containing 10 mMol CaCl 2 ) prior to lyophilization exhibit the so-called pH-

memory effect. The synthesis was carried out in solvents such as benzene, toluene

and chloroform, which did not affect the activity of the PLA 2. The highest degree of

transesterification was 6.5 % obtained withNaja najaPLA 2. Adding methanol to the

reaction mixture causes a suppression of the PC synthesis, which gives rise to the

assumption (stated earlier by others) that the small amount of water left after lyo-

philization is essential for the catalytic effect, and that this water is removed from the

microenvironment of the enzyme in the presence of small amounts of polar solvents.

Lin et al. (1993) also investigated the behavior of PLA 2 in apolar organic solvents

such as chloroform with low water content. With regard to the hydrolysis of 1,2

dimyristoyl-sn-glycerophosphocholine (DMPC), they found that bee-venom PLA 2

showed the highest activity followed byNaja najaandNaja mocambiquevenom

PLA 2 , whereas pancreatic PLA 2 yielded only poor activities. A small amount of

methanol added to chloroform with 1.7 % water increased the rate of the bee venom

enzyme-catalyzed hydrolysis by a factor of 4, in contrast to the normally observed

effect of this solvent on phospolipase activity. This is possibly due to the fact that

removal of residual water from the microenvironment of the enzyme takes time, and

that the positive influence of methanol on the solubility of the substrate is initially

predominant. Similar findings were reported by Campanella et al. (1998a) in con-

nection with the use of immobilized PLD for analytical purposes (see Section

13.4.2). The course of hydrolysis rate as a function of temperature had a minimum

at T¼ 458 C due to conformational changes of DMPC in 2-position, as proved by

NMR spectroscopy. Furthermore, the authors described a novel route for a venom

PLA 2 -catalyzed transacylation ofL-lyso-PC with long-chain acyl donors. The best

results (37 % yield) were obtained with palmitic anhydride in chloroform-water

(100/1, v/v) at 37 8 C. Ha ̈rro ̈d and Elfman (1995) developed a high-pressure reaction

unit for the synthesis of new phospholipids in isooctane, carbon dioxide and propane

as solvents. The products were obtained by the reaction of lyso-PC with polyunsa-

turated fatty acids (PUFA) of fish oil origin (mainly consisting of eicosapentaenoic

and docosahexaenoic acids) and PLA 2 from porcine pancreatic glands immobilized

to the polysiloxane matrix Deloxan as catalyst. Optimum results with a yield of 25 %

were achieved in a solvent mixture of 91 % PUFA and 9 % propane.

280 13 Preparation and Application of Immobilized Phospholipases