Front Matter

days’ storage under the same conditions), and a considerable broadening of the pH-

optimum compared to the soluble PLD resulting in near-equal activities in pH range

between 5 and 7 (Lambrecht and Ulbrich-Hofmann, 1993).

A similar stabilization effect on PLD by attaching the enzyme to a solid support

was observed by Takami and Suzuki (1995) when they bound PLD fromStrepto-

mycessp. to different cation-exchange resins. In contrast to strong acid types

with sulfonic acid exchange groups mainly catalyzing the hydrolysis to PA, weak

acid types such as Amberlite IRC-50 proved to be excellent supports with respect

to the activity towards the transphosphatidylation of 1,2-dipalmitoyl-3-sn-phospha-

tidylcholine to 4-methoxyphenol in non-polar solvents. The yield of 1,2-dipalmitoyl-

3-sn-phosphatidyl-4-methoxyphenol (DPP-PMP) was 45 % after 20 h and the

amount of the corresponding PA below 2 %. The immobilization was performed

by simply adding 4 units of PLD in 10ll of a 0.2 M acetate buffer (pH 5.6) to

a stirred suspension of 50 mg Amberlite IRC-50 in 1 mL benzene. After repeated

stirring, and sonication until the benzene phase became clear, the immobilized bio-

catalyst could be used immediately after removal of the solvent. As the resin absorbs

water up to 50 % of its own weight, all the enzyme, together with the aqueous buffer

solution, can be assumed to be entrapped within the porous matrix of the carrier.

Appropriate solvents for the synthesis of DPP-PMP were benzene, toluene, and

methylene chloride. In the presence of water-soluble organic solvents, no catalytic

activity with respect to DPP-PMP formation was observed because of the reasons

already discussed. Under comparable conditions (0.1 % acetate buffer, 1 mL ben-

zene) native PLD was inactive. An increase in the amount of buffer of up to 5 %

of the reaction volume enhanced the production rate of DPP-PMP.

With respect to an economical large-scale production of PG and PS, the use of

immobilized PLD is of special interest. Earlier investigations into this topic with

PLD attached to octyl-Sepharose CL-4B revealed biocatalysts with a low opera-

tional stability (Juneja et al., 1987; 1988). Recently, Wang and co-workers

(1997) tested different carrier materials (and immobilization procedures) for the

immobilization of PLD fromPseudomonassp., such as Amberlite XAD-2, con-

trolled pore glass (CPG), polyethyleneimine (PEI)-cellulose, and calcium algi-

nate-enveloped PEI-glutaraldehyde. Surprisingly, the latter component turned out

to be the most suitable for the synthesis of PG by head group exchange of refined

soybean lecithin (40 % PC, 31.2 % PE, 17.6 % PI, and 10.1 % PA). The optimum

composition of the support was obtained with 1.39 % calcium alginate, 7.78 % PEI,

and 1.22 % glutaraldehyde. The optimum reaction parameters with respect to the

yield of PG (which was maximally 85 %) were a reaction temperature of 25 8 C

to 30 8 C, a diethyl ether to water ratio of between 1.5 and 2.5, and a pH of 8.2,

which is astonishing in so far as normally Ca-alginate itself is not stable under alka-

line conditions. As far as the operational stability is concerned, PLD immobilized to

the Ca-alginate/PEI/glutaraldehyde system could be used for 15 repeated batches

without significant loss of activity, after which the degree of conversion declined

significantly to about 10 % after 40 repeated batch operations. Similar observations

concerning the long-term stability of the biocatalyst were made in an earlier inves-

tigation by Lee et al. (1985) who used PLD from cabbage in a microporous mem-

brane reactor for the continuous production of PG.

284 13 Preparation and Application of Immobilized Phospholipases