Front Matter

the adsorption of PLA 2 from aqueous solutions to a polysiloxane carrier with a hy-

drophobic surface (Deloxan HAP) was examined. The hydrolysis of PC was per-

formed in the presence of SDS (Texapon K12; Henkel) in a solution containing

4 mM CaCl 2 , 20 vol% of an organic solvent, and Tris/HCl to adjusting the pH to

9. The data in Figure 7A reconfirm that the immobilization of PLA 2 by including

e-amino residues in this process when GDA is taken as a spacer, leads to low residual

activities. The activity values increase for preparations obtained by adsorption, and

are the highest for PLA 2 bound covalently via the azo linkage. The sensitivity of the

hydrolysis rate to the design of the reaction medium becomes obvious from the

results shown in Figure 7B. As a consequence of the exchange of ethanol by ethyl

acetate, the catalytic activities are raised by a factor of 4 to 5, independently of the

type of binding. The PLA 2 -Deloxan catalysts revealed residual activities of above

70 % in the case of ethanol/water, and more than 85 % when the PC-hydrolysis was

carried out in the ethyl acetate/water mixture, compared to the native enzyme under

corresponding reaction conditions. PLA 2 immobilized by diazotation can be stored at

48 C for several months without considerable loss of activity. The high operational

stability of these products has been proven by repeated use in a column reactor, so

that in principle these biocatalysts could be applied for the continuous production of

lysolecithins, or in a FIA device for the determination of PC concentrations (Grun-

wald, unpublished results). As reported by Maderoy et al. (1995), PLA 2 from bee

venom could be also successfully immobilized through adsorption to the weakly acid

cation-exchange resin carboxymethyl Sephadex (CM-Sephadex, Serva). The activity

retention was above 80 % and the operational stability – obviously due to additional

electrostatic interactions between the enzyme and the surface of the support – was

demonstrated by eight successive applications without activity loss. The activity

proved to be strongly dependent on the amount of enzyme bound. Indeed, it de-

creased from 115lmol (mgmin)–1for 15 mg fixed enzyme per g carrier to 10

lmol (mgmin)–1when 45 mg g–1were attached to the support. This was the result

of increasing steric hindrance and/or diffusion limitation with increasing enzyme

loading. Compared to the soluble enzyme, the pH/activity profile was considerably

broadened. Similar results were obtained by Lambrecht and Ulbrich-Hofmann

(1993) for PLD immobilized to octyl-Sepharose.

Shen and Cho (1995) found that the acylation of Lys7 and Lys10 ofAgkistrodon

piscivorus piscivorusPLA 2 as well as ofNaja naja najaPLA 2 prior to their covalent

immobilization to beaded carbonyldiimidazol-activated crosslinked agarose (Pierce)

yielded high activities compared to those of nonacylated PLA 2 towards defined sub-

strates such as large unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-

phocholine. This holds true also for the hydrolysis of phospholipids on the surface of

low-density lipoproteins, a point which is of interest in connection with the applica-

tion of immobilized PLA 2 in an extracorporeal shunt for the treatment of hyperch-

olesterolemia (Labeque et al., 1993). A detailed report of this procedure, including

the synthesis of 4-nitro-3-octanoyloxybenzoic acid, the Lys7 and Lys10 acylation of

PLA 2 , and its immobilization was published recently (Cho and Shen, 1999). These

findings again corroborate the importance of the chemistry of enzyme binding for the

resulting catalytic activity, and also indicate the importance of these lysine residues

for the catalytic action of the two venom PLA 2 s.

278 13 Preparation and Application of Immobilized Phospholipases