Front Matter

A special type of application of immobilization techniques is that of enzyme pur-

ification by affinity chromatography. Bernal and Pidgeon (1996) used so-called im-

mobilized artificial membranes (IAMs) for the enrichment of PLA 2 fromCrotalus

atrox. IAMs (Qiu et al., 1994) are prepared by immobilizing anx-carboxyphospha-

tidylcholine ligand on silica propylamine surfaces. The ligand density corresponds

approximately to that of a monolayer. Remaining amino groups are end-capped with

decanoic and propionic anhydride. An interesting aspect of this investigation is that

IAMs containing the glycerol backbone, as well as those lacking it, were used. It

transpired that these differences were not important for the purification process,

which suggests that for the interaction of the PLA 2 -surface with the ‘membrane’

the polar head group of the substrate is the decisive factor. This example demon-

strates once more that immobilization can be a useful tool to enlighten theoretical

questions – in this case the mechanism of enzyme/substrate interaction. Moreover, it

was possible – starting from a crude venom protein mixture with 4.5 % PLA 2 –to

enrich the enzyme content up to 70 % with this technique.

Mustranta et al. (1994) investigated thetransesterification of synthetic dimyris-

toyl-PC with oleic acid catalyzed by commercially available lipase preparations

fromAspergillus niger(immobilized by adsorption on diatomaceous earth) andRhi-

zomucor miehei(bound to an anion-exchange resin (Lypozyme; Novo) and its de-

pendence on different reaction conditions. The enzymes exhibited lipase and phos-

pholipase activity, both being considerably higher in theAspergillus nigerlipase.

Nevertheless, theRhizomucor mieheienzyme was more active in transesterification

and 35 % of modified PC was the maximum yield. The reactions were carried out in

solvent-free medium (oleic acid) and in toluene. The optimum water content related

to the obtained amount of oleyl myristoyl-PC was 0.5 % and 1 % in case ofAsper-

gillus nigerandRhizomucor mieheiin toluene, and 1 % and 2.5 %, respectively in

the solvent-free reaction mixture. On the basis of the experience that transesterifica-

tion occurs mainly at the 1-position of phospholipids, and that no dioleyl-PC was

found, it can be assumed that the production of 1-oleyl 2-myristoyl-PC was cata-

lyzed by lipase. A further example of research in the field of transesterification

of phospholipids with lipase preparations was published by Hara and Nakashima

(1996), who used eight different acetone-driedRhizopusspecies. The cells were

immobilized on biomass support particles (BSP, HR-40, Bridgestone) where they

formed a dense film. Compared with the immobilization of extracellular lipase,

this procedure is advantageous as the cultivation of the lipase producer and its ad-

hesion onto the BSP-surface occurs simultaneously (Nakashima et al., 1988). With

the incorporation of heptanoic acid into PC it could be shown that most of the im-

mobilizedRhizopusspecies exceeded the transesterification potential of commer-

cially available immobilized lipases (Lipozyme IM20 and IM60 from Novo).

BSP of polyurethane foam have also proven to be useful carriers for the immobiliza-

tion ofStreptoverticillium cinnamoneum, with the aim of producing extracellular

PLD (Fukuda et al., 1998). The work of Haas et al. (1995) also dealt with the in-

vestigation of lipases concerning their ability to hydrolyze phospholipids apart from

triglycerides. With respect to this, commercially available, immobilized lipases were

tested. For example, the data obtained with Lipozyme IM20 showed that triglycer-

ides in water-saturated hexane as solvent were hydrolyzed completely within 20 h at

13.4 Immobilization of phospholipases 279