13 Preparation and Application of Immobilized
Phospholipases
Peter Grunwald
This chapter provides a short introduction into the field of phospholipids and phos-
pholipases, followed by a brief survey on immobilization techniques, suitable carrier
materials and some fundamental aspects of the kinetics of immobilized biocatalysts.
The section about immobilized phospholipases deals mainly with the immobilization
of phospholipase A 2 (PLA 2 ) and phospholipase D, but also includes some data on
lipases exhibiting phospholipase A 1 activity.
13.1 Introduction
Nowadays, the immobilization of enzymes, which means transferring them from a
water-soluble to a water-insoluble state, is a well-established biotechnological meth-
od, and the application of immobilized biocatalysts has gained increasing signifi-
cance in fields such as medicine, pharmacy, analytical chemistry, organic synth-
esis, food and environmental technology. The main advantages of using immobi-
lized enzymes are that they are re-usable – an essential prerequisite for economic
production if the applied enzymes are expensive (e.g., phospholipase C and phos-
pholipase D) – and that the products obtained are not spoiled by catalytically active
protein since immobilized biocatalysts can be separated easily from the reaction
mixture by filtration or centrifugation. Furthermore, they can be used in different
types of reactors for continuous production, as well as in flow injection analysis
(FIA) devices. Immobilization techniques also offer the opportunity of obtaining
information about the biocatalyst itself. They have been employed to identify
whether single subunits of an oligomeric enzyme are catalytically active, or in
the study of the mechanisms of protein refolding (Chan, 1976; Mosbach 1980; Mar-
tinek and Mozhaev, 1985). Ferreira et al. (1994) immobilized the dimericCrotalus
atroxvenom PLA 2 by single-subunit attachment to a support. From the finding that
the catalysts obtained exhibited comparatively low specific activities, these workers
concluded that only the dimeric form of this PLA 2 was the fully active form. Further
examples for this type of application are provided in connection with the immobi-
lization of phospholipases (see Section 13.4).
13.2 Phospholipids and phospholipases
Phospholipids – together with other polar lipids – are vital constituents of mem-
branes in animal and vegetable organisms. All the natural representatives can be
Enzymesin LipidModification.Editedby UweT. Bornscheuer
Copyright 2000 Wiley-VCHVerlagGmbH& Co. KGaA,Weinheim.ISBN:3-527-30176-3
