Oxidation of azaheterocycles by free and immobilized xanthine oxidase and xanthine dehydrogenase

Research output: Thesisinternal PhD, WU


The objective of the study described in this thesis was to show that enzymes, especially immobilized enzymes, can be advantageously used in synthetic organic chemistry.
In Chapter 1 enzymes are introduced and the advantages discussed when these highly active and specific biocatalysts are immobilized, i.e. , attached to a solid support. Criteria for general acceptation of a specific enzyme as a routine catalyst in the organic-chemistry laboratory are stated. The reasons for the choice of xanthine oxidase as a model enzyme and some relevant properties are given.
The solid support used for the immobilization determines to a large extent the ultimate characteristics of the immobilized enzyme. In Chapter 2 properties of an ideal support and guidelines for the evaluation of a specific support are
discussed. Various supports are treated in some detail.
The mode of immobilization is also very important, especially with respect to the eventual specific activity of the immobilized enzyme. In Chapter 3 the various methods of immobilization are discussed. Just as with the supports (Chapter 2), special attention is given to the ones investigated in this study.
In Chapter 4 various properties of immobilized milk xantine oxidase are described, as well as the stabilization by coimmobilization with protein, superoxide dismutase and catalase. It is concluded that conversion of substrate on a preparative
scale can be performed conveniently, but that for efficient application a more stable enzyme preparation is desired.
Chicken-liver xanthine dehydrogenase is an enzyme very similar to milk xanthine oxidase. In Chapter 5 the reasons are given why it was expected that the liver enzyme would be more stable and practical for synthetic application. The immobilization and the properties of the free and immobilized enzyme are described. Analogous to the milk enzyme, the operational stability of xanthine dehydrogenase is much lower than the storage stability.
In order to determine the substrate limits of xanthine oxidase more systematical ly, so that more-reliable predictions would be possible, series of substrates were synthesized for this purpose. In Chapter 6 the synthesis of 7-(p-X-phenyl) pteridin-4-ones and the oxidation by free and immobilized xanthine oxidase are presented. As X becomes more electron withdrawing, the rate of oxidation decreases. It is therefore concluded that the electron donation from substrate to enzyme must be the rate-limiting step.
Convenient oxidation of the above substrates and easy product isolation was possible on a small preparative scale. The limited stability of xanthine oxidase demanded however a relatively large amount of enzyme. In Chapter 7 an easy and mild immobilization procedure, using gelatin as support and glutaraldehyde as cross-linking agent, is presented. Whole milk can be used as starting enzyme solution and no isolation is required. The resulting preparation of immobilized xanthine oxidase is very cheap, highly active, relatively stable and suitable for application in organic synthesis in a continuous manner.
In Chapter 8 the improvement of the immobilization method with gelatin is described and the extension to the immobilization of whole cells, i.e. , Arthrobacter cells containing xanthine-oxidase activity. These cells were chosen because of their high specific activity, their substrate activation instead of inhibition and their expected higher stability. Immobilized Arthrobacter xanthine
oxidase is indeed highly active, more stable and has a different substrate specificity than milk xanthine oxidase. Therefore, a wider spectrum of substrates can be conveniently and efficiently oxidized.
In the final Chapter 9 additional information, mostly gathered at a later stage than the time of publication, is given and discussed.
The end conclusion is: Xanthine oxidase, either from whole milk or Arthrobacter, when immobilized in glutaraldehyde-crosslinked gelatin, can be conveniently used for the oxidation of a wide range of substrates in a continuous fashion.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • van der Plas, H.C., Promotor
Award date19 Dec 1979
Place of PublicationWageningen
Publication statusPublished - 19 Dec 1979


  • fermentation
  • immobilization
  • oxidoreductases
  • heterocyclic nitrogen compounds
  • xanthine oxidase
  • xanthine dehydrogenase
  • oxidation
  • organic chemistry


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