[Influence of polymerization of D-amino acid oxidase on the behavior of the enzyme immobilized on chitosan by covalent fixation].
D-Amino-acid oxidase is a flavoprotein using FAD as cofactor. The enzyme has been immobilized in the presence of FAD on a non-porous matrix: chitosan. This support is covalently bound to the enzyme with glutaraldehyde as cross-linking reagent. It is characterized by a good mechanical resistance to mechanical stirring. The enzymatic assays have been performed in batch reactor with D-phenylglycine as substrate by a spectrophotometric method which is based on the variation of the absorbance at 252 or 280 nm. The behaviour of the biocatalysts has been studied during repeated assays of 1 h at 25 degrees C in the absence of exogenous FAD. The experimental results have been compared with those obtained with the soluble enzyme tested in the presence or in the absence of FAD. The dependence of D-amino-acid oxidase on FAD concentration has been studied. Immobilized enzyme on chitosan appears to be less sensitive to the association-dissociation equilibrium of FAD. This property and the capacity of the enzyme to polymerize spontaneously in solution according to the experimental conditions have been established. The fact that the enzyme can exist in various oligomeric forms is of major importance because its catalytic expression is dependent of this phenomenon. The polymerization is known to be responsible for a decrease of the maximal rate V of the enzyme. It has also been shown that in the same way this decrease was accompanied by an improvement of the affinity of enzyme for substrates. Furthermore, the value of the dissociation constant of the apoenzyme-FAD complex is significantly smaller as the degree of polymerization is high. The conclusion is that the dissociation of the cofactor can be avoided if the immobilization step is carried out at high concentration of enzyme which is favourable to its polymerization.
D002686:Chitin / Q000031:analogs & derivatives*; D048271:Chitosan; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004800:Enzymes, Immobilized; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007700:Kinetics; D011108:Polymers / Q000494:pharmacology*
D004800:Enzymes, Immobilized; D011108:Polymers; D002686:Chitin; D005182:Flavin-Adenine Dinucleotide; D048271:Chitosan; D003605:D-Amino-Acid Oxidase
Influence de la polymérisation de la D-aminoacide oxidase sur le comportement de l'enzyme immobilisée sur chitosane par fixation covalente.