RESUMO
Isochorismate hydroxymutase (isochorismate synthase, E.C. 5.4.99.6) catalyzes the interconversion of chorismic acid [1] and isochorismic acid [2]. The enzyme was extracted from a Flavobacterium K3-15 that overproduces vitamin K2 (i.e., menaquinones) and was purified to homogeneity. The N-terminal amino acid sequence and the mol wt (36,240 +/- 100 daltons) were determined by ms following SDS PAG electrophoresis. The enzyme was characterized (stability, cofactor requirement, isoelectrical point), and antibodies were raised which showed no cross reactivity with isochorismate hydroxymutase from Escherichia coli and Enterobacter aerogenes 62-1. The kinetic data of the enzyme are different from those observed for the corresponding enzyme from Escherichia coli and Galium mollugo.
Assuntos
Proteínas de Bactérias/química , Flavobacterium/enzimologia , Transferases Intramoleculares , Isomerases/química , Sequência de Aminoácidos , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos , Western Blotting , Ácido Corísmico/metabolismo , Escherichia coli/enzimologia , Flavobacterium/química , Cinética , Dados de Sequência Molecular , Peso MolecularRESUMO
Partially purified isochorismate hydroxymutase (isochorismate synthase, E.C. 5.4.99.6) from Flavobacterium K3-15, a vitamin K overproducer, was immobilized on CNBr-activated Sepharose 4B, alkylamine glass substituted with glutardialdehyde, and aminohexyl Sepharose 4B substituted with glutardialdehyde. The immobilized enzyme exhibited a lower specific activity but a broader pH tolerance and a higher thermostability than the soluble enzyme. The stability of the enzyme was greatly increased by immobilization. Isochorismic acid, which is not commercially available, was prepared by a constant flow incubation.
