title: Vanadate substituted phytase: immobilization, structural characterization and performance for sulfoxidations.
abstract: A cross-linked enzyme aggregate (CLEA) of 3-phytase (EC 3.1.3.8) was synthesised, which was incubated with vanadate and tested as a biocatalyst in the asymmetric sulfoxidation of thioanisole using hydrogen peroxide as the oxidant. The results show that the 3-phytase-CLEA demonstrates a similar efficiency (ca. 95% conversion) and asymmetric induction (ca. 60%) as the free enzyme. Moreover, the 3-phytase-CLEA can be reused at least three times without significant loss of activity. The activity of the 3-phytase in the presence of organic solvents is however still limited. Studies were undertaken to elucidate the role of vanadate on the active site and on the effect of organic solvents on the conformation of the enzyme. The incorporation of vanadate in the active sites of two different phytases could be followed using (51)V NMR and circular dichroism (CD) spectroscopies. (51)V NMR spectra show the incorporation of vanadate into the active site at pH 5.0 and 7.6, and suggest coordination to oxygen functions at two different binding sites, which probably explains the poor enantioselectivity found in the catalytic studies. After addition of H(2)O(2), only at pH 5.0 and with the 3-phytase a V-phytase-peroxide complex could be observed, which is the active species responsible for the oxidation reactions. CD studies showed that the alpha-helical content of the enzyme decreased upon coordination of vanadate, but in the concentration range used in the catalytic studies (<30 microM) the secondary conformation of the enzyme was unchanged. Acetonitrile decreases the alpha-helical content of both phytases from 59% to 51% and from 42% to 34%, in the 3- and 6-phytases, respectively, this being in agreement with the activity loss in the catalytic experiments.
authors.affiliation: Centro Química Estrutural, Instituto Superior Técnico, TU Lisbon, Av. Rovísco Pais, 1049-001 Lisboa, Portugal. icorreia@ist.utl.pt
authors.forename: Isabel
authors.initials: I
authors.lastname: Correia
authors.forename: Seda
authors.initials: S
authors.lastname: Aksu
authors.forename: Pedro
authors.initials: P
authors.lastname: Adão
authors.forename: João Costa
authors.initials: JC
authors.lastname: Pessoa
authors.forename: Roger A
authors.initials: RA
authors.lastname: Sheldon
authors.forename: Isabel W C E
authors.initials: IW
authors.lastname: Arends
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D014638:Vanadates / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D014638:Vanadates; D010832:6-Phytase