title: Stabilization of native and double D-amino acid oxidases from Rhodosporidium toruloides and Trigonopsis variabilis by immobilization on streptavidin-coated magnetic beads.
abstract: Double D: -amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k(cat)/K(M)) of immobilized DAOs toward D: -alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward D: -alanine was decreased by 56%. After immobilization, the T(m) value for RtDAO was shifted 15 degrees C higher to 60 degrees C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5-8 degrees C to 56, 60 and 60 degrees C, respectively. In the presence of 10 mM H(2)O(2), immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin-streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.
authors.affiliation: Department of Bioengineering, Tatung University, Taipei, 10452, Taiwan, ROC.
authors.forename: Shih-Juei
authors.initials: SJ
authors.lastname: Wang
authors.forename: Chi-Yang
authors.initials: CY
authors.lastname: Yu
authors.forename: I-Ching
authors.initials: IC
authors.lastname: Kuan
mesh_terms: D001487:Basidiomycota / Q000201:enzymology*; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D008280:Magnetics; D008863:Microspheres; D004718:Saccharomycetales / Q000201:enzymology*; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D019809:Streptavidin; D003605:D-Amino-Acid Oxidase