title: Subunit fusion of two yeast D-amino acid oxidases enhances their thermostability and resistance to H2O2.
abstract: D-amino acid oxidases from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) are both yeast homodimeric flavoenzymes. Two of their cDNA genes were connected by a hexanucleotide linker and heterologously expressed in E. coli to produce the corresponding double DAOs (dRtDAO and dTvDAO) with two subunits fused into a single polypeptide. The specific activities of double DAOs remained similar to those of native dimeric DAOs, although the catalytic efficiencies (k(cat)/K(M)) were decreased due to higher K(M) values. The T(m) value for dRtDAO was shifted 5 degrees C higher while that for dTvDAO was increased only by 2 degrees C, in comparison with the corresponding native counterparts. In the presence of 10 mM H(2)O(2), dRtDAO and dTvDAO exhibited half-lives of about 60 and 40 min, respectively, which were 2- and 1.5-fold, respectively, longer than their native DAOs. These yeast DAOs can therefore be thermally and oxidatively stabilized by linking their subunits together.
authors.affiliation: Department of Bioengineering, Tatung University, Taipei 10452, Taiwan.
authors.forename: Shih-Juei
authors.initials: SJ
authors.lastname: Wang
authors.forename: Chi-Yang
authors.initials: CY
authors.lastname: Yu
authors.forename: Cheng-Kang
authors.initials: CK
authors.lastname: Lee
authors.forename: Ming-Kai
authors.initials: MK
authors.lastname: Chern
authors.forename: I-Ching
authors.initials: IC
authors.lastname: Kuan
mesh_terms: D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D010084:Oxidation-Reduction / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
chemical_list: D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase