Subunit fusion of two yeast D-amino acid oxidases enhances their thermostability and resistance to H2O2.
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.
Wang
Shih-Juei
SJ
Department of Bioengineering, Tatung University, Taipei 10452, Taiwan.
Yu
Chi-Yang
CY
Lee
Cheng-Kang
CK
Chern
Ming-Kai
MK
Kuan
I-Ching
IC
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
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase