Wednesday, November 11, 2015
Exhibit Hall 1 (Salt Palace Convention Center)
Xanthine dehydrogenase (EC 188.8.131.52, XDH) catalyzes the oxidative metabolism of purines and aldehydes on CH or CH2 groups with NAD+ or NADP+ as the electron acceptor. XDHs have wide applications, such as in clinical diagnosis for enzymatic determination of inorganic phosphorus, 5`-nucleotidase and adenosine deaminase by coupling with purine-nucleoside phosphorylase and uricase. Here, we described cloning, expression and characterization of a novel alkaline XDH from Acinetobacter baumannii in Escherichia coli. A 4867-bp gene cluster consisting of 3 genes (xdhA, xdhB and xdhC) was cloned using PCR from the chromosomal DNA of Acinetobacter baumannii ATCC 19606. The 3 genes showed the highest homology of 91.4%~95.5% to the putative genomic genes of Acinetobacter baumannii retrieved in the GenBank, but only less than 52% homology to the experimentally confirmed XDHs genes of other species. The xdhABC gene cluster was functionally expressed in E. coli under the control of the trc promoter in pTrc99A plasmid, and the recombinant enzymes were purified to homogeneity by Ni-NTA affinity chromatography, anion exchange chromatography on Q-sepharose and hydrophobic interaction chromatography on phenyl-sepharose. The purified recombinant XDH was composed of two subunits with the respective molecular weights of 87 kDa(XDHB) and 56 kDa (XDHA), while the chaperone XDHC with a deduced molecular weight of 37 kDa was not confirmed by SDS-PAGE. The purified enzyme showed the maximum activity at pH 8.5 and high enzyme stability after incubation under the alkaline conditions (from pH 7.0 to pH 11.0) for 24 hours at 25. The optimum temperature was 40to 45, and the half-inactivation temperature () was 59. The enzyme exhibited a turnover number (kcat) of 69s-1 and apparent Michaelis constant (Km) of 25.3μmol.l-1 for xanthine, resulting a catalytic efficiency (kcat/Km) of 2.74 l.μmol-1.s-1. These enzymatic properties were the best among the reported results. The high catalytic activity, thermal stability and alkaline stability of the recombinant XDH enable its application to be feasible.