Studies On Expression And Purification Of D-amino Acid Oxidase And The Enzymatic Characterization

D-amino acid oxidase (D-AAOs, EC1.4.3.3) is a FAD-dependent enzyme that widely in nature and plays an important role in metabolism of endogenous D-amino acids in living cells. The D-Amino acid oxidase (DAAO) catalyzes oxidative deamination of D-amino acids yielding hydrogen peroxide, ammonium and the corresponding a-keto acids. In2006, researchers reported on the biochemical characterization of a bacterial D-AAO from the Arthrobacter protophormiae for the first time and till now this was the only one paper about the prokaryotic DAAO..In this paper, the gene of DAAO was cloned from Arthrobacter protophormiae (DSM20168) and four mutants DAAO and investigated the enzyme characterization of the wild-type and mutant proteases. This is not only the supplement of the D-amino acid oxidase from prokaryotes, but also making a preliminary exploration and laying a theoretical foundation to build more conducive enzyme preparation for the life and production.The results are as follows:Firstly, in order to obtain a DAAO gene from Arthrobacter protophormiae (DSM20168), the primers of daao gene which with an additional Ndel and Xhol restriction sites were designed according to the known gene sequences dao(GeneBank accession No: AY306197),and amplified Apdaao gene by PCR,then cloned into pMD-19T,the sequence was comfirmed by DNA sequencing and compared with other DAAO genes from Arthrobacter protophormiae reported in literature,their homology was more than98%.The DAAO gene digested with NdeI and XhoI was inserted into a prokaryotic expression vector pET22b+, the recombinant plasmid pET-Apdaao was obtained and then transformed into the expression host E.coli BL21(DE3). It was induced by0.5mM IPTG when the initial bacterial concentration was OD6oo=0.5, and induced at30℃for10-12h.Secondly, we designed four pairs of primers of site-directed mutagenesis and obtained the four mutant DAAO by using OE-PCR(overlap extension PCR) and one-step PCR,the sequene is comfirmed by DNA sequencing.The recombinant mutant-plasmids,named as pET-E115A, pET-N119D,pET-T256K, pET-T286Awere then trasformed into expression host E.coli BL21(DE3) respectively.They were induced by IPTG with the same induction conditions of wild-type ApDAAO.The crude extract from the recombinant strain was purified by Ni-NTA affinity chromatography. The result of SDS-PAGE showed that the relative molecular weight of the protease was about39kD.The studies of the enzyme characterization of the protease DAAO including substrate specificity,optinal temperature range and pH range.The results showed that the proteases were stable at20-40℃, their optimal reactive temperature were30℃;the optimal pH of wild-type ApDAAO was10.but the mutant ApDAAO were stable at8-9;compared with the ApDAAO reported in literature,both the wild-type and mutants were in differently substrate specificity,especcially with D-Ala,D-Phe and D-Lys.