Sugano Yasushi, Ishii Yosuke, Shoda Makoto
Chemical Resources Laboratory, Tokyo Institute of Technology, R1-29-4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan.
Biochem Biophys Res Commun. 2004 Sep 10;322(1):126-32. doi: 10.1016/j.bbrc.2004.07.090.
The expression system of a unique dye-decolorizing peroxidase DyP in Escherichia coli has been constructed. The molecular mass of the expressed DyP (eDyP) is 47kDa, indicating no any modification with saccharides. The characteristics of eDyP were almost the same as those of native DyP from a fungus Thanatephorus cucumeris Dec 1 and recombinant DyP with Aspergillus oryzae except thermostability. As H164 was suggested to be the proximal histidine based on the preliminary X-ray crystallographic analysis of DyP, the site-directed mutations H164A and H166A (residue near H164) were introduced into the gene encoding DyP. The specific activity and RZ value of the purified H164A were 1.52U/mg and 0.11, respectively, which were 99.8% and 95% lower than those of eDyP, respectively. On the contrary, those of H166A were not different from those of eDyP. Therefore, H164 was confirmed to be the proximal histidine.
已构建了一种独特的染料脱色过氧化物酶DyP在大肠杆菌中的表达系统。所表达的DyP（eDyP）的分子量为47kDa，表明其没有任何糖基化修饰。eDyP的特性与来自瓜亡革菌Dec 1的天然DyP以及米曲霉重组DyP的特性几乎相同，除了热稳定性。基于DyP的初步X射线晶体学分析，H164被认为是近端组氨酸，因此将定点突变H164A和H166A（H164附近的残基）引入到编码DyP的基因中。纯化后的H164A的比活性和RZ值分别为1.52U/mg和0.11，分别比eDyP低99.8%和95%。相反，H166A的比活性和RZ值与eDyP没有差异。因此，H164被确认为近端组氨酸。
