Huang Huoqing, Luo Huiying, Yang Peilong, Meng Kun, Wang Yaru, Yuan Tiezheng, Bai Yingguo, Yao Bin
Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, PR China.
Biochem Biophys Res Commun. 2006 Dec 1;350(4):884-9. doi: 10.1016/j.bbrc.2006.09.118. Epub 2006 Sep 29.
A Yersinia intermedia strain producing phytase was isolated from glacier soil. The phytase gene, appA, was isolated by degenerate PCR and TAIL-PCR. The full-length fragment contained 2354bp with a 1326-bp open reading frame encoding 441 amino acids. APPA contained the active site RHGXRXP and HD sequence motifs that are typical of histidine acid phosphatases. To our knowledge, this is the first report of the detection of phytase activity and cloning of the relevant gene from Y. intermedia. The gene was overexpressed in Pichia pastoris, and the purified recombinant APPA had a specific activity for sodium phytate of 3960U/mg, which is higher than that of the Citrobacter braakii phytase (previously the highest specific activity known). Recombinant APPA had high activity from pH 2 to 6 (optimum 4.5) and optimal temperature of 55 degrees C; the enzyme was resistant to pepsin and trypsin. These characteristics suggest that APPA may be highly suitable for use in the feed industry.
从冰川土壤中分离出一株产植酸酶的中间耶尔森菌。通过简并PCR和热不对称交错PCR分离出植酸酶基因appA。全长片段为2354bp，包含一个1326bp的开放阅读框，编码441个氨基酸。APPA含有组氨酸酸性磷酸酶典型的活性位点RHGXRXP和HD序列基序。据我们所知，这是首次关于从中间耶尔森菌中检测到植酸酶活性并克隆相关基因的报道。该基因在毕赤酵母中过表达，纯化后的重组APPA对植酸钠的比活性为3960U/mg，高于布氏柠檬酸杆菌植酸酶（此前已知的最高比活性）。重组APPA在pH2至6（最适pH4.5）时具有高活性，最适温度为55℃；该酶对胃蛋白酶和胰蛋白酶具有抗性。这些特性表明APPA可能非常适合用于饲料工业。
