Gu Weina, Huang Huoqing, Meng Kun, Yang Peilong, Fu Dawei, Luo Huiying, Wang Yaru, Yao Bin, Zhan Zhichun
Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People's Republic of China.
Appl Biochem Biotechnol. 2009 May;157(2):113-23. doi: 10.1007/s12010-008-8329-6. Epub 2008 Aug 5.
A novel phytase gene, appA, was isolated by degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR from Dickeya paradisiaca. The full-length appA comprises 1278 bp and encodes 425 amino acid residues, including a 23-residue putative N-terminal signal peptide. The deduced amino acid sequence of appA reveals the conserved motifs RHGXRXP and HD, which are typical of histidine acid phosphatases; significantly, APPA shows maximum identity (49%) to a phytase from Klebsiella pneumoniae. To characterize the properties of APPA, appA was expressed in Escherichia coli and purified. The purified recombinant APPA has two pH optima at pH 4.5 and 5.5, optimum temperature at 55 degrees C, specific activity of 769 U/mg, and good pH stability. The K(m) value for the substrate sodium phytate is 0.399 mM with a Vmax of 666 U/mg. To our knowledge, this is the first report of a phytase or phytase gene isolated from Dickeya.
通过简并聚合酶链反应（PCR）和热不对称交错PCR从迪氏果胶杆菌中分离出一个新的植酸酶基因appA。appA全长1278 bp，编码425个氨基酸残基，包括一个23个残基的推定N端信号肽。appA推导的氨基酸序列显示出保守基序RHGXRXP和HD，这是组氨酸酸性磷酸酶的典型特征；值得注意的是，APPA与肺炎克雷伯菌的植酸酶具有最高的同一性（49%）。为了表征APPA的特性，appA在大肠杆菌中表达并纯化。纯化的重组APPA在pH 4.5和5.5时有两个最适pH值，最适温度为55℃，比活性为769 U/mg，并且具有良好的pH稳定性。底物植酸钠的K(m)值为0.399 mM，Vmax为666 U/mg。据我们所知，这是首次报道从迪氏果胶杆菌中分离出的植酸酶或植酸酶基因。
