Cloning, expression, and characterization of a new phytase from the phytopathogenic bacterium Pectobacterium wasabiae DSMZ 18074.
The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072+/-47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50 degrees C, respectively. The Km value was 0.17 mM, with a Vmax of 1,714 micromol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium.
Shao
Na
N
Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, PR China.
Huang
Huoqing
H
Meng
Kun
K
Luo
Huiying
H
Wang
Yaru
Y
Yang
Peilong
P
Yao
Bin
B
D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018556:Crops, Agricultural / Q000382:microbiology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D044043:Pectobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology*; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D010832:6-Phytase