Gene cloning, expression, and characterization of a novel phytase from Dickeya paradisiaca.
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.
Gu
Weina
W
Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People's Republic of China.
Huang
Huoqing
H
Meng
Kun
K
Yang
Peilong
P
Fu
Dawei
D
Luo
Huiying
H
Wang
Yaru
Y
Yao
Bin
B
Zhan
Zhichun
Z
D010832:6-Phytase / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004755:Enterobacteriaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010833:Phytic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D008670:Metals; D011994:Recombinant Proteins; D010833:Phytic Acid; D010832:6-Phytase