Cloning, expression, and enzyme characterization of an acid heat-stable phytase from Aspergillus fumigatus WY-2.
A novel thermostable phytase gene was cloned from Aspergillus fumigatus WY-2. It was 1459 bp in size and encoded a polypeptide of 465 amino acids. The gene was expressed in Pichia pastoris GS115 as an extracellular enzyme. The expressed enzyme was purified to homogeneity and biochemically characterized. The purified enzyme had a specific activity of 51 U/mg with an approximate molecular mass of 88 kDa. The optimum pH and temperature for activity were pH 5.5 and 55 degrees C, respectively. After incubation at 90 degrees C for 15 min, it still remained at 43.7% of the initial activity. The enzyme showed higher affinity for sodium phytate than other phosphate conjugates, and the K(m) and K(cat) for sodium phytate were 114 microM: and 102 s(-1), respectively. Incubated with pepsin at 37 degrees C for 2 h at the ratio (pepsin/phytase, wt/wt) of 0.1, it still retained 90.1% residual activity. These exceptional properties give the newly cloned enzyme good potential in animal feed applications.
Wang
Yan
Y
Department of Bioscience and Biotechnology, School of Environment and Life, Dalian University of Technology, Dalian, 116024, P.R. China.
Gao
Xiaorong
X
Su
Qiao
Q
Wu
Wei
W
An
Lijia
L
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D005656:Fungal Proteins; D011994:Recombinant Proteins; D010833:Phytic Acid; D010832:6-Phytase; D010434:Pepsin A