Characterization of a HAP-phytase from a thermophilic mould Sporotrichum thermophile.
The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of approximately 456kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 degrees C with a T(1/2) of 16h at 60 degrees C and 1.5h at 80 degrees C. The activation energy of the enzyme reaction is 48.6KJmol(-1) with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg(2+) exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3mM. The maximum hydrolysis rate (V(max)) and apparent Michaelis-Menten constant (K(m)) for sodium phytate were 83nmolmg(-1)s(-1) and 0.156mM, respectively. The catalytic turnover number (K(cat)) and catalytic efficiency (K(cat)/K(m)) of phytase were 37.8s(-1) and 2.4x10(5)M(-1)s(-1), respectively. Based on the N-terminal and MALDI-LC-MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.
Singh
Bijender
B
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110 021, India.
Satyanarayana
T
T
D010832:6-Phytase / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013175:Sporothrix / Q000201:enzymology*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013696:Temperature
D010832:6-Phytase