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Purification, characterization and properties of phytase from Shigella sp. CD2.

Indian journal of biochemistry & biophysics (2012-10-20)
Moushree Pal Roy, Madhumita Poddar, Kamal Krishna Singh, Shilpi Ghosh
ABSTRACT

Phytases catalyze the release of phosphate from phytic acid. In this study, a phytase producing bacterial strain Shigella sp. CD2 was isolated from the wheat rhizosphere. Phytase production started from the exponential phase of bacterial growth, showing the highest activity during the stationary phase. The enzyme activity was detected in both periplasmic and intracellular fractions. The enzyme was purified by about 133-fold with specific activity 780 U mg(-1) protein. The optimum pH and temperature of the enzyme was 5.5 and 60 degrees C, respectively. The enzyme was thermostable and retained 100% and 75% of its activity on pre-incubation at 70 degrees and 80 degrees C for 30 min, respectively. The Km value for the substrate sodium phytate was 0.25 mM. The enzyme was highly specific to substrate phytate, and no activity was detected in presence of other phosphorylated substrates, such as ATP, ADP, glucose 6-phosphate, fructose 6-phosphate and p-nirophenyl phosphate. The activity declined dramatically in presence of Cu2+, Zn2+ and Fe2+ and SDS, whereas Mg2+ and Co2+ slightly enhanced the enzyme activity. The addition of other metal ions or chemicals had little or no effect on phytase activity. The enzyme was resistant to both pepsin and trypsin. Due to high specific activity, substrate specificity, good pH profile, protease insensitivity and thermostability, phytase encoding gene from Shigella sp. CD2 could be an interesting candidate for industrial applications. Further studies on cloning and expression of Shigella phytase gene are currently in progress.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Pepsin from porcine gastric mucosa, lyophilized powder, ≥2,500 units/mg protein (E1%/280)
Sigma-Aldrich
Pepsin from porcine gastric mucosa, tested according to Ph. Eur.
Sigma-Aldrich
Pepsin from porcine gastric mucosa, powder, slightly beige, 1200-2400 U/mg
Sigma-Aldrich
Pepsin from porcine gastric mucosa, powder, slightly beige, ≥500 U/mg
Sigma-Aldrich
Pepsin from porcine gastric mucosa, powder, ≥250 units/mg solid
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Pepsin from porcine gastric mucosa, powder, ≥400 units/mg protein
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Pepsin−Agarose from porcine gastric mucosa, lyophilized powder, ≥30 units/mg dry solid
Sigma-Aldrich
Phytase from wheat, ≥0.01 unit/mg solid
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Pepsin from porcine gastric mucosa, lyophilized powder, ≥3,200 units/mg protein
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Pepsin from porcine gastric mucosa, Vetec, reagent grade