Purification and Characterization of a Phytase from Pseudomonas syringae MOK1

Abstract

A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40 degrees C. The Michaelis constant (Km) and maximum reaction rate (Vmax) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu2+, Cd2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.

Purification, sequencing and evaluation of a divergent phytase from <i>Penicillium oxalicum</i> KCTC6440

“…phytase activity of samples collected from each purification step was measured on phytic acid as described by Heinonen and Lahti et al with minor modifications (Cho et al, 2003;Heinonen and Lahti, 1981;Sanikommu et al, 2013;Yoon et al, 1996). A unit (U) of phytase activity was defined as the amount of enzyme required to release 1 µmol of inorganic phosphate per min under the assay conditions.…”

Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil

“…Phytase activity was measured in an assay mixture containing 100 l of sodium phytate (0.5 w/v) prepared in 0.2 M sodium acetate buVer pH 5.5 [1,5,7,8] and 100 l of suitably diluted enzyme. The reaction was stopped by adding an equal volume (200 l) of 15% trichloroacetic acid after 30 min of incubation at 50°C [3].…”

Purification, biochemical characterization, and genetic cloning of the phytase produced by <i>Burkholderia</i> sp. strain a13

“…The activity of this enzyme was inhibited by thiolacting agents, such as Zn 2+ , Cu 2+ , Hg 2+ , and iodoacetic acid, and activated weakly by the disulfide bridge-reducing agents, DTT and 2-mercaptoethanol (Table 3), clearly indicating the requirement of free thiol group(s) of Cys residue(s) for the activity. The activity of some other HAP phytases of bacterial origin were also inhibited by Cu 2+ and Zn 2+ , though the level of inhibition was different (Cho et al, 2003;Fu et al, 2008;Huang et al, 2006). The alignment showed that the mature portion of this enzyme contains eight Cys residues, of which five are conserved in bacterial HAP phytases examined and the remaining three are conserved in six phytases, including H. alvei phytase.…”