Calcium-Dependent Catalytic Activity of a Novel Phytase from Bacillus amyloliquefaciens DS11

Abstract

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.

Screening of phytate hydrolysis Bacillus sp. isolated from soil and optimization of the certain nutritional and physical parameters on the production of phytase

“…There are many such reports on the effect of metal ions on phytase production by bacteria and fungi. Similar result was reported that CaCl 2 was an important salt for phytase production by B.amyloliquefaciens [30], Bacillus sp. DS11 [23], Bacillus subtilis (natto) [19], Bacillus subtilis [24].…”

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

“…CC1295 has a phytase domain. The protein with this domain was found to bind to Ca 2ϩ and organic phosphate (myo-inositol hexakisphosphate) (49). Further investigations are needed to prove or disprove the hypothesis that the active site of this enzyme Uranium stress also induced transcription of 48 transcripts which were up-regulated at least twofold (see the supplemental data); however, the response to uranium does not appear to overlap substantially with other heavy metal stresses evaluated in this study.…”

Characterization and Identification of Gut-Associated Phytase-Producing Bacteria in Some Freshwater Fish Cultured in Ponds

“…Similar ranges of temperature and pH tolerance have been reported in other strains of Bacilli isolated from fish gut (Ghosh et al 2002a, Saha et al 2006, Kar et al 2008. Because of neutral pH condition in the small intestine of fish, phytic acid might change from the protonated form into phytates, predominantly Ca-phytate, which is true substrate of Bacillus phytase (Oh et al 2001). Thus, Bacillus phytase appears to be most promising for phytase activity within the small intestine of fish.…”