A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

Abstract

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100 degrees C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20-30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed-batch cultivation, glucose concentration was maintained at 2-3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.

Characterization of an extremely salt-tolerant and thermostable phytase from Bacillus amyloliquefaciens US573

“…Similar to the phytases previously characterized from Bacillus species [29,[31][32][33], the PHY US573 showed dependence toward calcium for its catalytic activity. Increasing the concentration of this metal ion enhanced the enzyme activity, which attains its maximal level in the presence of 1 mM CaCl 2 .…”

“…This temperature was superior to that of the phytases purified from B. subtilis VTTE 68013 [27] and B. subtilis US417 [25], but comparable to those produced by B. amyloliquefaciens DS11 and Bacillus sp. MD2 which have been reported to be optimally active at 70 • C and 67-73 • C, respectively [29,31]. Study of the effect of temperature on the activity of Ronozyme PL and Natuphos showed that the optimum of these commercial enzymes was 55 • C (Fig.…”

“…For comparison, TS-Phy and the recombinant phytase from Bacillus sp. MD2 (expressed in Escherichia coli), which are considered as the highest thermostable Bacillus phytases so far characterized, retained 50 and 40% of activity following incubation for 10 min in the presence of 5 mM CaCl 2 at 90 and 100 • C, respectively [29,31]. Pertaining to the commercial enzymes, Ronozyme PL recovered 45 and 33%, while Natuphos retained 53 and 16.04% subsequent to heat treatment for 10 min at 75 and 90 • C, respectively.…”

“…The highest identity of 99% was observed with the phytases from B. amyloliquefaciens DS11 and B. sp. MD2 (3 and 5 residues difference, respectively) [29,31].…”

Complete signal peptide of Tk1884, an α-amylase from Thermococcus kodakarensis, is not necessary for extracellular secretion of the enzyme by Escherichia coli

“…Although the cell membrane and cell wall of archeons and bacteria are significantly different, the signal peptides from these organisms are significantly similar consisting of three regions: an N-region followed by a hydrophobic H-region and a carboxyl terminal C-region, which contains a signal peptidase cleavage site [24,25]. We have previously produced Tk1884, an α-amylase from hyperthermophilic archaeon T. kodakarensis, with the native signal sequence in E. coli and demonstrated that signal peptidase of the host cleaved the signal sequence of the archaeal origin and the mature protein was secreted in the extracellular medium [22].…”