The Extracellular Thermostable Phytase Gene From The Soil Bacteria Bacillus Subtilis Arrmk33 ( Bsphyarrmk33 )

3040 Words Aug 9th, 2014 13 Pages
The extracellular thermostable phytase gene was isolated from the soil bacteria Bacillus subtilis ARRMK33 (BsPHYARRMK33). The gene had an open reading frame of 1152 bp and it encoded 383 amino acid residues of a protein with a putative leader peptide of 27 amino acids. According to the insilico analysis of the phytase gene, the phytase from BsPHYARRMK33 is closely related to Bacillus subtilis sp. phytase proteins and has no similarities to other phytases. In silico analysis of this phytase disclosed β propellar structure of phytase. The Phytase encoding cDNA was subcloned into the pET-28a (+) expression vector. The recombinant plasmid pET-28 a (+) BsPHYARRMK33 was expressed in Escherichia coli BL21 (DE3). The expressed protein was analysed by SDS-PAGE and a specific band with a molecular mass of approximately 43 kDa was found. Recombinant phytase enzyme activity assay was verified, with sodium phytate substrate. The maximum phytase activity was 2.25 U ml-1 obtained from the cellular extract of E. coli BL21 (DE3) harbouring pET-28 (a) BsPHYARRMK33. The optimal pH and temperature of the crude recombinant phytase were 7.0 and 55 ᴼC, respectively.

Keywords: Bacillus subtilis, Phytase, thermostable, feed addtive.

Introduction

Phytic acid is the major storage form of phosphorus in plant tissues especially grains or seeds and bran. Plant seeds have 80% of the phosphorus in the form of phytic acid. The chemical description for phytic acid is myo-inositol (1, 2, 3, 4,…
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