Molecular Cloning of Partial Phytase Gene from Bacillus subtilis ATCC 6633 (GenBank: KT385665.1)

Phytase (myo-inositolhexakisphosphate phosphohydrolase) catalyzes the hydrolysis of myo-inositol hexakisphosphate (phytic acid) to inorganic monophosphate and lower myo-inositol phosphates, and in some cases to free myoinositol. Bacillus species are known to produce a thermostable phytase. The Bacillus subtilis strain ATCC 6633 was chosen for cloning of phy gene. Primers were designed for phy gene amplification using the phy gene sequence of B. subtilis (AF298179). A sequence of 904 bp characteristic of phy gene was obtained through PCR amplification. Sequencing of PCR amplified fragments and further analysis using NCBI-BLAST online homology search program revealed that phy gene of B.subtilis (ATCC 6633) and phy gene of B.subtilis are 94% similar. The phylogenetic analysis indicated that B.subtilis (ATCC 6633) had high similarities with the phytases from AF292103.1 Bacillus subtilis strain YMN2f phytase (phy) gene and HQ730912.1 Bacillus sp. B5 phytase PhyC1 gene. It was also found that B.subtilis (ATCC 6633) had relative high similarities with Bacillus sp. BAB-1 Phy (phy) gene and FJ541287.1 Bacillus subtilis strain spizizenii strain phytase gene. Further 904 bp fragment was cloned in Insta T/A cloning vector and E. coli was successfully transformed and confirmed by colony PCR.