Bacillus fitazinin klonlanmasi ve ekspresyonu

Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. Phosphorus is one of the necessary mineral nutrients for animals during their growth, reproduction and calcification of the bones. In this research, one native Bacillus sp. isolate containing phytase activity was identified as a Bacillus subtilis by biochemical and 16srDNA sequence analysis. Primer pairs were designed according to phytase gene sequence of Bacillus subtilis present in NCBI web site. After amplification by PCR the gene was ligated to pET22b vector as expression vector and transformed to E. coli Rosetta 2(DE3) pLysS by heat shock method. In preparation of PCR product and vector for ligation two restriction enzymes (Hind III and BamH1) were used. The optimum activity of recombinant phytase was emerged at 37oC and pH 7. Thermo stability with more than 73% residual activity at 60oC, and about 5% residual activity at 100∘C. The enzyme was stable over the pH range of 5.8 to 8. Recombinant phytase activity significantly stimulated with 5mM CoCl2, but decreased activity with MgCl2, CuCl2, HgCl2, NiCl2, MnCl2, FeCl2 and EDTA. Molecular weight of recombinant phytase was estimated as 45 kDa on SDS-PAGE analysis. These results of this study showed that, recombinant phytase could be an interesting candidate for feed applications. Key words: phytase, pET22b, Bacillus subtilis, cloning