Cloning, Overexpression And Characterization Of The Phytase Gene (PhyA)from Aspergillus Fumigatus Sp.

Phytate, myo-inositol1,2,3,4,5,6-hexakisphosphate, is the primary source of inosi-tol and the major storage form phosphorus in agricultural feedstuffs, such as cereals, leg-umes, and oilseeds. Phytase, myo-inositol hexakisphosphate hydrolase (EC3.1.3.8for3-phytase and3.1.8.26for6-phytase), catalyzes the hydrolysis of phytate into myo-inositol and inorganic phosphates. Monogastric animals such as pigs and chickens are incapable of digesting phytate phosphorus due to the lack of, or low levels of, phytase activity in their digestive systems. Supplementation of phytase in these animals’feedstuffs enhances not only the nutritional quality of phytate-rich feed, but also the growth performance of the animals, thus decreasing phosphorus pollution in areas of intensive animal agriculture. The potential industrial applications of phytase increase the interest of isolation of new bacte-rial strains producing novel and efficient phytases is increasing.In this thesis, a phytase producing Aspergillus fumigatus sp (A. fumigatus sp) specie, was isolated and identified, the gene phyA encoding its thermostable phytase was cloned and heterologously expressed in E.coli, active recombinant phytase was purified from in-clusion bodies and then its thennostability and the pH dependence were assayed in detail.Firstly, isolation of A. fumigatus phytase producing was selected by size of clear zone on the plate with modified phytase screening medium.Secondly, total RNA was extracted from A. fumigatus grown on modified phytase screening medium, the cDNA libraries were generated by RevertAidTM First Strand cDNA Synthesis kit. The gene encoding phytase of A. fumigatus without signal peptide and intron, PhyA, which comprises1,329bp and encodes443amino-acid residues, was PCR amplified and sequenced. The nucleotide sequence of A. fumigatus phytase gene showed99%identities to that of A. fumigatus Af293phytase and67%homology to that of A. niger phytase. The deduced amino acid sequence of A. fumigatus sp. phytase showed65%and41%identities to those of A. terreus A91phytase and A. oryzae RIP40phytase, respectively. The phytase of A. fumigatus was predicted to be a novel member of the his-tidine-acid phosphatase family with its conserved motifs active site septa-peptide RHGXRXPT and catalytically active dipeptide HD in the amino-acid sequence.Thirdly, expression plasmid pET21a (+)-phyA was constructed by cutting PhyA of A. fumigatus without original leader peptide and intron from cloning vector pMD18T-phyA with Nde Ⅰ and EcoR Ⅰ and religated into the same restriction sites of prokaryotic ex- pression vector pET21a (+). The recombinant phytase was overexpressed in E. coli BL21(DE3) as an inclusion body after IPTG induction. However, numerous attempts, such as changing IPTG concentration, Ca2+concentration, or induction temperature, to improve the soluble expression of recombinant PhyA in E.coli were not successful. The molecular weight of expressed phytase was estimated as45kDa by SDS-polyacrylamide gel electro-phoresis.At last, inclusion body renaturation conditions and partial characterics of recombi-nant phytase were studied. Ca2+ion was essential for obtaining active enzyme. Recombi-nant A. fumigatus phytase with specific activity of625UmL-1was obtained by refolding at pH5.5acetate buffer including1mM Ca2+. Purified phytase exhibited optimal activity at60℃. The enzyme retained70%of its original activity after20min incubation at80℃. Optimum pH was5.5-6.0. Recombinant phytase was more stable at acid conditions than neutral and alkaline conditions. It remained fairly stable over pH range of5.5to6.5.The broad pH optima and high thermostability of the phytase makes it a promising candidate for feed-pelleting application.