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PHAN TRUNG HIẾU. 2012. Cloning, overexpression and characterization of the phytase gene (PhyA) from Aspergillus fumigatus. MSc. Thesis. East China University of Science and Technology – CHINA. English Abstract. trunghieu1108@yahoo.com
Thứ năm, 19-07-2012 | 13:31:51

ABSTRACT

 

Phytate, myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the primary source of inositol and the major storage form phosphorus in agricultural feedstuffs, such as cereals, legumes, and oilseeds. Phytase, myo-inositol hexakisphosphate hydrolase (EC 3.1.3.8 for 3-phytase and 3.1.8.26 for 6-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 bacterial 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 inclusion bodies and then its thermostability 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 comprises 1,329bp and encodes 443 amino-acid residues, was PCR amplified and sequenced. The nucleotide sequence of A. fumigatus phytase gene showed 99% identities to that of A. fumigatus Af293 phytase and 67% homology to that of A. niger phytase. The deduced amino acid sequence of A. fumigatus sp. phytase showed 65% and 41% identities to those of A. terreus A91 phytase and A. oryzae RIP40 phytase, respectively. The phytase of A. fumigatus was predicted to be a novel member of the histidine-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 expression 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 as 45kDa by SDS-polyacrylamide gel electrophoresis.

At last, inclusion body renaturation conditions and partial characterics of recombinant phytase were studied. Ca2+ ion was essential for obtaining active enzyme. Recombinant A. fumigatus phytase with specific activity of 625 UmL-1 was obtained by refolding at pH 5.5 acetate buffer including 1mM Ca2+. Purified phytase exhibited optimal activity at 60oC. The enzyme retained 70% of its original activity after 20 min incubation at 80oC. Optimum pH was 5.5 - 6.0. Recombinant phytase was more stable at acid conditions than neutral and alkaline conditions. It remained fairly stable over pH range of 5.5 to 6.5.

The broad pH optima and high thermostability of the phytase makes it a promising candidate for feed-pelleting application.

 

Key words: A. fumigatus ; phytase gene (PhyA gene); cloning; expression; Inclusion bodies; Refolding

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