Cloning And Overexpression Of Phytase PhyA Gene In Pichia Pastoris

Phytic acid (myo-inositol hexaphosphate) is the main storage form of phosphorus in plant feedstuff. However, monogastric animals, such as swine and poultry, are unable to utilize phytic acid due to the low levels of phytase activity in their digestive tracts.Phytase, myo-inositol hexaphosphate phosphohydrolase, which can be used as feed additive to improve the utility of phosphate in plant feedstuffs and the absorption of nutrients. It also lessens the crisis of pollution caused by phosphorus. Therefore, phytase has wide prospect in the feedstuff industry. Phytase can be found in many plants, animals and microorganisms.However, the yield is very low. Genetically engineered strains are constructed by genetic recombination technology in order to enhance the yield.Researchers also modified the phytase gene to obtain new phytases which are well suited for catalyzing in animals'bodies, such as better thermostability, pH optima, activity and so on.In this paper, phyA gene was cloned from a selected A. niger strain which produces extracellular phytase. And then this gene was inserted into yeast secretive expressional vector pPICZαA and the recombinant plasmid pPICZαA/phyA was transformed into Pichia pastoris by electroporation. The transformants were found to produce extracellular phytase and its characteristics were studied. The recombinant phytase holds great promise for use in the animal feed industry. The following results were achieved: 1. Isolation and identification of phytase-producing strains The different soil samples were isolated on initial screening media containing calcium phytate as the sole phosphorus source. The positive-strain can hydrolyze the calcium phytate immobilized in the agar and produce a clear zone surrounding growing strain. The positive-strain was identified as Aspergillus niger by method of microbiology and named Aspergillus niger N-J.2. The cloning of A. niger N-J phytase phyA gene The genomic DNA of A. niger N-J was obtained by benzyl chloride method and amplified by PCR. The amplified fragment was approximately 1.4 kb. The analysis showed that the ORF comprises 1347 bp, encoding 448 amino acid residues of phytase mature protein. The homology of phyA gene sequence between A. niger N-J and A. niger NRRL3135 (GenBank Accession No.Z16414) was 96.44% and the homology of amino acid sequence was 97.77%. In the amino acid sequence, there 11 potential N-glycosylation sites. It has 11 potential glycosylation site and the conservative motif RHGXRXP (+62~+68).3. The overexpression of A. niger phytase N-J phyA gene The plasmid pMD-T/phyA was double-digested and inserted into yeast secretive expressional vector pPICZαA. After being linearized by Pme I, the recombinant plasmid pPICZαA/phyA was transformed into Pichia pastoris GS115 by electroporation. Several positive strains were obtained after the selections of Zeocin and PCR. Five strains were selected and the excellent strain ZαA-phyA-2 was obtained. After being induced (methanol, 2%) for 96 h, the phytase activity in fermented broth was approximately 242,000 U/mL, 30,000-fold higher than that of original A. niger N-J phytase, with specific activity of 503,000 U/mg of protein. It indicated that the phyA gene was overexpressed in Pichia pastoris.4. Properties of recombinant phytase The analysis of SDS-PAGE showed that its molecular weight was approximately 66~80 kD. For the substrates phytate and pNPP, the values of Km were 0.196 mM and 18.15 mM, respectively; the values of kcat/Km were 2.0×106 M-1s-1and 104.7 M-1s-1, respectively. The optimum pH was 5.5. The recombinant phytase showed high activity in the range of pH 2.5 to 5.5. The optimal temperature at pH 5.5 was 55℃when the reaction time was 10 min. After exposure to 90℃for 15 min, the recombinant phytase can retain 68.8% of its activity, which was 13% higher than the best-known commercial phytase additive NatuphosR. The assay of fluorescence emission spectra showed that red-shift of recombinant phytase was 4 nm after exposure to 90℃for 60 min, while, the red-shift of completely denatured phytase treated by 6 M urea was 8 nm. The results indicated that the recombinant owned stable tertiary structure.