Aspergillus Phya Gene Cloning And Expression In Pichia Pastoris Secretion

Phytate (myo-inositol hexophosphate), the major source of inositol and the main storage form of phosphorus in plant seeds, serves as an antinutritional factor in monogastric animals as well as a significant source of phosphorus pollutants in animal manures. Phytase (EC 3.1.3.8), a subfamily of histidine acid phosphatases (HAPs), can hydrolyze phytate to liberate inositol and inorganic phosphorus. Because of its desirable catalytic properties, considerable efforts have been made to produce economical phytase enzymes as feed additives. A number of sources including plants, animals and microorganisms (especially of fungal and bacterial origin) were screened for the ability to secrete phytase. Many phytase genes have been isolated, and some strains containing two phytase genes have been found, such as Aspergillus niger, Escherichia coli and Ceriporia sp.At present, many of the reported phytase genes were isolated by screening DNA libraries or cDNA libraries. A hybridization probe is usually designed either by amplifying a fragment of the target gene or by determining partial amino acid sequence of the protein. However, this method is comparatively tedious and time-consuming. To date, many studies focus on isolating new phytase genes encoding phytases with preferable characteristics, such as high specific activity, excellent thermostability and appropriate pH optima. Thus, a simple and efficient method for cloning phytase genes is of great importance.An effective strategy for cloning the phytase gene (phyA) from different strains was developed using CODEHOP PCR and TAIL-PCR. Based on the conserved regions recognized by ClustalW online, CODEHOP primers were designed and used to amplify a partial sequence of the phytase gene. TAIL-PCR was then employed to clone the 5' and 3' flanking sequences. An essential step of screening E. coli transformants for the CODEHOP PCR product was added to isolate different genes encoding the homologous protein in a specific strain. The phytase genes from Aspergillus niger and Aspergillus oryzae were cloned using this strategy.The coding sequences of the cloned genes were acquired by RT-PCR and then transformed into Pichia pastoris by the method of PEG1000. Methol were added tothe concentration of 1% (v/v) to induce the expression of phyA. The properties of the crude recombinant phytase enzymes were characterized.In the range of pH from 4.5 to 6.0, the recombinant phytases (rPhyAs) showed high activity (>60% of maximum). The optimal temperature for the rPhyAs was approximately 55℃. After heat treatment for 15 minutes at more than 55℃, A. niger PhyA and A. oryzae rPhyA maintained 50% of their initial activities. SDS-PAGE analysis showed that the molecular weight of A. niger rPhyA varied from 70kDa to 90 kDa while A. oryzae rPhyA were approximately 70kDa in size. However, after deglycosylation by endoglycosidase Hf, the molecular weights of the two rPhyAs decreased to approximately 50 kDa, which was in accordance with the calculated molecular size.Sequence analysis and characterization of the recombinant enzymes revealed that they belonged to the family of histidine acid phosphatases.