Cloning Of Phytase Gene PhyA From Aspergillus Fumigatus And Its Expression In Pichia Pastoris

Phytase belongs to one of the acid enzymes that hydrolyze phytate, myo-inositol hexakisphosphate, into myo-inositol and inorganic phosphate. That phytase is added into feed can result in the following advantages: increasing the absorptive efficiency of phosphate in animal body, decreasing the phosphate pollution in environment, eliminating the chelateding reaction between phytate and metal ion, amino acid. So the additive, phytase, can improve the efficiency of feed.Most phytates characterized high activity exist in molds. A lot of phytase genes have been successfully cloned and expressed by researchers, which generated industrially in high yield of phytate. In the field of industry, phytase genes integrated into engineering microorganisms were from Aspergillus niger most times for that Aspergillus niger is able to synthesis phytase possess the highest avtivity. But phytase from Aspergillus niger can not endure high temperature and the activity decreases drastically during drying and particle-formation. Phytase from Aspergillus fumigatus, however, can endure high temperature ranged from centigrade 90 to 100 and disassemble phytate in a wide pH value. So it has a potential force on occupying market.In this study, the fragment of phyA about 1.3 kb absent signal sequence and intron was amplified by PCR with primers designed according to published phyA sequences from Aspergillus fumigatus CCTCC AF93024, it was also cloned and sequenced. The plasmid pPlC9-phyA for expression was constructed with the phyA fragment and transformed into Pichia pastoris GS115. Enzyme activity assay and SDS-PAGE of expression product showed that phyA gene was expressed and secreted in recombinant efficiently. Activity of flask ferment supernatant induced for 48 hours achieved 113 u/mL, the product is active under pH 2.0-8.0 while it reaches the highest activity under pH6.0; the best temperature for activity is centigrade 60; it remains 74% activity after 10 minutes under centigrade 90, which means preferably tolerance to high temperature.