Cloning, Expression And Characterization Of Phytase AppA Gene

Phytate is the major storage form of phosphate in phytophagous feed and accounts for over 80% of the total phosphorus in legumes and cereals, and it's one of the indispensable and important elements for growth and development in animal and plant. Phytases belong to phosphatase family, which can hydrolyze phytic acid releasing phosphorus, exsisting in plant and microorganism widely, and it was well known as feed additive in monogastric animals all over the world. Monogastric animals use phytate poorly or not at all, because they lack the digestive enzyme that hydrolyzes phytate and don't release inorganic phosphorus. Phytate can decrease the nutritional factors'value by chelating them forming complexes. So, we must add those factors to feed for meeting monogastric animals'needs, what would result in high cost and environmental pollution, but those problems can be alleviated by adding phytase to animal feed.AppA phytase from E.coli. is a new phytase. There is high adaptability and ability of hydrolyzing phytate in acid environment of monogastric animals'gastrointestinal tract. What's more, the activity of appA phytase is the strongest, up to now.This research get a high activity E. coli. strain by vitamin C and molybdenum blue method from pig manure of no feed additive, and then design a pair of primer based on those sequences which have been reported, and get this phytase appA gene. The result of this DNA sequence shows that the homologous sequence between the phytase appA gene and those genes reported have a very high similarity, and the full-length gene consists of an open reading frame of 1233 bp and encodes 411 amino acids. The theoretical molecular weight(MW) and isoelectric point (pI) of this gene expressing product were predicted about 44.821 kDa and 6.09, respectively. And the content of (G+C)% is 54.60%. The structure analysis of amino acid shows that there is a conservative sequence of RHGxRxP and a catalytic centre site of HD, which was consistent with acid phosphatase, and 3 N-Glycosylation sites.This appA gene was cloned into expression vector pPIC9K and transformed into methylotropic yeast Pichia pastoris GS115 for obtaining more phytase, and then induced expression. A gene engineering strain transformant GS115/appA of the strongest phytase appA activity was selected by PCR analysis and phytase activity determination. The result of SDS-PAGE analysis shows a specific band about 55～60 kDa. The purified appA phytase showed a specific activity of 2669.46U/mg with a molecular mass about 57.2 kDa. The phytase characterization exhibits that the optimal pH and temperature for this recombinant phytase was about 3.0 and 55℃, respectively. And the thermostability shows that the activity of appA phytase begin to decrease when the temperature of thermal treatment is up to 60℃, and extending the treatment time would make the phytase activity unstability obvious.