Molecular Cloning of Phytase Gene from ASUIA279 and Its Expression in Pichia pastoris System

Phytases catalyze the hydrolysis of phytate (myo-inositol hexakisphosphate), one of the major storage form of phosphate in plants, with subsequent release of myo-inositol, phosphate and phytate-bound minerals. Non-ruminant animals such as chicken, swine and fish can't use the organic phosphorus and minerals from their diet because there is no phytase activity in their digestive tract. Phytate degrading enzyme is added to the animal feed diet to improve phosphorus availability from the dietary phytate and at the same time this lessen the phosphate pollution level in areas of intensive animal production as the phosphate would not be excreted out to the environment. ASUIA279, a bacterial strain isolated from Malaysian soil has potentially shown good phytase activity. In the present work, the gene encoding for phytase has been amplified from the plasmid DNA of recombinant ASUIA279(5) obtained from a previous study (unpublished data) by a polymerase chain reaction (PCR) methodology. The amplified phytase gene was extracted, purified then cloned into the pPICZA plasmid and transformed into Pichia pastorisX-33 strain for enzyme production.ABSTRAK: Fitase pemangkinan hidrolisis fitat (myo-inositol hexakisphosphate), merupakan salah satu cara penyimpanan utama fosfat dalam tumbuhan, dengan pelepasan berturut myo-inositol , fosfat dan galian terikat fitat. Haiwan bukan ruminan seperti ayam, khinzir dan ikan tidak dapat memanfaatkan fosforus organik serta galian yang diperolehi daripada makanan kerana tidak mempunyai aktiviti fitase di dalam saluran pencernaan mereka. Enzim pengecilan fitase dicampurkan ke dalam pemakanan haiwan untuk mempertingkatkan keperolehan fosforus dari fitat diet. Pada masa yang sama ia dapat mengurangkan tahap pencemaran fosfat di kawasan yang terdapat penternakan haiwan secara intensif agar fosfat tidak dikumuhkan ke persekitaran. ASUIA279, satu strain bakteria yang diasingkan daripada tanih di Malaysiamenunjukkan aktiviti fitase yang memberansangkan. Buat masa kini, pengekodan gen fitase telah diperkuatkan dengan plasmid DNA dari ASUIA279(5), rekombinan yang diperolehi daripada kajian terdahulu (data tak diterbitkan) berdasarkan kaedah tindak balas berantai polimerase (polymerase chain reaction (PCR)). Gen fitase yang diperkuatkan, ditulenkan dan kemudian diklonkan menjadi plasmid pPICZαA dan berubah menjadi strain Pichia pastorisX-33 untuk penghasilan enzim.KEYWORDS:  Phytase, animal feed, pollution, polymerase chain reaction (PCR), Pichia pastoris