Cloning Of The Tannase Gene From Aspergillus Oryzae And Its Expression In Escherichia Coli And Pichia Pastoris

Tannin acyl hydrolase (tannase) is an enzyme that hydrolyzes the ester bonds of tannic acid to produce gallic acid and some other compounds. Tannase is widly used in beverage, food, leather and chemical industry. In China, tannase is obtained normally by the fermentation of natural Aspergillus strains. The cell wall structure of this microorganism is very complex and the tannase was fixed tightly between the cell wall and the plasma membrane, which made the extraction and purification of this enzyme very difficult.The gene encoding tannase was amplified from the total DNA of Aspergillus oryzae by PCR and was expressed in E.coli and Pichia pastoris.The gene was insered in the expression plasmid pSE380. The recombinant plasmid pSE380-tan was transformed into E. coli strains DH5a, Top10, BL21 (DE3). The three recombinant strains were induced to express with IPTG. The tannase activity was detected by Ultravillet Spectroscopy and the target protein was found in SDS-PAGE with the correct size of 63KD.The gene was also insered in the plasmid pPIC9k of Pichia pastoris. The Pichia pastoris recombinant strain was induced by methanol addition to the culture medium. The activity of tannase was not detected. The target product was not found by the SDS-PAGE.