Cloning Of The Tannase Gene From Aspergillus Oryzae And Its Expression In Aspergillus Niger

Tannin acyl hydrolase (tannase) i s an enzyme that hydrolyzes the ester bonds of tann i c ac i d to produce gallic acid and some other compounds. Tannase is widely used in beverage, food, leather, chemical and pharmic industry. It was reported tannase is obtained normally by the fermentat ion of natural Aspergillus strains. There is a little report to improve its production by gene recombination technology. Aspergillus expression system have particular excellence in contrast to E.coli and yeast, such as high secrete capacity, high intensity promoter, perfect post-translation process and mature ferment technics. Nowadays it is extensively applying to expression heterologous protein by using Aspergillus niger expression system in fremdness, but those reports was absent in nation.The gene was cloned from Aspergillus oryzae at first, and then was inserted in the shuttle plasmid ANEP2-SP2. The recombinant plasmid ANEP2-SP2-tan was transformed into Aspergillus higher strain ST31 by protoplast transformation technology. These recombinant strains were induced to express with 1% maltose. Ultraviolet Spectroscopy detected the tannase activity. We get a strain whose have the highest tannase activity up to104.02 U/ml, and average level is 84.53U/ml. It is 2-3 times as original strain.The optimum conditions for preparation of protoplasts from Aspergillus ngier ST31 were investigated too. The result was obtained after Aspergillus niger ST31 was cultured in CM containing 1% CMC. Max imum protoplast yield could be obtained 20% by using 2% cellulase and 2% snailase , stabilizing with NaCl and sucrose and digesting for 2. 5h at 30℃. Furthermore, the configuration of protoplast by microscope was observed. At last, 20-30 transformant /ug piasmid DNA of transformant efficiency was attained by PEG/CaCl₂ methods . It can concluded the high efficiency transformation system of Aspergillus niger was constructed.