CLONING AND SEQUENCING OF THE GENE ENCODING TANNASE AND A STRUCTURAL STUDY OF THE TANNASE SUBUNIT FROM ASPERGILLUS-ORYZAE

We cloned the Aspergillus oryzae tannase gene using three oligodeoxyri bonucleotide (oligo) probes synthesized according to the tannase N-ter minal and an internal amino acid (aa) sequence. The nucleotide (nt) se quence of the tannase gene was determined and compared with that of a tannase DNA complementary to RNA (cDNA) by means of reverse transcript ase PCR. The results indicated that there was no intron in the tannase gene and that it coded for 588 aa with a molecular weight of about 64 000. The tannase low-producing strain A. oryzae AO1 was transformed w ith the plasmid pT1 which contained the tannase gene, and tannase acti vities of the transformants increased in proportion to the number of c opies. Tannase consisted of two kinds of subunits, linked by a disulfi de bond(s) with molecular weights of about 30 000 and 33 000, respecti vely. We purified these subunits and determined their N-terminal aa se quences. The large and small subunits of tannase were encoded by the f irst and second halves, respectively. Judging from the above results, the tannase gene product is translated as a single polypeptide that is cleaved by post-translational modification into two tannase subunits linked by a disulfide bond(s). We concluded that native tannase consis ted of four pairs of the two subunits, forming a hetero-octamer with a molecular weight of about 300 000.