Abstract
Background and Objective: Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a partial gene of tannase from Aspergillus niger as an important bioreactor has been cloned and sequenced.
Materials and Methods: Genomic DNA was extracted from Aspergillus niger using a modified version of the method described by Mennium (1977). Degenerated primers were designed against highly conserved amino acid sequences of tannase from Aspergillus awamori, Aspergillus aculeatus and Aspergillus oryzae and used for PCR after determining optimum annealing temperature and magnesium concentration. Southern analysis was carried out with a number of restriction enzymes using DIG labelled tannase probe and a single band between 1.5-6 kb were observed depending on the restricting enzymes.
Results: A PCR product of the predicted size (about 990 bp) were obtained and ligated into pGEMT- Easy vector and cloned into E. coli Top-10F’ (Stratigen) using the standard cloning procedures. Following transformation of competent cells, blue/white selection performed on ampicillin agar plates containing x-gal and IPTG. Plasmid DNA was extracted from transformant cells restricted with EcoR I and digested DNA run out by gel electrophoresis. Three colonies containing the expected insert size for tannase gene(s) were sent for sequencing.
Conclusion: All three nucleotide sequences were identical and shared a high degree of homology to Aspergillus awamori, Aspergillus aculeatus and Aspergillus oryzae tannase sequences (64-52% identity at the nucleotide level). Result of southern blotting indicating probability of single copy of tannase gene in the genome of A. niger.