Identification, isolation, cloning and sequencing of tannase gene from Aspergillus niger

Abstract

Introduction and objective: Tannin acyl hydrolase (tannase E. C. 3.1.1.20), commonly referred to as tannase, hydrolyses the 'ester' bond (galloyl ester of an alcohol moiety) and the 'depside' bond (galloyl ester of gallic acid) in substrates such as tannic acid, methylgallate and m-digallic acid. This is an inducible enzyme produced by various filamentous fungi. The aim of present research was to isolate, clone and sequence partial gene of tannase from Aspergillus niger. Materials and methods: Aspergillus niger was grown in a selective medium and then genomic DNA was directly extracted from fungal mycelium. Using designed primers, PCR carried out and a 950bp expected band obtained that was ligated into cloning vector and then cloned into Escherichia coli Top-10F'. In order to screen transformed cells, the blue/white bacterial colony selection was carried out and expected the inserted DNA was extracted from putative transformants cells. Results: Extracted DNA was sequenced using ABI automatic system and a 908bp intronlees motif was obtained using BlastX analaysing software that showed 36% identity with tannase precursor of A. oryzae and 29% with Debaryomyces hansenii tannase gene in amino acid level. Conclusion: In conclusion, the identification, isolation, cloning and sequencing of a 908bp partial sequence of tannase gene from A. niger which is considered as an important bioreactor and industrial fungus were reported.