Cloning of family 11 Xylanase, xynG1 from locally isolated aspergillus fumigatus RT-1 & its expression in escherichia coli

Abstract

Locally isolated fungi are screened for xylanase activity via Remazol Brilliant Blue-xylan agar plate for hydrolysis of 1,4-beta-D-xylosidic linkages and dinitrosalicylic acid (DNS) method for release of xylose. Two of the fungi which showed the highest xylanase activity is identified as Aspergillus fumigatus RT-1, with activity 8.1 U/ml, followed by Aspergillus fumigatus RT-2, with activity 7.8 U/mL. Family 11 xylanase consensus fragments are amplified from genomic DNA by PCR using consensus degenerate primers that exhibited the conserved motifs within fungal family 11 xylanase genes. Two full-length xylanase genes, named xlnA and xynG1 are obtained by PCR and RT-PCR using specific primers designed based on the sequences in the database. The full-length xlnA contained 739 bp, including one intron of 52 bp while the full-length xynG1 contained 712 bp, with one intron of 46 bp. The mature peptide of xynG1 is cloned into pET-21a and pET-22b and expressed in Escherichia coli BL21 (DE3).