Identification of new GH 10 and GH 11 xylanase genes from Aspergillus versicolor MKU3 by genome-walking PCR

Abstract

Xylanases randomly clear the backbone of xylans, which are hemicelluloses representing a considerable source of fixed carbon in nature. Consequently, these enzymes have important industrial applications. To characterize the genes responsible for producing these enzymes, we cloned xylanase genes belonging to the GH11 and GH10 families from Aspergillus versicolor MKU3 using a 2-step polymerase chain reaction (PCR) protocol involving degenerate PCR and genome-walking PCR (GWPCR). We amplified a family 10 xylanase consensus fragment using degenerate PCR primers exhibiting specificity for conserved motifs within fungal family 10 xylanase genes. We identified a single family 10 xylanase gene (xynv10) and determined its entire gene sequence during the second step of GWPCR, which was used to amplify genomic DNA fragments upstream and downstream of xynv10. The xynv10 sequence contains a 1,378-bp open reading frame separated by 8 introns with an average size of 49 bp. We also amplified a partial GH11 xylanase gene sequence (xynv11) using degenerate PCR and genome-walking methods. Amplification of the C-terminal region of xynv11 using a degenerate primer designed from sequences revealed strong homology with the partial GH11 xylanase gene of A. versicolor MKU3. The structural region in xynv11 was approximately 680 bp and has one intron that is approximately 64 bp in length. Further expression and characterization of these genes will give better understanding of the role of these genes in xylan degradation by A. versicolor.