Construction of alpha-L-arabinofuranosidase cDNA from aspergillus niger by MOE-PCR

Abstract

Alpha-L-arabinofuranosidases (a-L-AFases) (EC 3.2.1.55) are among key enzymes of the hemicellulase system which is tremendously useful in bio-bleaching of paper pulp, bioconversion of lignocelluloses material to fermentative products and improve of animal feed-stock digestibility, a- L-AFases are exo-type enzymes, catalyse the hydrolysis of a-1,2-, a-1,3- and a-1,5-L-arabinofuranosidic bonds in hemicelluloses such as arabinoxylan, L-arabinan and other L-arabinose-containing polysaccharides. a-L-AFases with their synergistic action with other lignocelluloses degrading enzymes are the promising tools in agroindustrial processes. Owing to their industrial importance, a variety of a-L.-AFases have been purified from various sources such as bacteria, fungi and plants. Many genes encoding a-L-AFases have been cloned and sequenced. In this study, the a-L-AFase gene was cloned from Aspergillus niger ATCC Strain No. 120120. The cDNA was successfully amplified using Modified Overlap Extension-PCR (MOEPCR), The a-L-AFase gene carried eight exons interrupted by seven introns and had an open reading frame of 1887-bp coding for a protein of 628 amino acid residues. Based on the Signal-SL prediction, the cDNA sequence predicted that the mature enzyme is preceded by a 25 amino acids signal sequence and the molecular mass was predicted to be 65 245 Da.