Cloning and sequence analysis of a novel xylanase gene, Auxyn10A, from Aspergillus usamii

Abstract

A full-length cDNA sequence, encoding a novel endo-1,4-β-D: -xylanase (AuXyn10A) of Aspergillus usamii, was obtained by using rapid amplification of cDNA ends (RACE) methods and cloned into the pUCm-T vector, followed by DNA sequencing. The cDNA gene, designated as Auxyn10A, is 1,235 bp in length harboring 5'- and 3'-non-encoding regions, as well as an ORF of 984 bp that encodes a 19-aa signal peptide, a 6-aa propeptide and a 302-aa mature peptide with a calculated MW of 32,756 Da. The AuXyn10A displays high similarity to the xylanases of Aspergillus niger, Aspergillus kawachii and Aspergillus niger, members of the glycoside hydrolase family 10. Its three-dimensional structure was predicted using http://swiss-model.expasy.org/on-line programs based on the crystal structure of Penicillium simplicissimum xylanase (1B30_A) from the family 10. The complete DNA gene was cloned from the genomic DNA of A. usamii using conventional PCR and hairpin structure-mediated PCR techniques. The DNA gene is 2,255 bp in length, containing a 510 bp of 5'-flanking promoter region and a 1,745 bp of downstream fragment that consists of ten exons and nine short introns ranging from 52 to 62 bp.

Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris

“…In our previous studies, a filamentous fungus of A. usamii E001 strain, closely related to A. niger in taxonomy and physiological properties, was isolated from the soil in China and preserved in our lab. This strain can produce a series of acidophilic glycoside hydrolases such as xylanases, b-mannanases and cellulases by solid-state fermentation, but the cellulolytic activity was much lower [21]. Therefore, enhancing the cellulolytic activity by means of genetic engineering is highly desirable.…”

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S

“…Many kinds of xylanases have been cloned [12][13][14][15][16][17], and expressed in heterologous hosts [13][14][15][16][17][18][19]. Although many xylanase genes have been described from diverse microorganisms, relatively little is known about xylanase gene from Aspergillus niger XZ-3S.…”

Optimized expression, purification and characterization of a family 11 xylanase (AuXyn11A) from Aspergillus usamii E001 in Pichia pastoris

“…Xylanase activity was assayed by measuring the amount of reducing sugars released from dissolved birchwood xylan using the 3,5-dinitrosalicylic acid (DNS) method as reported previously. 2 One unit (U) of xylanase activity was defined as the amount of enzyme liberating 1 µmol L −1 reducing sugar min −1 under the assay conditions (pH 4.6, 50 • C, 15 min). Protein concentration was assayed with a BCA-200 Protein Assay Kit (Pierce, Rockford, IL, USA).…”

“…Xylanases (endo‐ β ‐1,4‐ d ‐xylanases, EC 3.2.1.8) can catalyze the hydrolysis of internal β ‐1,4‐ d ‐xylosidic linkages of xylans, major hemicellulosic constituents in plant cell walls . Based on their primary structure homology alignment and hydrophobic cluster analysis, all xylanases hitherto reported have been classified into glycoside hydrolase families 5, 7, 8, 10, 11, 43 and 62 (http://www.cazy.org/fam/acc_GH.html), with most belonging to families 10 and 11 . Family 11 comprises only ‘true xylanases’, which are catalytically active exclusively on d ‐xylose‐containing substrates and are inactive on cellulosic substrates .…”