Cloning and expression of a family 10 xylanase gene (Aoxyn10) from Aspergillus oryzae in Pichia pastoris.
A full-length cDNA sequence of Aoxyn10, a gene encoding a glycoside hydrolase (GH) family 10 xylanase from Aspergillus oryzae, was amplified from the total RNA by 3' and 5' rapid amplification of cDNA ends. The cDNA sequence is 1,689 bp, containing 5', 3' untranslated regions and a 1,422 bp open reading frame (ORF) that encodes a 21-aa signal peptide and a 452-aa mature peptide (designated AoXyn10). Multi-alignment revealed that AoXyn10 contains two regions: a catalytic domain (CD) and a family 1 carbohydrate-binding module (CBM1). The three-dimensional (3-D) structure of the CD was predicted by multiple template-based homology modeling. A 2,308-bp complete DNA sequence of Aoxyn10 was obtained from the genomic DNA by both pUCm-T vector-mediated and conventional PCRs, harboring 5', 3' flanking regulatory regions, five exons and four introns. Moreover, Aoxyn10 was extracellularly expressed in Pichia pastoris. One transformant labeled as P. pastoris GS/Xyn4-11 was selected, expressing the highest recombinant AoXyn10 (named reAoXyn10) activity of 45.0 U/ml. SDS-PAGE analysis revealed that reAoXyn10, a glycoprotein with an apparent molecular weight (M.W.) of about 56.0 kDa, was secreted into the cultured medium. The purified reAoXyn10 displayed the maximum activity at pH 5.5 and 60°C. It was stable at a pH range of 4.0-7.0, and at 50°C or below. Its activity was not affected by an array of metal ions or EDTA, but was inhibited by Mn(2+) and Ba(2+). The Km and Vmax of reAoXyn10 were 1.7 mg/ml and 817 μmol/min/mg, respectively.
School of Biotechnology and Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, Jiangnan University.
Xin
X
Yin
Yan-Yan
YY
Gong
Jun-Qing
JQ
Wang
Cun-Duo
CD
Tang
Min-Chen
MC
Wu
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
D018076:DNA, Complementary; D005656:Fungal Proteins; D006023:Glycoproteins; D021382:Protein Sorting Signals; D043364:Endo-1,4-beta Xylanases