Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris

• Published in: Protein expression and purification Vol. 46; no. 1; pp. 143 - 149
• Main Authors: Chantasingh, Duriya, Pootanakit, Kusol, Champreda, Verawat, Kanokratana, Pattanop, Eurwilaichitr, Lily
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2006
• Subjects: Amino Acid Sequence; Aspergillus - enzymology; Aspergillus terreus; Base Sequence; Cloning, Molecular; Conserved Sequence; DNA Primers; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - isolation & purification; Endo-1,4-beta Xylanases - metabolism; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; Glycosyl hydrolase 10; Kinetics; Molecular Sequence Data; Pichia - enzymology; Pichia pastoris; Pichia pastoris expression; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Xylan; Xylanase; Pichia pastoris expression; Xylan; Glycosyl hydrolase 10; Xylanase; Aspergillus terreus
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2005.09.013

A full-length xylanase gene, encoding 326 amino acids belonging to the fungal glycosyl hydrolase family 10, from Aspergillus terreus BCC129 was cloned and sequenced. Sequence analysis suggested that the first 25 amino acids of this enzyme is the signal peptide. Therefore, only the mature xylanase gene of 906 bp was cloned into a yeast expression vector, pPICZαA, for heterologous expression in Pichia pastoris. A band of approximately, 33 kDa was observed on the SDS–PAGE gel after one day of methanol induction. The expressed enzyme was purified by gel filtration chromatography. The purified recombinant xylanase demonstrated optimal activity at 60 °C, pH 5.0 and a K m of 4.8 ± 0.07 mg/ml and a V max of 757 ± 14.54 μmol/min mg, using birchwood xylan as a substrate. Additionally, the purified enzyme demonstrated broad pH stability from 4 to 10 when incubated at 40 °C for 4 h. It also showed a moderate thermal stability since it retained 90% of its activity when incubated at 50 °C, 30 min, making this enzyme a potential use in the animal feed and paper and pulp industries.