Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris.
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, pPICZalphaA, 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 degrees C, pH 5.0 and a Km of 4.8 +/- 0.07 mg/ml and a Vmax of 757 +/- 14.54 micromol/min mg, using birchwood xylan as a substrate. Additionally, the purified enzyme demonstrated broad pH stability from 4 to 10 when incubated at 40 degrees C for 4 h. It also showed a moderate thermal stability since it retained 90% of its activity when incubated at 50 degrees C, 30 min, making this enzyme a potential use in the animal feed and paper and pulp industries.
Chantasingh
Duriya
D
BIOTEC Central Research Unit, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani, 12120, Thailand.
Pootanakit
Kusol
K
Champreda
Verawat
V
Kanokratana
Pattanop
P
Eurwilaichitr
Lily
L
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases