Identification and expression of cellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp. (BCC4124) in Pichia pastoris.
A gene encoding a cellobiohydrolase (CBHI) was isolated from Fusicoccum sp. (BCC4124), an endophytic fungus belongs in phylum Ascomycota, using 5' and 3' rapid amplification of cDNA end (RACE) technique. This CBHI gene contains 1395 nucleotides and encodes a 465-amino acid protein with a molecular weight of approximately 50 kDa. The deduced amino acid sequence showed significant similarity to those of other fungal CBHI belonging to family 7 of glycosyl hydrolase. Interestingly, the result from the amino acid alignment revealed that this CBHI does not contain the cellulose binding domain nor the linker region. The CBHI gene was successfully expressed in Pichia pastoris KM71. The purified recombinant CBHI has ability to hydrolyze Avicel, filter paper and 4-methylumbelliferyl beta-d-cellobioside (MUC) but not carboxymethylcellulose (CMC). It showed an optimal working condition at 40 degrees C, pH 5 with K(m) and V(max) toward MUC of 0.57 mM and 3.086 nmol/min/mg protein, respectively. The purified enzyme was stable at pH range of 3-11. The enzyme retained approximately 50% of its maximal activity after incubating at 70-90 degrees C for 30 min. Due to its stability through wide range of pH, and moderately stable at high temperature, this enzyme has potential in various biotechnology applications.
Kanokratana
Pattanop
P
Molecular and Enzyme Screening Laboratory, BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand.
Chantasingh
Duriya
D
Champreda
Verawat
V
Tanapongpipat
Sutipa
S
Pootanakit
Kusol
K
Eurwilaichitr
Lily
L
D001203:Ascomycota / Q000201:enzymology*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D005800:Genes, Fungal; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase