Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris.
Endoglucanase is a major cellulolytic enzyme produced by Syncephalastrum racemosum (BCC18080). Preliminary results showed that this endoglucanase is thermotolerant as it retained more than 50% of its activity after incubation at 80 degrees C for an hour. As this property may be of industrial use, we have cloned the full-length BCC18080 endoglucanase gene of 1020 nucleotides. Sequence analysis suggested that it belonged to the glycosyl hydrolase family 45. N-terminal sequencing and analysis by SignalP program suggested that the first 32 amino acid residues encoded the signal peptide. Expression of the recombinant clones with and without its own signal peptide in Pichia pastoris demonstrated that P. pastoris produced active 55 and 30 kDa secreted proteins. N-terminal sequencing suggested that the 55 kDa band was the mature protein while the 30 kDa band was the truncated protein. Glycoprotein analysis showed that the 55 kDa protein was glycosylated; while the smaller protein was not. All recombinant endoglucanases showed optimal temperature of 70 degrees C and optimal pH of 5-6. They retained more than 50% activity for 4h at 70 degrees C. In addition, high k(cat) and low apparent K(m) of these recombinant proteins indicated good properties of this enzyme against carboxylmethylcellulose.
Wonganu
Benjamaporn
B
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhorn-Pathom 73170, Thailand.
Pootanakit
Kusol
K
Boonyapakron
Katewadee
K
Champreda
Verawat
V
Tanapongpipat
Sutipa
S
Eurwilaichitr
Lily
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D008969:Molecular Sequence Data; D009090:Mucorales / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D002480:Cellulase