Publication: Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris
Issued Date
2008-03-01
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ISSN
10465928
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2-s2.0-38549088955
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Mahidol University
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SCOPUS
Bibliographic Citation
Protein Expression and Purification. Vol.58, No.1 (2008), 78-86
Suggested Citation
Benjamaporn Wonganu, Kusol Pootanakit, Katewadee Boonyapakron, Verawat Champreda, Sutipa Tanapongpipat, Lily Eurwilaichitr Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris. Protein Expression and Purification. Vol.58, No.1 (2008), 78-86. doi:10.1016/j.pep.2007.10.022 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/18958
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Title
Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris
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Abstract
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 °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 °C and optimal pH of 5-6. They retained more than 50% activity for 4 h at 70 °C. In addition, high kcatand low apparent Kmof these recombinant proteins indicated good properties of this enzyme against carboxylmethylcellulose. © 2007 Elsevier Inc. All rights reserved.