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Cloning and characterization of a thermostable and pH-stable cellobiohydrolase from Neocallimastix patriciarum J11.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2013 Aug; Vol. 90 (2), pp. 153-9. Date of Electronic Publication: 2013 Jun 13. - Publication Year :
- 2013
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Abstract
- An 1888-bp cDNA designated celA, isolated from a cDNA library of Neocallimastix patriciarum J11 was cloned. The celA had an open reading frame of 1530 bp encoding J11 CelA of 510 amino acids. The primary structure analysis of J11 CelA revealed a complete cellulose-binding domain at the N-terminal, followed by an Asn, Ala, Gly, Gln and Pro-rich linker and ending with a C-terminal glycosyl hydrolase family 6 catalytic domain. The mature J11 CelA was overexpressed in Escherichia coli and purified to homogeneity. This enzyme had high specific activities towards barley β-glucan and lichenan, low toward carboxymethyl cellulose (CMC), Avicel, and H3PO4-swollen Avicel (PSA). The product of Avicel hydrolysis was cellobiose indicating that J11 CelA is a typical cellobiohydrolase. The recombinant J11 CelA had an optimal pH of 6.0 and was stable over a wide range of pH (5.2-11.3). The enzyme showed an optimal temperature of 50°C and was still maintained approximately 50% of the maximum activity in response to the treatment at 70°C for 1h. Cobalt and Fe(3+) at 1 mM greatly activated the enzyme activity. As a thermostable and pH stable enzyme with crystalline cellulose-degrading activity, J11 CelA is a potential candidate for the bioethanol industry.<br /> (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Carboxymethylcellulose Sodium chemistry
Carboxymethylcellulose Sodium metabolism
Cellulose 1,4-beta-Cellobiosidase metabolism
Cloning, Molecular
DNA, Complementary chemistry
DNA, Complementary metabolism
Enzyme Stability
Escherichia coli genetics
Fungal Proteins chemistry
Fungal Proteins metabolism
Hot Temperature
Hydrogen-Ion Concentration
Molecular Sequence Data
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Cellulose 1,4-beta-Cellobiosidase chemistry
Cellulose 1,4-beta-Cellobiosidase genetics
Fungal Proteins genetics
Neocallimastix enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0279
- Volume :
- 90
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 23770555
- Full Text :
- https://doi.org/10.1016/j.pep.2013.06.004