title: Cloning and characterization of a thermostable and pH-stable cellobiohydrolase from Neocallimastix patriciarum J11.
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.
authors.affiliation: Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan.
authors.forename: Hui-Chang
authors.initials: HC
authors.lastname: Wang
authors.forename: Yo-Chia
authors.initials: YC
authors.lastname: Chen
authors.forename: Ching-Tsan
authors.initials: CT
authors.lastname: Huang
authors.forename: Ruey-Shyang
authors.initials: RS
authors.lastname: Hseu
mesh_terms: D000595:Amino Acid Sequence; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D020088:Neocallimastix / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase; D002266:Carboxymethylcellulose Sodium
keywords: Anaerobic fungi; Cellobiohydrolase; Cellulase; Neocallimastix patriciarum; Renewable energy