論文 - 詳細
| RRC ID | 28669 |
|---|---|
| 著者 | Inoue T, Moriya S, Ohkuma M, Kudo T. |
| タイトル | Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus. |
| ジャーナル | Gene |
| Abstract |
The endo-beta-1,4-glucanase gene was cloned from a cDNA library constructed from the mixed population of symbiotic protists in the hindgut of the lower termite, Coptotermes formosanus, using the lambda ZAP II vector. The recombinant phage library was screened for cellulolytic activity by the Congo red staining procedure. The nucleotide sequence comprised 941 nucleotides including a polyA tail sequence and showed high sequence similarity with endoglucanase genes belonging to glycosyl hydrolase family 5. Determination of the 5' end of the cellulase gene using the 5'RACE method showed that the full-length cDNA comprised a 921-bp ORF, encoding a putative 33,620 Da protein. The organismal source of this cellulase gene was identified using PCR with gene-specific primers and whole-cell in situ hybridization as the smallest symbiotic hypermastigote protist, Spirotrichonympha leidyi. The optimal pH and temperature of the cellulase heterologously expressed in Escherichia coli were 5.8-6.0 and 70 degrees C, respectively. The Km and Vmax values on carboxymethyl cellulose (CMC) substrate were 1.90 mg/ml and 148.2 units/mg protein, respectively. |
| 巻・号 | 349 |
| ページ | 67-75 |
| 公開日 | 2005-4-11 |
| DOI | 10.1016/j.gene.2004.11.048 |
| PII | S0378-1119(04)00724-3 |
| PMID | 15777663 |
| MeSH | Amino Acid Sequence Animals Base Sequence Catalytic Domain Cellulase / analysis Cellulase / chemistry* Cellulase / genetics* Cellulase / isolation & purification Cloning, Molecular* Escherichia coli / genetics Eukaryota / enzymology Eukaryota / genetics* Eukaryota / physiology Gene Library Genes, Protozoan* Hydrogen-Ion Concentration Hydrophobic and Hydrophilic Interactions In Situ Hybridization Intestines / microbiology Isoptera / parasitology* Kinetics Molecular Sequence Data Molecular Weight Open Reading Frames Phylogeny Polymerase Chain Reaction Protein Structure, Tertiary Sequence Homology, Amino Acid Sequence Homology, Nucleic Acid Substrate Specificity Symbiosis* Temperature |
| IF | 2.984 |
| 引用数 | 59 |
| WOS 分野 | GENETICS & HEREDITY |
| リソース情報 | |
| 遺伝子材料 | HmEG1 (clone 03B33) (RDB08982) Cellulase gene of a termite-gut flagellate (RDB08983) PgCBH (clone 03A77) (RDB08984) pET21c-HAT-CFP-eg1 (RDB12217) pET25b-CFP-eg1-His (RDB12218) |