Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9127261 | Gene | 2005 | 9 Pages |
Abstract
The endo-β-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 5â² end of the cellulase gene using the 5â²RACE 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 °C, respectively. The Km and Vmax values on carboxymethyl cellulose (CMC) substrate were 1.90 mg/ml and 148.2 units/mg protein, respectively.
Keywords
CMCGHFIPTGHCAcDNADNA complementary to RNASDS–PAGEsodium dodecyl sulfate–polyacrylamide gel electrophoresisisopropyl β-D-thiogalactopyranosideHydrophobic cluster analysisCellulose degradationBase pair(s)glycosyl hydrolase familyLuria–Bertani (medium)plaque-forming unit(s)polymerase chain reactionPCRpfucDNA librarycarboxymethylcelluloseGlycosyl Hydrolase
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Authors
Tetsushi Inoue, Shigeharu Moriya, Moriya Ohkuma, Toshiaki Kudo,