Molecular cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus.
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.
Inoue
Tetsushi
T
JST Bio-Recycle Project/Environmental Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. i-tetsu@riken.jp
Moriya
Shigeharu
S
Ohkuma
Moriya
M
Kudo
Toshiaki
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002480:Cellulase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D056890:Eukaryota / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D015723:Gene Library; D017125:Genes, Protozoan; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D017403:In Situ Hybridization; D007422:Intestines / Q000382:microbiology; D020049:Isoptera / Q000469:parasitology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013559:Symbiosis; D013696:Temperature
D002480:Cellulase