Characterization of a Neocallimastix patriciarum cellulase cDNA (celA) homologous to Trichoderma reesei cellobiohydrolase II.
The nucleotide sequence of a cellulase cDNA (celA) from the rumen fungus Neocallimastix patriciarum and the primary structure of the protein which it encodes were characterized. The celA cDNA was 1.95 kb long and had an open reading frame of 1,284 bp, which encoded a polypeptide having 428 amino acid residues. A sequence alignment showed that cellulase A (CELA) exhibited substantial homology with family B cellulases (family 6 glycosyl hydrolases), particularly cellobiohydrolase II from the aerobic fungus Trichoderma reesei. In contrast to previously characterized N. patriciarum glycosyl hydrolases, CELA did not exhibit homology with any other rumen microbial cellulases described previously. Primary structure and function studies in which deletion analysis and a sequence comparison with other well-characterized cellulases were used revealed that CELA consisted of a cellulose-binding domain at the N terminus and a catalytic domain at the C terminus. These two domains were separated by an extremely Asn-rich linker. Deletion of the cellulose-binding domain resulted in a marked decrease in the cellulose-binding ability and activity toward crystalline cellulose. When CELA was expressed in Escherichia coli, it was located predominantly in the periplasmic space, indicating that the signal sequence of CELA was functional in E.coli. Enzymatic studies showed that CELA had an optimal pH of 5.0 and an optimal temperature of 40 degrees C. The specific activity of immunoaffinity-purified CELA against Avicel was 9.7 U/mg of protein, and CELA appeared to be a relatively active cellobiohydrolase compared with the specific activities reported for other cellobiohydrolases, such as T. reesei cellobiohydrolases I and II.
Denman
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CSIRO Division of Tropical Crops and Pastures, St. Lucia, Queensland, Australia.
Xue
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GP
Patel
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005658:Fungi / Q000201:enzymology* / Q000235:genetics*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012417:Rumen / Q000382:microbiology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics*
D018076:DNA, Complementary; D004271:DNA, Fungal; C074775:CelA endoglucanase; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase