An endoglucanase from the anaerobic fungus Orpinomyces joyonii: characterization of the gene and its product.
An endoglucanase gene (celA) was isolated from a genomic library of the ruminal fungus Orpinomyces joyonii. DNA sequence analysis of celA revealed an intronless gene encoding a typical signal sequence, an N-terminal catalytic domain, two repeated regions linked by a short Ser/Thr-rich linker and a domain of unknown function. The deduced amino acid sequence of the catalytic domain showed homology with the family 5 cellulases. While the catalytic domain of CelA was not homologous to the catalytic domain of the endoglucanase gene (EG3) from the ruminal bacterium Fibrobacter succinogenes, the repeated regions of CelA were very similar to the noncatalytic domain of EG3. This suggests that evolutionary shuffling of endoglucanase domains might occur among bacteria and fungi within the anaerobic ecosystem of the rumen. The celA gene was expressed in Escherichia coli, and the periplasmic endoglucanase was used for the characterization studies of the enzyme. CelA exhibited both endoglucanase and xylanase activities. Its pH optimum was 4 and the temperature optimum was 40 degrees C. Deletion analysis showed that the repeated sequences and C-terminal domain of CelA were not required for enzyme activity.
Liu
J H
JH
Lethbridge Research Centre, Agriculture and Agri-Food Canada, Canada. jhliu@acs.ucalgary.ca
Selinger
L B
LB
Hu
Y J
YJ
Moloney
M M
MM
Cheng
K J
KJ
Beauchemin
K A
KA
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D000818:Animals; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005658:Fungi / Q000201:enzymology / Q000235:genetics*; D005800:Genes, Fungal; D015698:Genomic Library; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012417:Rumen / Q000382:microbiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005656:Fungal Proteins; C074775:CelA endoglucanase; D002480:Cellulase