Comparison of gene structures and enzymatic properties between two endoglucanases from Humicola grisea.
We have cloned two endoglucanase genes (egl3 and egl4) from a thermophilic fungus, Humicola grisea. The coding region of the egl3 gene was interrupted by an intron of 56-bp, and the deduced amino acid sequence of the egl3 gene was 305 amino acids in length and showed 98.4% identity with Humicola insolens EGV. The coding region of the egl4 gene was also interrupted by an intron of 173-bp, which contains 34 TTC repeated sequence units, and the deduced amino acid sequence of the egl4 gene was 227 amino acids in length and showed 61.5% identity with H. grisea EGL3. The typical hinge and the cellulose-binding domain were observed in the C-terminal region of EGL3, but they were not observed in EGL4. In the 5' upstream region of both genes, there were a TATA box or its similar sequence, CAAT motifs, and 6-bp sites which are identical or similar to the consensus sequence for binding a catabolite repressor CREA in Aspergillus nidulans. The egl3 and the egl4 genes were expressed in Aspergillus oryzae, and the translation products were purified. The fusion protein, EGL4CBD, which consists of a catalytic domain of EGL4 and the C-terminal region of EGL3, was also constructed and produced by A. oryzae, and purified. These enzymes showed relatively high activity toward carboxymethyl cellulose (CMC) and could not hydrolyze p-nitrophenyl-beta-D-glucoside and p-nitrophenyl-beta-D-cellobioside. The positive effect of substituting the C-terminal region of EGL4 with that of EGL3 was observed in the hydrolysis of CMC.
Takashima
S
S
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
Iikura
H
H
Nakamura
A
A
Hidaka
M
M
Masaki
H
H
Uozumi
T
T
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004271:DNA, Fungal; D005800:Genes, Fungal; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid