Characterization of an endoglucanase belonging to a new subfamily of glycoside hydrolase family 45 of the basidiomycete Phanerochaete chrysosporium.
The wood decay fungus Phanerochaete chrysosporium has served as a model system for the study of lignocellulose conversions, but aspects of its cellulolytic system remain uncertain. Here, we report identifying the gene that encodes the glycoside hydrolase (GH) family 45 endoglucanase (EG) from the fungus, cloning the cDNA, determining its heterologous expression in the methylotrophic yeast Pichia pastoris, and characterizing the recombinant protein. The cDNA consisted of 718 bp, including an open reading frame encoding a 19-amino-acid signal peptide, a 7-amino-acid presequence at the N-terminal region, and a 180-amino-acid mature protein, which has no cellulose binding domain. Analysis of the amino acid sequence revealed that the protein has a low similarity (<22%) to known fungal EGs belonging to the GH family 45 (EGVs). No conserved domain of this family was found by a BLAST search, suggesting that the protein should be classified into a new subdivision of this GH family. The recombinant protein has hydrolytic activity toward amorphous cellulose, carboxylmethyl cellulose, lichenan, barley beta-glucan, and glucomannan but not xylan. Moreover, a synergistic effect was observed with the recombinant GH family 6 cellobiohydrolase from the same fungus toward amorphous cellulose as a substrate, indicating that the enzyme may act in concert with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.
Igarashi
Kiyohiko
K
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan. amsam@mail.ecc.u-tokyo.ac.jp
Ishida
Takuya
T
Hori
Chiaki
C
Samejima
Masahiro
M
D000595:Amino Acid Sequence; D002480:Cellulase / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000235:genetics*; D008969:Molecular Sequence Data; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013379:Substrate Specificity
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D002480:Cellulase