Molecular cloning and characterization of two intracellular beta-glucosidases belonging to glycoside hydrolase family 1 from the basidiomycete Phanerochaete chrysosporium.
cDNAs encoding two glycoside hydrolase family 1 beta-glucosidases (BGL1A and BGL1B) were cloned from the basidiomycete Phanerochaete chrysosporium, and the substrate specificities of the recombinant enzymes and the expression patterns of the two genes were investigated in relation to cellobiose metabolism by the fungus. The cDNA sequences contained open reading frames of 1,389 base pairs (bp) (bgl1A) and 1,623 bp (bgl1B), encoding 462 and 530 amino acids, respectively. Although high sequence identity (65%) was observed between the deduced amino acid sequences of the two enzymes, an apparent difference was observed at the C-terminal region: BGL1B has a 63-amino acid extension, which has no similarity with any known protein. Both recombinant enzymes expressed in Escherichia coli showed hydrolytic activity towards several beta-glycosidic compounds. However, the substrate recognition patterns of the two enzymes were quite different from each other. In particular, cellobiose was hydrolyzed more effectively by BGL1B than by BGL1A. The expression of the two genes in the fungus was monitored by reverse transcription-PCR, which showed that bgl1A was expressed constitutively in both glucose- and cellobiose-containing culture, whereas bgl1B was expressed in cellobiose culture but was repressed in glucose culture, possibly because of carbon catabolite repression. We conclude that BGL1B contributes to cellobiose metabolism during cellulose degradation by P. chrysosporium.
Tsukada
Takeshi
T
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Igarashi
Kiyohiko
K
Yoshida
Makoto
M
Samejima
Masahiro
M
D000595:Amino Acid Sequence; D002475:Cellobiose / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D006027:Glycosides / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012331:RNA, Fungal / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D001617:beta-Glucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D018076:DNA, Complementary; D004271:DNA, Fungal; D006027:Glycosides; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002475:Cellobiose; D001617:beta-Glucosidase