Molecular cloning and characterization of two intracellular β-glucosidases belonging to glycoside hydrolase family 1 from the basidiomycete Phanerochaete chrysosporium

• DOI: 10.1007/s00253-006-0526-z
• Publication Date: 2006-08-08T09:11:45Z
• Authors: Takeshi Tsukada; Kiyohiko Igarashi; Makoto Yoshida; Masahiro Samejima
• Keywords: 0303 health sciences; Cellobiose; DNA, Complementary; Sequence Homology, Amino Acid; Reverse Transcriptase Polymerase Chain Reaction; beta-Glucosidase; Molecular Sequence Data; RNA, Fungal; Sequence Analysis, DNA; Phanerochaete; Recombinant Proteins; Protein Structure, Tertiary; Kinetics; Open Reading Frames; 03 medical and health sciences; Gene Expression Regulation, Fungal; Amino Acid Sequence; Glycosides; RNA, Messenger; Cloning, Molecular; DNA, Fungal

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.