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

Abstract

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.

Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase GH61D from the Basidiomycota Fungus Phanerochaete chrysosporium

“…This fungus contains up to nine LPMOs known to be expressed when grown on lignocellulosic substrates (12,59). Because extensive work has been done on other model glycoside hydrolase (65)(66)(67)(68) and dehydrogenase enzymes (69,70) from P. chrysosporium, the P. chrysosporium LPMOs offer an excellent model system to understand the need for multiple oxidative activities to degrade biomass. The PchGH61D structure revealed potentially important residues around the active site and putative binding surface that may impart differences in LPMO specificity and activity.…”

Cellotriose and Cellotetraose as Inducers of the Genes Encoding Cellobiohydrolases in the Basidiomycete Phanerochaete chrysosporium

“…In the case of cel7C, for example, expression was upregulated by 2 h of cultivation in the presence of cellobiose, cellotriose, or cellotetraose, and at that time point, larger amounts of glucose residue had been assimilated in cellotriose or cellotetraose culture than in cellobiose culture. In P. chrysosporium, hydrolysis of cellooligosaccharide may be mainly catalyzed by intracellular BGL (BGL1B) rather than extracellular BGL (BGL3A) (49). The catalytic efficiency (k cat / K m ) of BGL1B is much greater for cellotriose (21 ϫ 10 1 s Ϫ1 mM Ϫ1 ) and cellotetraose (16 ϫ 10 1 s Ϫ1 mM Ϫ1 ) than for cellobiose (75 s Ϫ1 mM Ϫ1 ) (T. Tsukada et al, unpublished data).…”

Volatile Glycosylation in Tea Plants: Sequential Glycosylations for the Biosynthesis of Aroma β-Primeverosides Are Catalyzed by Two Camellia sinensis Glycosyltransferases

“…Calculation of the cLogP (low value indicates high water solubility) revealed that the two sequential glycosylation reactions supporting the conversion of geraniol into geranyl-pri are associated with a stepwise increase in hydrophilicity, from 2.97 to 2.00 (geranyl-glc) and from 2.00 to 0.46 (geranyl-pri). The cLogP value significantly depends on the sugar type at the nonreducing end as well as the sugar number (Tsukada et al, 2006). Furthermore, the existence of exoglycosidases that cleave disaccharide primeverose into Glc and Xyl has not been established in tea plant or other plants.…”