title: A GHF7 cellulase from the protist symbiont community of Reticulitermes flavipes enables more efficient lignocellulose processing by host enzymes.
abstract: Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7-3, GHF7-5, and GHF7-6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full-length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus-insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7-3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β-glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell-1), beta-glucosidase (β-glu), and laccase (LacA). GHF7-3 was the only GHF7 to enhance glucose release by Cell-1 and β-glu. Finally, GHF7-3, Cell-1, and β-glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β-glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7-3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases.
authors.affiliation: Department of Entomology, Purdue University, West Lafayette, Indiana.
authors.forename: Amit
authors.initials: A
authors.lastname: Sethi
authors.forename: Elena S
authors.initials: ES
authors.lastname: Kovaleva
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Slack
authors.forename: Susan
authors.initials: S
authors.lastname: Brown
authors.forename: George W
authors.initials: GW
authors.lastname: Buchman
authors.forename: Michael E
authors.initials: ME
authors.lastname: Scharf
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056890:Eukaryota / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism; D041981:Gastrointestinal Tract / Q000201:enzymology / Q000469:parasitology; D015971:Gene Expression Regulation, Enzymologic; D020049:Isoptera / Q000201:enzymology* / Q000469:parasitology*; D008031:Lignin / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D059467:Transcriptome
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; C036909:lignocellulose; D008031:Lignin; D044602:Cellulases
keywords: CAZyme; biomass; cellobiohydrolase; cellulose; endoglucanase; exoglucanase; glucose; protist; symbiont; termite