Cloning, Production and Characterization of a Glycoside Hydrolase Family 7 Enzyme from the Gut Microbiota of the Termite Coptotermes curvignathus

Abstract

Coptotermes curvignathus is a termite that, owing to its ability to digest living trees, serves as a gold mine for robust industrial enzymes. This unique characteristic reflects the presence of very efficient hydrolytic enzyme systems including cellulases. Transcriptomic analyses of the gut of C. curvignathus revealed that carbohydrate-active enzymes (CAZy) were encoded by 3254 transcripts and that included 69 transcripts encoding glycoside hydrolase family 7 (GHF7) enzymes. Since GHF7 enzymes are useful to the biomass conversion industry, a gene encoding for a GHF7 enzyme (Gh1254) was synthesized, sub-cloned and expressed in the methylotrophic yeast Pichia pastoris. Expressed GH1254 had an apparent molecular mass of 42 kDa, but purification was hampered by its low expression levels in shaken flasks. To obtain more of the enzyme, GH1254 was produced in a bioreactor that resulted in a fourfold increase in crude enzyme levels. The purified enzyme was active towards soluble synthetic substrates such as 4-methylumbelliferyl-β-D-cellobioside, 4-nitrophenyl-β-D-cellobioside and 4-nitrophenyl-β-D-lactoside but was non-hydrolytic towards Avicel or carboxymethyl cellulose. GH1254 catalyzed optimally at 35 °C and maintained 70% of its activity at 25 °C. This enzyme is thus potentially useful in food industries employing low-temperature conditions.

Cellobiohydrolase B ofAspergillus nigerover-expressed inPichia pastorisstimulates hydrolysis of oil palm empty fruit bunches

“…Other reports of CBHs derived from P. pastoris mentioned decreased activity towards insoluble crystalline substrates such as Avicel ® or bacterial microcrystalline cellulose (BMCC) ( Boer, Teeri & Koivula, 2000 ; Bronnenmeier, Rücknagel & Staudenbauer, 1991 ; Kanokratana et al, 2008 ; Woon et al, 2017 ). Most probably, this decreased activity is linked to the high glycan content (both N - and O -linked) of CBHs that potentially perturbs folding of the enzyme, substrate binding and enzyme activity ( Gao et al, 2012 ; Woon et al, 2016b ).…”

Harnessing microbial wealth for lignocellulose biomass valorization through secretomics: a review

“…[ 185 – 187 ]. In addition, several cellulase and hemicellulose-encoding genes derived from the insect gut microbiomes have been heterologously expressed [ 184 , 188 ]. However, integrated metagenomic, metatranscriptomic and metaproteomic analyses are required to analyse the metabolic pathways, enzymes involved in biomass degradation and their regulation at the community level [ 189 – 192 ].…”

Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris

“…Cellulases of flagellate origin have also been identified as members of GHF5, GHF7 and GHF45 from hindgut flagellates of Coptotermes formosanus , C. lacteus , Mastotermes darwiniensis and Reticulitermes speratus [ 7 ]. CBHs are only found in the hindgut of lower termites [ 7 , 16 ], whereas both EGLs and BGLs are found in the midgut and hindgut of lower termites [ 11 ]. In comparison, higher termites, which do not have flagellates in their hindguts, account for over 75% of termite species.…”