A novel glucose-tolerant β-glucosidase from the salivary gland of the termite Nasutitermes takasagoensis (original) (raw)
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Applied and Environmental Microbiology, 2012
ABSTRACTTermites are well-known cellulose decomposers and can give researchers insights into how to utilize lignocellulosic biomass in the actual scenario of energy consumption. In this work, an endogenous β-glucosidase from the midgut of the higher termiteNasutitermes takasagoensiswas purified to homogeneity by Ni2+affinity chromatography and its properties were characterized. This β-glucosidase (G1mgNtBG1), which belongs to glycoside hydrolase family 1, is a homotrimer in its native form, with a molecular mass of 169.5 kDa, as demonstrated by gel filtration chromatography. The enzyme displayed maximum activity at pH 5.5 and had broad substrate specificities toward several saccharides, including cellobiose. G1mgNtBG1 showed a relatively high temperature optimum of 65°C and one of the highest levels of glucose tolerance among several β-glucosidases already characterized, with aKiof 600 mM glucose. To examine the applicability of G1mgNtBG1 in biomass conversion, we compared the therm...
Digestive enzymes from workers and soldiers of termite Nasutitermes corniger
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2014
The digestive apparatus of termites may have several biotechnological applications, as well as being a target for pest control. This report discusses the detection of cellulases (endoglucanase, exoglucanase, and β-glucosidase), hemicellulases (β-xylosidase, α-L-arabinofuranosidase, and β-D-xylanase), α-amylase, and proteases (trypsinlike, chymotrypsin-like, and keratinase-type) in gut extracts from Nasutitermes corniger workers and soldiers. Additionally, the effects of pH (3.0-11.0) and temperature (30-100°C) on enzyme activities were evaluated. All enzymes investigated were detected in the gut extracts of worker and soldier termites. Endoglucanase and β-xylanase were the main cellulase and hemicellulase, respectively. Zymography for proteases of worker extracts revealed polypeptides of 22, 30, and 43 kDa that hydrolyzed casein, and assays using protease inhibitors showed that serine proteases were the main proteases in worker and soldier guts. The determined enzyme activities and their response to different pH and temperature values revealed that workers and soldiers contained a distinct digestive apparatus. The ability of these termites to efficiently digest the main components of lignocellulosic materials stimulates the purification of gut enzymes. Further investigation into their biotechnological potential as well as whether the enzymes detected are produced by the termites or by their symbionts is needed.
Insect Biochemistry and Molecular Biology, 2010
This research focused on digestive beta-glucosidases from glycosyl hydrolase family (GHF) 1 from the gut of the lower termite Reticulitermes flavipes. In preceding studies on R. flavipes, we characterized betaglucosidase activity across the gut and its inhibition by carbohydrate-based inhibitors, and subsequently we identified two partial beta-glucosidase cDNA sequences from a host gut cDNA library. Here, we report on the full-length cDNA sequence for one of the R. flavipes beta-glucosidases (RfBGluc-1), the expression of its mRNA in the salivary gland and foregut, the production of recombinant protein using a baculoviruseinsect expression system, optimal recombinant substrate specificity profiles and parameters, and significant inhibition by the established beta-glucosidase inhibitor cellobioimidazole. We also report the partial cDNA sequence for a second gut beta-glucosidase (RfBGluc-2), and show that like RfBGluc-1 its mRNA is localized mainly in the salivary gland. Other results for RfBGluc-1 showing activity against laminaribose, a component of microbial cell walls, suggest that RfBGluc-1 may serve dual functions in cellulose digestion and immunity. These findings provide important information that will enable the testing of hypotheses related to collaborative hostesymbiont lignocellulose digestion, and that contributes to the development of next-generation termiticides and novel biocatalyst cocktails for use in biomass-to-bioethanol applications.
Microbiological research, 2011
β-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying β-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose.
Insect Biochemistry and Molecular Biology, 1997
Two endo-/3-1,4-glucanase components (YEGI and YEG2) of the endogenous cellulase from the Japanese subterranean termite, Reticulitermes speratus, were purified to homogeneity using gel filtration and hydroxylapatite chromatography, and their enzymatic properties were investigated. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), YEGI and YEG2 had Mr of 42 kDa and 41 kDa, respectively. Both components had an optimal pH of 6.0, an optimal temperature of 50°C and were stable at 40°C fop at least 30 min. Both components showed high activity on sodium carboxymethylcelluiose (CMC), 73.6 U/mg protein for YEGI and 83.4 U/mg protein for YEG2. The Km values of YEGI and YEG2 on CMC were 1.83 mg/ml and 1.48 mg/ml, respectively. YEGI did not hydrolyse cellotetraose or ceHotriose, whereas YEG2 hydrolysed cellotetraose to ceUobiose and cellotriose to cellobiose and glucose. Both YEGI and YEG2 hydrolysed cellopentaose to cellotriose and cellobiose. Neither component hydrolysed cellobiose. The hydrolytic products from crystalline cellulose (Sigmacell type 20) by YEGI and YEG2 were cellobiose and a trace amount of glucose. Polyclonal mouse anti-serum raised against YEG2 crossreacted with YEGI, suggesting a common origin for both components. Using this anti-serum, Western blotting and immunohistochemistry showed the presence of YEGI and YEG2 in the salivary glands, but not in the midgut epithelium. The data suggest that the salivary glands are the site of secretion of endo-~-l,4glucanase in R. speratus.
FEMS microbiology letters, 2011
The gut of the termite Reticulitermes santonensis contains an interesting diversity of prokaryotic and eukaryotic microorganisms not found elsewhere. These microorganisms produce many enzyme-digesting lignocellulosic compounds, probably in cooperation with endogenous enzymes. Regarding cellulose and hemicellulose digestion in the termite gut, much remains to be learned about the relative contributions of termite enzymes and enzymes produced by different microorganisms. Here we grew bacterial colonies from termite gut suspensions, identifying 11 of them after PCR amplification of their 16S rRNA genes. After constructing in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained, we performed functional screening for α-amylase, xylanase, β-glucosidase, and endoglucanase activities. This screen revealed a clone producing β-glucosidase activity. Sequence analysis showed that the cloned genomic DNA fragment contained three complete ORFs (bglG, bglF, and bglB) organized in a putative bgl operon. The new β-glucosidase (BglB), identified with its regulators BglG and BglF, belongs to glycoside hydrolase family 1. The new β-glucosidase was expressed in E. coli and purified by affinity chromatography. The purified enzyme shows maximal activity at pH 6.0 and 40 °C. It also displays β-xylosidase activity.