Site of secretion and properties of endogenous endo-β-1,4-glucanase components from Reticulitermes speratus (Kolbe), a Japanese subterranean termite (original) (raw)
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Applied Biochemistry and Biotechnology, 2010
RsSymEG, an endoglucanase of glycosyl hydrolase family (GHF) 7 encoded by a transcript isolated from the symbiotic protist of the termite Reticulitermes speratus, is expressed in Aspergillus oryzae. Interestingly, purified RsSymEG1 has a relatively higher specific activity (603 μmol min−1 mg−1 protein) and V max value (769.6 unit/mg protein) than previously reported data for GHF7 endoglucanase of Trichoderma ressei. It also has the same K m value (1.97 mg/ml) with Clostridium cellulolyticum enzymes that contain cellulose binding module, a property indicative of high affinity to substrate, though no cellulose binding module is found within it. Thin-layer chromatography analysis revealed that RsSymEG1 preferentially hydrolyzes the β-1,4-cellulosic linkage of cellodextrins into cellobiose and glucose.
Throughout the history of studies on cellulose digestion in termites, carboxymethyl-cellulose has been preferably used as a substrate for measuring cellulase activity in termites due to its high solubility. However, carboxymethyl-cellulose degradation is not directly related to digestibility of naturally occurring cellulose because many noncellulolytic organisms can also hydrolyse carboxymethyl-cellulose. To address this issue, a comparative study of microcrystalline cellulose digestion is performed in diverse xylophagous termites, using gut homogenates. For those termites harbouring gut flagellates, the majority of crystalline cellulose appears to be digested in the hindgut, both in the supernatant and the pellet. For Nasutitermes takasagoensis, a termite free of gut flagellates, crystalline cellulose is degraded primarily in the midgut supernatant, and partially in the pellet of the hindgut. The fungus-growing termite Odontotermes formosanus, which also does not possess intestinal flagellates, shows only a trace of crystalline cellulose hydrolysis throughout the gut. Comparison of levels of activity against crystalline cellulose with previously reported levels of activity against carboxymethyl-cellulose in the gut of each termite reveals significant differences between these activities. The results suggest that the hindgut flagellates produce commonly cellobiohydrolases in addition to endo-b-1,4-glucanases, which presumably act synergistically to digest cellulose. Preliminary evidence for the involvement of bacteria in the cellulose digestion of N. takasagoensis is also found.
Tropical Life Sciences Research, 2021
Insects of the taxonomic order Coleoptera are recognised for considerable cellulolytic activity in their digestive fluid. The cellulolytic activity of the gut fluid in Hoplasoma unicolor, a member of Coleoptera, however, remains unexplored. In this study, we, for the first time, report the qualitative and quantitative analysis of cellulolytic activity in the digestive fluid of this insect. The cellulolytic endo-1,4-β-D-glucanase activity was confirmed in the supernatant of the insect’s digestive fluid by agar plate assay using carboxymethyl cellulose as the substrate. To determine the optimum pH, enzyme activity was further assessed in an acidic pH range of 5 to 6, and the highest activity was observed at pH 5.3. For quantitative analysis, endoglucanase activity was measured using 3,5-dinitrosalicylic acid method which revealed that the specific activity of the gut sample was 0.69 (±0.01) units per mg of protein. For further characterisation of the cellulases in the sample, SDS-PAGE and zymogram analysis were carried out. Two active cellulolytic bands were detected on the zymogram suggesting the presence of two distinct endoglucanases which completely disappeared upon heating the sample at 55°C. Our study, therefore, highlights prospect of the gut fluid of H. unicolor as an important source of cellulase enzymes that merits further investigations into their extensive characterisation for potential industrial applications.
Marine Biology, 2004
The soluble fraction of crude tissue homogenates prepared from intact specimens of the shipworm Lyrodus pedicellatus Quatrefages was observed to increase the reducing sugar content of solutions of carboxymethylcellulose (CMC). The predominant CMCase activity detected in these lysates by zymography corresponds to a polypeptide of approximately 40 kDa. This protein, designated Lp-egl-1, was purified and characterized. Experiments described in this study were performed in 1997 using L. pedicellatus from a culture maintained at the Woods Hole Oceanographic Institution. Purified Lp-egl-1 increases reducing sugar content of solutions of CMC and suspensions of phosphoric acid-swollen cellulose, decreases viscosity of CMC solutions, and cleaves the oligosaccharides cellopentaose and cellotetraose internally to release cellotriose and cellobiose. It has no detectable activity against microcrystalline cellulose, cellotriose, cellobiose, or p-nitrophenyl b-D-glucopyranoside and does not release glucose from any tested substrate. These results are consistent with the conclusion that Lp-egl-1 is an endo-1, 4-b-D glucanase (E.C. 3.2.1.4). The K m value of Lp-egl-1 for CMC is 20 mg ml )1 . The enzymatic activity is inhibited by cellobiose and cellotriose, but not by glucose. Lp-egl-1 differs in molecular mass as well as kinetic and immunological properties from a previously described endoglucanase secreted by a cultivable endosymbiont of shipworms, Teredinibacter turnerae Distel et al., 2002. It has not been determined whether Lp-egl-1 is a product of the L. pedicellatus nuclear genome or of an associated symbiotic microbe.
Pakistan J. Zool, 2011
Mylabris pustulata (blister beetle) was studied for the enzymes involved in hydrolysis of cellulose. Carboxy methyl cellulose hydrolyzing activity (endo-β-1, 4-D-glucanase) was detected in the salivary glands and fore gut, very little activity was present in the hind gut. The multiple forms of the enzyme activity were detected on zymogram after non-denaturing PAGE. One of the fractions was purified by gel filtration and preparative native PAGE. The purified protein appeared as single band on SDS-PAGE with a molecular weight of 150 kilo Dalton. The characteristics of the enzyme showed two optimum pH values, one acidic and one neutral 2.0 and 7. The optimum temperature for endo-β -D-1, 4-glucanase was 50 °C. The enzyme was maximum activity against carboxy methyl cellulose. Km and Vmax of the enzyme was determined as 0.6g/l and 0.3, respectively. To our knowledge this is first report on the digestive cellulose hydrolyzing activity of Mylabris pustulata.
Journal of Animal & Plant …, 2010
A novel polysaccharidase from major soldier of the termite Macrotermes subhyalinus was purified to homogeneity by a three-step procedure consisting of ion-exchange, sizeexclusion and hydrophobic interaction chromatographies in order to elucidate its contribution to the degradation of plant material. The only substrates that were hydrolyzed by the purified enzyme were xylans and carboxymethylcellulose. The specific activities towards carboxymethylcellulose and xylan from Birchwood were respectively 2.00 and 2.75 U/mg of protein. The molecular weight was measured to be 78.90 kDa by gel filtration and 76.95 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme behaved as a monomer. The optimum temperatures of the enzyme were found to be 45°C using carboxymethylcellulose and 60°C using xylan from Birchwood as substrates which pointed out that this enzyme had separate sites for each activity. The pH-activity optimum was pH 5.0 for both substrates. The enzyme was capable of hydrolyzing both beta-1, 4-glucosidic and beta-1, 4-xylosidic bonds in cellulose and xylan respectively. Based on thin-layer chromatographic analysis of the degradation products, the cellulase activity produced cellobiose and cellodextrins from carboxymethylcellulose as the substrate. When xylan from Birchwood was used, end products were xylose, xylobiose and xylodextrins. The catalytic efficiency values for carboxymethylcellulose and xylan from Birchwood were respectively 13.60 and 31.74 U.ml/mg 2. The polysaccharidase purified is an endo-beta-xylanase with endo-beta-glucanase activity. The enzyme catalyzed both hydrolysis and transglycosylation. It appears to be distinct from the other termite and Termitomyces sp cellulases and xylanases so far reported in terms of substrate specificity and low activity values against carboxymethylcellulose and xylans. The role of the purified enzyme in the digestive tract is the hydrolysis of amorphous cellulose and xylans from the plant material.
Insect Biochemistry and Molecular Biology, 2000
Two β-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl β-d-glucoside (NPβGlu) (79% of the total activity in saturated enzyme), cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG (galactosidase site) hydrolyses p-nitrophenyl β-d-galactoside (NPβGal) better than NPβGlu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0.
Insect Biochemistry and Molecular Biology, 2003
Three β-glycosidases, named βGly1, βGly2 and βGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The β-glycosidases prefer β-glucosides to β-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose β-1,3 linkages better than β-1, 4-or β-1,6 linkages. βGly1 and 2 were completely purified, whereas βGly3 was isolated with a contaminant peptide that has no activity upon β-glycosides.
Endogenous cellulase enzymes in the stick insect (Phasmatodea) gut
High cellulase (endo-beta-1,4-glucanase) activity was detected in the anterior midgut of the walking stick (Phasmatodea) Eurycantha calcarata. The enzyme was isolated and analyzed via mass spectrometry. RT-PCR revealed two endoglucanase genes, EcEG1 and EcEG2. Mascot analysis of the purified enzyme confirms it to be the product of gene EcEG1. Homologous cDNAs were also isolated from a distantly related species, Entoria okinawaensis, suggesting a general distribution of cellulase genes in phasmids. Phasmid cellulases showed high homology to endogenously-produced glycoside hydrolase family 9 (GH9) endoglucanases from insects, especially to those of termites, cockroaches, and crickets. The purified E. calcarata enzyme showed clear antigency against an anti-serum for termite GH9 cellulase, which, together with the sequence homology, further suggests an endogenous origin of the enzyme. This discovery suggests a possible nutritive value for cellulose in the leaf-feeding phasmids, unlike in herbivorous Lepidoptera.