Digestive Cellulose Hydrolyzing Enzyme Activity (endo-β1, 4-D-glucanase) in the Gut and Salivary Glands of Blister Beetle, Mylabris pustulata (original) (raw)
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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.
International Biodeterioration & Biodegradation, 2003
The foregut, proximal and distal midgut, and hindgut of free living larvae of a bark runner, the longhorn beetle Rhagium inquisitor L., have been investigated for the presence of enzymes hydrolyzing a number of aryl-glycosides and polysaccharides representative of wood components. Speciÿc activity was highest in the proximal midgut. No hydrolytic activity was found in control tissue, the extra-intestinal fat bodies. Activity was detected with CM-chitin, xylan, phosphoric-acid swollen cellulose, soluble starch, -mannoside, -glucoside, -cellobioside and -arabinoside. The highest activity was found on -1,4-glucan and -fucoside. No hydrolytic activity could be observed with crystalline cellulose. An identical molecular mass of 70 kDa for -glucosidase and cellobiosidase was determined, corresponding to the major protein band. This suggests the absence of a cellobiohydrolase activity and thus a lack of ÿber degradation ability. The endo--glucanase had a molecular mass of 32 kDa and an extremely broad pH-activity. ?
Cellulase Activity and Kinetics in Rice Grasshopper Hieroglyphus banian (Orthoptera: acrididae)
Indian Journal of Science and Technology, 2012
Grasshopper (Hieroglyphus banian), an orthopteran species is considered to be a serious pest of rice plant in India. To study the activity and kinetics of cellulase in grasshopper this experiment is undertaken and accordingly foregut and midgut homogenates were prepared. The cellulase enzyme activity and kinetics was measured by DNSA method of Miller,(1959) by taking different concentrations of crystalline cellulose substrate in spectrophotometer at 540nm. Cellulase activity in foregut and midgut were found to be 0.482 u/mg of tissue and 0.687u/mg of tissue respectively. K m values were determined separately both in foregut and midgut and were found to be 3 mg/dl and 2 mg/dl respectively. Experimental data indicates the presence of high cellulolytic activity in the midgut which may suggest that cellulases of endogenous origin are present in this organism. It can be expected that in future, detailed study of these efficient lignocellulolytic systems will help in identification of novel enzymes possessing features that optimize biotechnological applications for the biofuel industry. Moreover, identification of crucial insect cellulases may help in the development of insecticidal technologies aimed at inhibiting their vital digestive role. Considering that this grasshopper species is a pest of paddy and grass, characterization of insect cellulolytic systems may aid in reducing the grasshopper pest attack in Indian subcontinent.
Prospecting for cellulolytic activity in insect digestive fluids
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2010
Efficient cellulolytic enzymes are needed to degrade recalcitrant plant biomass during ethanol purification and make lignocellulosic biofuels a cost-effective alternative to fossil fuels. Despite the large number of insect species that feed on lignocellulosic material, limited availability of quantitative studies comparing cellulase activity among insect taxa constrains identification of candidate species for more targeted identification of effective cellulolytic systems. We describe quantitative determinations of the cellulolytic activity in gut or head-derived fluids from 68 phytophagous or xylophagous insect species belonging to eight different taxonomic orders. Enzymatic activity was determined for two different substrates, carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), approximating endo-β-1,4-glucanase and complete cellulolytic activity, respectively. Highest CMC gut fluid activities were found in Dictyoptera, Coleoptera, Isoptera, and Orthoptera, while highest MCC gut fluid activities were found in Coleoptera, Hymenoptera, Lepidoptera, and Orthoptera. In most cases, gut fluid activities were greater with CMC compared to MCC substrate, except in Diptera, Hymenoptera, and Lepidoptera. In contrast, cellulolytic activity levels in most head fluids were greater on the MCC substrate. Our data suggests that a phylogenetic relationship may exist for the origin of cellulolytic enzymes in insects, and that cellulase activity levels correlate with taxonomic classification, probably reflecting differences in plant host or feeding strategies.
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.
2015
Aims: The present study aimed at isolating new source of uricase producers from Malaysian hot springs together with partial purification and characterization of thermophilic uricase from novel strain. Methodology and results: A bacteria strain, designated as SN4, was found to have the ability to degrade uric acid. 16S rRNA analysis identified SN4 as Pseudomonas otitidis. Uricase was then extracted from SN4 and purification was performed via ammonium sulphate precipitation. The effects of temperature, pH and metal ions on partially purified uricase were evaluated. Results showed that 70% ammonium sulphate concentration gave the highest uricase activity at 4.18 U/mL compared to other concentrations. The molecular weight of the partially purified uricase was 33 kilodalton (kDa). The optimum temperature for uricase was 45 °C and its activity was highest at pH 8.0. Calcium ions and copper ions enhanced uricase activity while cobalt ions reduced uricase activity. Conclusion, significance and impact of study: Isolation and investigation of uricase producers from new sources such as thermophiles would increase availability and thermal stability of the uricase that could be used for significant purposes such as in biochemical and clinical applications.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2010
Previous screening of head-derived and gut fluid extracts of Carolina grasshoppers, Dissosteira carolina (L.) revealed relatively high activity against cellulase substrates when compared to other insect groups. In this work we report on the characterization and identification of enzymes involved in cellulolytic activity in digestive fluids of D. carolina. In zymograms using carboxymethylcellulose (CMC) as substrate, we detected four distinct cellulolytic protein bands in D. carolina gut fluids, common to all developmental stages. These cellulolytic enzymes were localized to foregut and midgut regions of the D. carolina digestive tract. Cellulases were purified from D. carolina head and gut fluid extracts by liquid chromatography to obtain N-terminal amino acid sequence tags. Database searches with sequence tags from head fluids indicated high similarity with invertebrate, bacterial and plant β1,4-endoglucanases, while no homologues were identified for the gut-derived protein. Our data demonstrate the presence of cellulolytic activity in the digestive system of D. carolina and suggest that cellulases of endogenous origin are present in this organism. Considering that this grasshopper species is a pest of grasses, including switchgrass that has been suggested bioethanol feedstock, characterization of insect cellulolytic systems may aid in developing applications for plant biomass biodegradation for biofuel production.
Canadian Chemical Transactions, 2015
Efficient and low-cost cellulolytic enzymes are urgently needed to degrade recalcitrant plant biomass during the industrial production of biofuels. Nevertheless, Blepharomastix is a serious pest of okra plant as it feeds on the leaves, soft stems, fruit of okra and eventually damage okra production. Therefore, the aim of this study is to characterize the total cellulase and endo-β-1, 4-glucanase (digestive enzymes) present in the larvae of Blepharomastix as it is a popular trend to apply enzymes in biofuels production and application of enzyme inhibitors to protect crops from damaging throughout the world. The result analysis showed that the whole gut from the larvae exhibited the activities of total cellulase and endo-β-1, 4-glucanase enzymes that hydrolyzed crystalline cellulose and carboxymethyle cellulose (CMC) to glucose and the activities in insect were 0.076 µmol min-1 ml-1 and 0.398 µmol min-1 ml-1 respectively. However, the optimum temperature for the activity of total cellulase and endo-β-1, 4-glucanase in Blepharomastix were 45˚C and 50˚C respectively. The pH optima of total cellulase and endo-β-1, 4-glucanase in Blepharomastix was 9.0. The cellulase activity was inhibited by ethylenediaminetetraaceticacid (EDTA), sodium dodecyl sulphate (SDS) and urea, whereas enhanced by NaCl, KCl and MgCl2.
Presence of cellulolytic and xylanolytic activities in the gut fluid of grasshopper Oxya velox
Biotechnologia, 2019
Insect guts, particularly of phytophagous insects, are considered as intriguing bioprospecting sources of cellulase and xylanase due to their use in biofuel industry. In this study, the activities of cellulase and xylanase were identified in the gut fluid of grasshopper, Oxya velox, and characterized. Qualitative assays of gut fluid carried out for endo-β-1,4-D-glucanase (EC 3.2.1.4) and endo-β-1,4-D-xylanase (EC 3.2.1.8) activities, using substrate-agar plate method, revealed clear transparent zones against the red-stained background. When measured by dinitrosali-cylic acid method, the gut fluid had 0.759 ± 0.005 U and 0.303 ± 0.002 U of endoglucanase and endoxylanase activities , respectively, per mg of protein. In the gel zymogram, four distinct cellulolytic protein bands and one xylano-lytic protein band were detected against substrates carboxymethyl cellulose and xylan, respectively. The optimal temperature and pH of both endo-β-1,4-D-glucanase and endo-β-1,4-D-xylanase were 55EC and 5, respectively. Pre-incubation at 70EC for 20 min resulted in almost complete loss of endo-β-1,4-D-glucanase activity as shown by zymography. The presence of both cellulase and xylanase activities suggested that O. velox could be considered as a model for studying the process of lignocellulose digestion in insect gut; the insect could also serve as a good source of enzymes for biofuel production.