Evaluation of Cellulolytic Endo-1,4-β-D-Glucanase Activity in the Digestive Fluid of Adult Phytophagous Beetle Hoplasoma unicolor (original) (raw)
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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.
The digestive system in Zygentoma as an insect model for high cellulase activity
PLOS ONE
The digestive system of selected phytophagous insects has been examined as a potential prospecting resource for identification of novel cellulolytic enzymes with potential industrial applications. In contrast to other model species, however, limited detailed information is available that characterizes cellulolytic activity and systems in basal hexapod groups. As part of a screening effort to identify insects with highly active cellulolytic systems, we have for the first time, identified species of Zygentoma that displayed the highest relative cellulase activity levels when compared to all other tested insect groups under the experimental conditions, including model species for cellulolytic systems such as termite and cockroach species in Rhinotermitidae (formerly Isoptera) and Cryptocercidae (formerly Blattodea). The goal of the present study was to provide a morphohistological characterization of cellulose digestion and to identify highly active cellulase enzymes present in digestive fluids of Zygentoma species. Morphohistological characterization supported no relevant differences in the digestive system of firebrat (Thermobia domestica) and the gray silverfish (Ctenolepisma longicaudata). Quantitative and qualitative cellulase assays identified the foregut as the region with the highest levels of cellulase activity in both T. domestica and C. longicaudata. However, T. domestica was found to have higher endoglucanase, xylanase and pectinase activities compared to C. longicaudata. Using nano liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS/MS) and a custom gut transcriptome we identified cellulolytic enzymes from digestive fluids of T. domestica. Among the identified enzymes we report putative endoglucanases matching to insect or arthropod enzymes and glucan endo-1,6-β-glucosidases matching bacterial enzymes. These findings support combined activities of endogenous and symbiont-derived plant cell wall degrading enzymes in lignocellulose digestion in Zygentoma and advance our understanding of cellulose digestion in a primitive insect group.
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.
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.
International Journal of Biological Macromolecules, 2017
A cellulolytic bacterium was obtained from the digestive tract of Osphranteria coerulescens. The breakdown of woody and cellulosic substances by this insect may be relative in part to its symbiont bacteria. Under optimal cultural conditions the novel isolate produced 5.35 U/ml cellulase after 72 h. The enzyme was purified to 36 fold with a 0.59% yield and showed a specific activity of 9.0 U/mg. It presented its maximum activity at 60 • C and pH 5, while it was stable in a wide range of temperature from 20 to 60 • C and pH from 5 to 10. The purified enzyme had a molecular weight of 42.50 kDa based on SDS-PAGE and zymogram analyses. It demonstrated high ions and solvent stability and its activity was stimulated by Mn 2+ , Na + , DMSO and chloroform. The enzyme could hydrolyze CMC, avicel, cellulose and sawdust. TLC analysis represented the cellobiose as the hydrolytic product of CMC. With regard to endo/exo glucanase activity and wide pH, temperature and solvent stability, it has potential for industrial application.
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.
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.
2011
serious pest and eri silkworm (Philosomia ricini) is a beneficial insect for human kind, feeding on cellulose rich substances. To have a better insight, how these insects are adapted to feed on cellulose rich diet ,an experiment was carried out where cellulase enzyme activity in all the three different insect species was determined both in foregut and midgut homogenates. The cellulase activity was measured by DNSA method using crystalline cellulose substrate. The highest amount of enzyme activity was recorded in both foregut and midgut of cockroach (foregut 18.5±4.29, midgut 125±15.82) followed by eri silkworm ( foregut 0.932±0.035, midgut 1±0.073 ) and termite ( whole gut 0.519±0.044). The higher cellulolytic activity in the gut of cockroach may be the result of its adaptation to almost all kinds of habitat which might have given them a better survival value than any other insects. Additionally, by enhancing the cellulose assimilation rate in silkworm the quality and quantity of er...
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.