Cellulase Assay Kit (CellG5 Method)

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00:05  Introduction
00:58  Principle
01:51    Reagent Preparation
08:41   Enzyme Extraction
11:22    Procedure
14:14    Calculations

Cellulase Assay Kit CellG5 Method K-CellG5 Schemes
   
Reference code: K-CellG5-4V
SKU: 700004272

Content:

120 / 240 assays (manual) / 480 assays (auto-analyser)

Content: (K-CellG5-4V)
120 / 240 assays (manual) / 480 assays (auto-analyser) or 
(K-CellG5-2V)
60 / 120 assays (manual) / 240 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: endo-Cellulase
Assay Format: Spectrophotometer, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 400
Signal Response: Increase
Limit of Detection: 1.2 x 10-3 U/mL
Reproducibility (%): ~ 3%
Total Assay Time: 10 min
Application examples: Fermentation broths, industrial enzyme preparations and biofuels research.
Method recognition: Novel method

The K-CellG5-2V pack size has been discontinued (read more).

Cellulase Activity Assay Kit.

The CellG5 assay reagent for the measurement of endo-cellulase (endo-1,4-β-glucanase) contains two components; 1) 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside (BPNPG5) and 2) thermostable β-glucosidase. The ketone blocking group prevents any hydrolytic action by the β-glucosidase on BPNPG5.  Incubation with an endo-cellulase generates a non-blocked colourimetric oligosaccharide that is rapidly hydrolysed by the ancillary β-glucosidase. The rate of formation of 4-nitrophenol is therefore directly related to the hydrolysis of BPNPG5 by the endo-cellulase. The reaction is terminated and the phenolate colour is developed on addition of Tris buffer solution (pH 9.0).

The CellG5 assay represents a huge step forward in the methodology for the measurement of cellulase that traditionally relied on substrates such as CM-cellulose, Avicel, cellooligosaccharides, filter paper or dyed polysaccharides including CMC Congo red or cellulose azure.

View Cellulase Activity Assay Protocol.

View our complete list of assay kits for enzyme activities.

Scheme-K-CellG5-2V CellG5 Megazyme

Advantages
  • Very cost effective 
  • All reagents stable for > 4 years 
  • Completely specific for cellulase (endo-1,4-glucanase) 
  • Generally applicable and highly sensitive 
  • Simple format. Well suited to automation 
  • Standard included
Documents
Certificate of Analysis
Safety Data Sheet
Assay Protocol Data Calculator Validation Report
Publications
Megazyme publication

Prediction of potential malt extract and beer filterability using conventional and novel malt assays.

Cornaggia, C., Evans, D. E., Draga, A., Mangan, D. & McCleary, B. V. (2019). Journal of Institute of Brewing, 125(3), 294-309.

Colourimetric assays were used to measure the activities of six key hydrolases endogenous to barley: β‐glucanase, xylanase, cellulase, α-amylase, beta‐amylase and limit dextrinase. The analysed barley malt samples were previously characterised by 27 conventional malt quality descriptors. Correlations between enzymatic activities and brewing parameters such as extract yield, fermentability, viscosity and filterability were investigated. A single extraction protocol for all six hydrolases was optimised and used for multi‐enzyme analysis using fully automatable assay formats. A regression analysis between malt parameters was undertaken to produce a relationship matrix linking enzyme activities and conventional malt quality descriptors. This regression analysis was used to inform a multi‐linear regression approach to create predictive models for extract yield, apparent attenuation limit, viscosity and filterability using the Small‐scale Wort rapId Filtration Test (SWIFT) and two different mashing protocols – Congress and a modified infusion mash at 65oC (MIM 65oC). It was observed that malt enzyme activities displayed significant correlations with the analysed brewing parameters. Both starch hydrolases and cell wall hydrolase activities together with modification parameters (i.e. Kolbach index) were found to be highly correlated with extract yield and apparent attenuation limit. Interestingly, it was observed that xylanase activity in malts was an important predictor for wort viscosity and filterability. It is envisaged that the automatable measurement of enzyme activity could find use in plant breeding progeny selection and for routine assessment of the functional brewing performance of malt batches. This analytical approach would also contribute to brewing process consistency, product quality and reduced processing times.

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Megazyme publication
A novel automatable enzyme-coupled colorimetric assay for endo-1,4-β-glucanase (cellulase).

Mangan, D., Cornaggia, C., McKie, V., Kargelis. T. & V. McCleary, B. V. (2016). Analytical and Bioanalytical Chemistry, 408(15), 4159-4168.

endo-1,4-β-Glucanase (endo-cellulase, EC 3.2.1.4) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In 2014, a novel enzyme-coupled assay that addressed many of the limitations of the existing assay methodology was reported. This involved the use of a bifunctional substrate chemically derived from cellotriose. Reported herein is a much improved version of this assay employing a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside.

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Megazyme publication
Novel substrates for the measurement of endo-1,4-β-glucanase (endo-cellulase).

McCleary, B. V., Mangan, D., Daly, R., Fort, S., Ivory, R. & McCormack, N. (2014). Carbohydrate Research, 385, 9-17.

A specific and sensitive substrate for the assay of endo-1,4-β-glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-β-cellotrioside (BzCNPG3) in the presence of thermostable β-glucosidase. Hydrolysis by exo-acting enzymes such as β-glucosidase and exo-β-glucanase is prevented by the presence of the benzylidene group on the non-reducing end D-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-β-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free D-glucose by the β-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-β-glucanase in general.

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Megazyme publication
Quantitative fluorometric assay for the measurement of endo-1,4-β-glucanase.

Mangan, D., McCleary, B. V., Liadova, A., Ivory, R. & McCormack, N. (2014). Carbohydrate Research, 395, 47-51.

There is a growing demand for research tools to aid the scientific community in the search for improved cellulase enzymes for the biofuel industry. In this work, we describe a novel fluorometric assay for cellulase (endo-1,4-β-glucanase) which is based on the use of 4,6-O-benzylidene-4-methylumbelliferyl-β-cellotrioside (BzMUG3) in the presence of an ancillary β-glucosidase. This assay can be used quantitatively over a reasonable linear range, or qualitatively as a solution screening tool which may find extensive use in the area of metagenomics.

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Publication

Liquid submerged fermentation by selected microbial strains for onion skins valorization and its effects on polyphenols.

Ramires, F. A., Bavaro, A. R., D’Antuono, I., Linsalata, V., D’Amico, L., Baruzzi, F., Pinto, L., Tarantini, A., Garbetta, A., Cardinali, A. & Bleve, G. (2023). World Journal of Microbiology and Biotechnology, 39(10), 258.

Onion skins, actually recycled as organic fertilizers, could be used as a substrate in environmental-friendly bioprocesses to recover high-value bioactive compounds and food ingredients. In this work, a bioprospecting method was carried out including 94 bacterial and 45 yeast strains from several agri-food and environmental niches to verify their ability to grow on onion skins as unique nutrients source. Red and yellow onion skins were assessed by newly selected starter-driven liquid submerged fermentation assays mainly aimed at the release and modification of polyphenols through microbial activities. Fermented onion skins were also investigated as a inexpensive favourable source of microbial enzymes (amylases, proteases, lipases, esterases, cellulases, xylanases). In red onion skins, the treatment with Lactiplantibacillus plantarum TB 11-32 produced a slight increase of bioactive compounds in terms of total phenolics, whereas with the yeast strain Zygosaccharomyces mrakii CL 30−29 the quercetin aglycone content increased of about 25% of the initial raw material. In yellow onion skins inoculated, the highest content of phenolic compounds was detected with the yeast strain Saccharomyces cerevisiae En SC, while quercetin aglycone increased of about 60% of the initial raw material in presence of the bacterial strain L. plantarum C 180-34. In conclusion, the proposed microbial pre-treatment method can be a potential strategy to re-use onion skins as a fermentation substrate, and as a first step in the development of a biorefinery process to produce value-added products from onion by-products.

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Publication

Wood Coloration and Decay Capabilities of Mycoparasite Scytalidium ganodermophthorum.

Van Court, R. C., Rogers, L., Robinson, S. C. & Presley, G. (2023). Journal of Fungi, 9(7), 738.

Scytalidium ganodermophthorum (telomorph: Xylogone ganodermopthora) Kang, Sigler, Lee & Yun is a destructive fungal pathogen that produces a yellow pigment that is used in sustainable product development. Similar pigmenting ascomycetes cause soft rot in woody substrates, however, the decay capabilities of S. ganodermophthorum have not been assessed or related to pigment production. A wood block decay test showed highly variable production of the expected bright yellow pigment and a secondary darker pigment when tested against multiple wood species and nutrient conditions. Microscopic examination showed cell wall erosion typical of type-2 soft rot in wood, although enzymatic analysis did not show detectible levels of endocellulase. Chitinase was detected in plate cultures but not wood cultures, indicating adaption of the fungus to a variety of environmental growth conditions. The high variability of pigmentation in wood cultures suggests that growth of S. ganodermophthorum on liquid media and use of extracted pigment is a superior method for obtaining consistent yellow coloration.

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Publication

Enhanced energy savings in enzymatic refining of hardwood and softwood pulp.

Nagl, M., Haske-Cornelius, O., Bauer, W., Nyanhongo, G. S. & Guebitz, G. M. (2023). Energy, Sustainability and Society, 13(1), 19.

Background: Pulp refining is an energy consuming, but integral part of paper production with the aim to increase tensile strength and smoothness of paper sheets. Commercial enzyme formulations are used to lower the energy requirements by pre-treatment of pulp before refining. However, a high number of different commercial enzyme products are available on the market containing enzymes of varying origin and composition, which complicates the prediction of their behavior, especially using different pulp types. Results: Endoglucanase-rich enzyme formulations were characterized regarding enzyme activity at different temperatures, resulting in a significant decrease of activity above 70°C. Some enzyme preparations additionally contained arabinosidase, xylanase and β-glucosidase activity consequently resulting in a release of xylose and glucose from pulp as determined by high-performance liquid chromatography. Interestingly, one enzyme formulation even showed lytic polysaccharide monooxygenase (LPMO) activity of 3.05 nkat mg−1. A correlation between enzyme activity using the endoglucanase specific derivatized cellopentaose (CellG5) substrate and enzyme performance in laboratory PFI (Papirindustriens forskningsinstitut) refining trials was observed on softwood pulp resulting in a maximum increase in the degree of refining values from 27.7°SR to 32.7°SR. When added to a purified endoglucanase enzyme (31.6°SR), synergistic effects were found for cellobiohydrolase II (34.7°SR) or β-glucosidase enzymes (35.7°SR) in laboratory refining. Comparison with previously obtained laboratory refining results on hardwood pulp allowed differences in enzyme performance based on varying pulp types to be elucidated. Conclusions: Interestingly, the individual enzymes indeed showed different refining effects on softwood and hardwood pulp. This difference could be predicted after development of an adapted enzyme activity assay by combination of the derivatized cellopentaose CellG5 substrate with either softwood or hardwood sulfate pulp.

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Publication

Biocontrol of Phytophthora xcambivora on Castanea sativa: Selection of Local Trichoderma spp. Isolates for the Management of Ink Disease.

Frascella, A., Sarrocco, S., Mello, A., Venice, F., Salvatici, C., Danti, R., Emiliani, G., Barberini, S. & Della Rocca, G. (2022). Forests13(7), 1065.

Ink disease is a devastating disease of chestnut (Castanea sativa) worldwide, caused by Phytophthora species. The only management measures of this disease are chemical and agronomic interventions. This work focuses on the evaluation of the in vitro antagonistic capacity of 20 isolates of Trichoderma spp. selected in a diseased chestnut orchard in Tuscan Apennines (San Godenzo, Italy) for the biocontrol of Phytophthora xcambivora. Each Trichoderma isolate was tested to investigate pathogen inhibition capability by antagonism in dual cultures and antibiosis by secondary metabolites production (diffusible and Volatile Organic Compounds). The six most performing isolates of Trichoderma spp. were further assessed for their aptitude to synthesize chitinase, glucanase and cellulase, and to act as mycoparasite. All six selected isolates displayed the capability to control the pathogen in vitro by synergistically coupling antibiosis and mycoparasitism at different levels regardless of the species they belong to, but rather, in relation to specific features of the single genotypes. In particular, T. hamatum SG18 and T. koningiopsis SG6 displayed the most promising results in pathogen inhibition, thus further investigations are needed to confirm their in vivo efficacy.

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Publication

Towards a better understanding of synergistic enzyme effects during refining of cellulose fibers.

Nagl, M., Haske-Cornelius, O., Bauer, W., Csarman, F., Ludwig, R., Nyanhongo, G. S. & Guebitz, G. M. (2022). Carbohydrate Polymer Technologies and Applications, 4, 100223.

Refining of cellulose fibers is essential for reaching desired paper properties, yet highly energy demanding. Enzymes like endoglucanases (e.g. EndoC) are increasingly used to reduce energy consumption during pulp refining. However, prediction of the enzyme effect is still a major concern, considering the high variety of commercially available enzyme formulations, containing a range of different enzymes. In this study, synergisms of xylanases and β-glucosidases in combination with endoglucanases purified from enzyme formulations were studied and related to their refining performance. Size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) of carboxymethylcellulose revealed that a narrow size distribution and a high reduction in molecular weight are beneficial characteristics for refining. SEC-MALLS of hardwood pulp resulted in pronounced formation of low molecular weight fractions (log MW 4.3) for most efficient refining enzymes. Application of enzyme formulations and combinations of endoglucanase EndoC with β-glucosidase or xylanase using Fourier-transform infrared spectroscopy (FTIR) revealed synergistic effects that promoted degradation of amorphous parts of cellulose. Laboratory refining trials on hardwood pulp confirmed the increase in degree of refining and tensile index after addition of xylanase and β-glucosidase. Surface plasmon resonance (SPR) analysis resulted in strong binding of endoglucanases to regenerated cellulose, which correlated to refining performance.

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Publication

Non-starch polysaccharide degradation in the gastrointestinal tract of broiler chickens fed commercial-type diets supplemented with either a single dose of xylanase, a double dose of xylanase, or a cocktail of non-starch polysaccharide-degrading enzymes.

Morgan, N., Bhuiyan, M. M. & Hopcroft, R. (2022). Poultry Science, 101(6), 101846.

The aim of this study was to examine non-starch polysaccharide (NSP) degradation in the gastrointestinal tract of chickens fed a range of commercial-type diets supplemented with a commercial dose of xylanase, a double dose of xylanase or a cocktail of NSP – degrading enzymes. Cobb 500 broilers (n = 1,080) were fed 12 dietary treatments; 4 diets with differing primary grain sources (barley, corn, sorghum, and wheat) and three different enzyme treatments (commercial recommended dose of xylanase (16,000 BXU/kg), a double dose of xylanase (32,000 BXU/kg) or an NSP-degrading enzyme cocktail (xylanase, β-glucanase, cellulase, pectinase, mannanase, galactanase, and arabinofuranosidase at recommended commercial levels). There were 108 pens, approximately 10 birds per pen, 9 replicates per dietary treatment. The diets were fed as 3 phases, starter (d 0-12), grower (d 12-23), and finisher (d 23-35). On bird age d 12, 23, and 35, performance (total pen body weight, feed intake, and feed conversion ratio corrected for mortality [cFCR]), litter and excreta dry matter content, and ileal and total tract soluble and insoluble NSP degradability and free oligosaccharide digestibility was determined. On d 35, the quantity of NSP in the gizzard, jejunum, ileum and excreta was determined. Results from this study showed that the double xylanase dose and NSP-ase cocktail had positive impacts on starter phase performance in birds fed the corn- and wheat-based diets. In the grower phase in birds fed the barley-based diet, these enzyme treatments improved cFCR and increased litter dry matter content. The NSP-ase cocktail had a negative impact on finisher phase cFCR in birds fed the sorghum-based diet. The double xylanase dose induced a positive impact on NSP degradability and free oligosaccharide digestibility. In conclusion, there appears to be advantages to feeding broilers a double xylanase dose, but lack of consistency when using an NSP-ase cocktail containing many enzymes.

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Publication

Mechanistic investigation of the effect of endoglucanases related to pulp refining.

Nagl, M., Haske-Cornelius, O., Skopek, L., Bausch, F., Pellis, A., Bauer, W., Nyanhongo, G. S. & Guebitz, G. M. (2022). Cellulose, 29(4), 2579-2598.

Endoglucanases are increasingly being touted as the ultimate solution for reducing energy consumption during the refining process in the pulp and paper industry. However, due to the high variety of endoglucanases in different enzyme formulations, these perform heterogeneously when applied to different pulps. In this study, the effect of four endoglucanases on softwood and hardwood pulp was studied using confocal laser scanning microscopy (CLSM) after addition of fluorescently labelled carbohydrate binding modules (CBMs). Nuclear magnetic resonance (NMR) analysis and high-performance liquid chromatography quantification of released oligo- and monosaccharides was performed for in-depth mechanistical investigation. Changes in the crystallinity levels caused by enzymatic degradation of amorphous regions were monitored by incubation with two different CBMs from Caldicellulosiruptor bescii and from Thermobifida fusca with high preference to either amorphous or crystalline regions of cellulose, respectively. When dosed at identical activity on the endoglucanase specific CellG5 substrate, CLSM analysis indicated the highest decrease of amorphous regions for those endoglucanases which were also most active in laboratory refining trials and which released highest amounts of cellooligomers from pulp. Using 13C-NMR analysis, an increase in para-crystalline cellulose caused by enzyme application was observed. Release of reducing sugars was determined at identical CellG5 dosage, indicating a high variance between the enzymes, especially when complex enzyme formulations were used. Scanning electron microscopy images were obtained for visualization of the endoglucanase activity. The results of mechanistical studies indicate that reduction of amorphous moieties of pulp by endoglucanases is especially beneficial for the refining process.

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Publication

The promoted hydrolysis effect of cellulase with ultrasound treatment is reflected on the sonicated rather than native brown rice.

Li, H., Xu, M., Yao, X., Wen, Y., Lu, S., Wang, J. & Sun, B. (2022). Ultrasonics sonochemistry, 83, 105920.

Brown rice is nutritionally superior to polished white rice, as it maintains a large content of external bran that involves a series of bioactive compounds. However, the presence of bran also restricts water diffusion and results in adverse quality of brown rice. In this work, ultrasound conditions were optimized for cellulase to improve its hydrolysis effect on rice bran, and combinations of enzymatic and ultrasound treatment in different manners were conducted on brown rice, to improve the textural attributes. The results showed significant improvements in the catalytic activity and efficiency of cellulase after ultrasonication at the optimal intensity of 1.67 W cm−3 and duration of 30 min, with the conformational variation of cellulase observed from the fluorescence spectra and circular dichroism (CD). Despite the enhanced activity of ultrasonicated cellulase, it leaded to a similar rice surface morphology and a comparable amount of released glucose, and equivalent textural parameters of brown rice treated by native cellulase. However, for the pre-sonicated brown rice, the ultrasonicated cellulase showed a significantly higher hydrolysis capacity than the untreated enzyme, suggesting the important influence of ruptured bran surface on amplifying the hydrolysis effect of cellulase. Compared to the successive ultrasound stimulation on both cellulase and brown rice, ultrasound-assisted cellulase treatment on brown rice produced less glucose from rice bran, but induced similar textural properties of brown rice, possibly resulting from the simultaneously promoting effect of ultrasonication on cellulase and water diffusion. Ultimately, this study highlighted that the mild rice surface rupture is a crucial factor to display the promoted hydrolysis effect of ultrasonicated cellulase on brown rice. Ultrasound-assisted cellulase treatment potentially provides an effective strategy to improve the edible quality of brown rice.

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Rapid Optimisation of Cellulolytic Enzymes Ratios in Saccharomyces Cerevisiae using in Vitro SCRaMbLE.

Kroukamp, H., Wightman, E. L. I., Pretorius, I. S., Paulsen, I. T. & Nevalainen, H. K. M. (2020). Biotechnology for Biofuels, 13(1), 182.

For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes; with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei . S. cerevisiae , transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that BPNPG5 (Megazyme®) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2 respectively (ratio of 1.6:1). Conclusions Here we describe a synthetic biology approach to rapidly optimize gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

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Light-regulated synthesis of extra-and intracellular enzymes related to wood degradation by the white rot fungus Cerrena unicolor during solid-state fermentation on ash sawdust-based medium.

Pawlik, A., Jaszek, M., Sulej, J. & Janusz, G. (2019). Acta Biochimica Polonic, 66 (4), 419-425.

The light-dependent metabolism of the white rot basidiomycete Cerrena unicolor FCL139 has already been demonstrated using transcriptomic and Biolog-based approaches. To further analyze the influence of light on C. unicolor wood degradation, we measured the activity of an array of CAZymes (carbohydrate-active enzymes) and enzymes involved in the redox system of fungal cells associated with lignolysis. Extra- and intracellular enzymatic extracts were obtained from solid-state ash sawdust C. unicolor cultures cultivated for 14 days under red, blue, green, or white light conditions, or in the dark. Light greatly influenced the synthesis of MnP, total cellulases, endo-1,4-β-glucanase, endo-1,4-β-xylanase, catalase, and superoxide dismutase. The production of MnP and catalase was evidently stimulated by white light. It is also worth noticing that blue light caused a gradual increase in the activity of total cellulases throughout the entire period of C. unicolor growth. Moreover, endo-1,4-β-glucanase showed the highest activity on day 13 of fungus cultivation and the production of laccase and β-glucosidase appeared to be the least influenced by light. However, the strongest activity of the endo-1,4-β-xylanase was observed in the dark. It seemed that light not only influenced the regulation of the synthesis of the wood-degrading enzymes at different levels, but also acted indirectly by affecting production of enzymes managing harmful lignin by-products causing oxidative stress. The ability of the fungus to decompose woody plant material is clearly influenced by environmental factors.

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The digestive system in Zygentoma as an insect model for high cellulase activity.

Pothula, R., Shirley, D., Perera, O. P., Klingeman, W. E., Oppert, C., Abdelgaffar, H. M., Johnson, H. M. Y. & Jurat-Fuentes, J. L. (2019). PloS One, 14(2), e0212505.

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 Tdomestica. 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.

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