Cellulase (endo-1,4-β-D-glucanase)
(Aspergillus niger)

Several procedures are available for the measurement of endo-1,4-β-glucanase (EG). Primary methods employ defined oligosaccharides or highly purified polysaccharides and measure the rate of hydrolysis of glycosidic bonds using a reducing-sugar method. However, these primary methods are not suitable for the measurement of EG in crude fermentation broths due to the presence of reducing sugars and other enzymes active on these substrates. In such cases, dyed soluble or insoluble substrates are preferred as they are specific, sensitive, easy to use, and are not affected by other components, such as reducing sugars, in the enzyme preparation.

Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.

Enzymatic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well-defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material (Bamforth, 1982). In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall-degrading enzymes are used to destroy the three-dimensional structure and water-binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate-degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial processes.

Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.

Banana production generates significant amounts of agricultural wastes, being fiber extraction one of the most relevant alternatives for their valorization. This process produces banana’s pseudostem pulp (BPP) as a byproduct, which shows an interesting composition for the biorefinery’s biochemical platform, with high polysaccharides (68%) and low lignin contents. This work deals with the enzymatic hydrolysis (EH) of raw and hydrothermally pre-treated BPP, focusing on the production of oligosaccharides (OS). Raw BPP hydrolysis with cellulase at different dosages rendered only 3.2% OS yields (OSY). Pectinase addition has not affected EH performance. On the other hand, EH of hydrothermally pre-treated BPP at 150°C and 170°C (P150 and P170) allowed to increase OSY up to 28% (P150, 1 FPU of cellulase/g dry biomass, 12 h), being 72% of the solubilized sugars in the form of cello-oligosaccharides. This last condition was subjected to a multi-stage EH strategy without improvements in OSY. An endo-glucanase was also tested, but obtained OSY were lower than cellulase results. Finally, obtained OS demonstrated to stimulate the growth of two Lactobacilli strains. The results show that BPP pre-treated under mild operational conditions is a good candidate for cello-oligosaccharides production by EH using 1 FPU/g DB of cellulase with a simple strategy.

Carbohydrate degradation by microbes plays an important role in global nutrient cycling, human nutrition, and biotechnological applications. Studies that focus on the degradation of complex recalcitrant polysaccharides are challenging because of the insolubility of these substrates as found in their natural contexts. Specifically, current methods to examine carbohydrate-based biomass degradation using bacterial strains or purified enzymes are not compatible with high-throughput screening using complex insoluble materials. In this report, we developed a small 3D printed filter device that fits inside a microplate well that allows for the free movement of bacterial cells, media, and enzymes while containing insoluble biomass. These devices do not interfere with standard microplate readers and can be used for both short- (24-48 h) and long-duration (> 100 h) experiments using complex insoluble substrates. These devices were used to quantitatively screen in a high-throughput manner environmental isolates for their ability to grow using lignocellulose or rice grains as a sole nutrient source. Additionally, we determined that the microplate-based containment devices are compatible with existing enzymatic assays to measure activity against insoluble biomass. Overall, these microplate containment devices provide a platform to study the degradation of complex insoluble materials in a high-throughput manner and have the potential to help uncover ecologically important aspects of bacterial metabolism as well as to accelerate biotechnological innovation.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

In order to develop cost-effective processes for conversion of lignocellulosic biomass, discovery of novel enzymes for enhanced lignocellulose hydrolysis is one of the main scientific and industrial goals. This could be achieved by applying proteomic strategies for identification of proteins secreted by filamentous fungi that are among the most powerful producers of biomass-degrading enzymes. Here a strategy for a comparative study of proteins differentially secreted on media inducing production of biomass-degrading enzymes (e.g., lignocellulosic biomass) and media repressing secretion of those enzymes (e.g., glucose) are presented. The protocols presented include preparation of samples for mass spectrometry and identification of cellulolytic and other carbohydrate-degrading enzymes using bioinformatics.

Mixed-linkage glucan : xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land-plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-D-glucan (MLG), with radioactive oligosaccharides of xyloglucan as acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography, HPLC and gel-permeation chromatography. Equisetum HTG consistently cleaved the MLG at the third consecutive β-( 1→4)-bond following (towards the reducing terminus) a β-( 1→3)-bond. It then formed a β-( 1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its XTH subfamily membership. Using size-homogeneous barley MLG as donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.

Continuous depletion in fossil fuel reserves and their contribution towards greenhouse gas emissions compelled the scientist to explore renewable sources of energy. Abundance of rice straw and its poor utilization is one major research question addressed through the present research work. The microwave assisted chemical treatment for Indian rice straw for bio-ethanol production has not been investigated so far and present study has provided insight in to the area of research. In the present research work, feasibility of microwave assisted alkali, acid and peroxide pretreatment has been investigated for rice straw. Mainly three chemicals NaOH, H₂SO₄ and H₂O₂ have been used. It has been found that the combination of microwave pretreatment with H₂O₂, H₂SO₄ and NaOH enhances the saccharification of rice straw, respectively by removing lignin and hemicelluloses in large quantity. Maximum reducing sugar is found through H₂O₂-microwave pretreatment (1453.64µg/mL). SEM images also confirmed that the surface of the samples treated with microwave assisted H₂O₂ were more ruptured than H₂SO₄ and NaOH. It becomes quite evident from experimental analysis that the enzymatic saccharification of rice straw can be assisted with microwave-chemical pretreatment.

The structure of ethanol precipitated fraction from 1 M KOH extracted flaxseed kernel polysaccharides (KPI-EPF) was studied for better understanding the molecular structures of flaxseed kernel cell wall polysaccharides. Based on methylation/GC–MS, NMR spectroscopy, and MALDI-TOF-MS analysis, the dominate sugar residues of KPI-EPF fraction comprised of (1,4,6)-linked-β-D-glucopyranose (24.1 mol%), terminal α-D-xylopyranose (16.2 mol%), (1,2)-α-D-linked-xylopyranose (10.7 mol%), (1,4)-β-D-linked-glucopyranose (10.7 mol%), and terminal β-D-galactopyranose (8.5 mol%). KPI-EPF was proposed as xyloglucans: The substitution rate of the backbone is 69.3%; R₁ could be T-α-D-Xylp-(1→, or none; R₂ could be T-α-D-Xylp-(1→, T-β-D-Galp-(1 → 2)-α-D-Xylp-(1→, or T-α-L-Araf-(1 → 2)-α-D-Xylp-(1→; R3 could be T-α-D-Xylp-(1→, T-β-D-Galp-(1 → 2)-α-D-Xylp-(1→, T-α-L-Fucp-(1 → 2)-β-D-Galp-(1 → 2)-α-D-Xylp-(1→, or none. The Mw of KPI-EPF was calculated to be 1506 kDa by static light scattering (SLS). The structure-sensitive parameter (ρ) of KPI-EPF was calculated as 1.44, which confirmed the highly branched structure of extracted xyloglucans. This new findings on flaxseed kernel xyloglucans will be helpful for understanding its fermentation properties and potential applications.

Xanthomonas axonopodis pv. Punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28°C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55°C while xylanase had highest activity at 45°C. Heat treatment of enzyme extract at 75°C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.

Biofuels are essentials as they can provide impending substitute for fossil fuels. Rice straw has gained much attention from researchers because of its usability as a potential feedstock for bioethanol production. Pre-treatments are crucial for enzymatic hydrolysis of rice straw. In this study, combination of microwave and hydrogen peroxide (H₂O₂) is employed for the delignification of rice straw. The Response surface methodology is used to optimize the pretreatment conditions with respect to H₂O₂ concentration, microwave power and irradiation time. Under optimum conditions, maximum reducing sugar obtained through microwave assisted H₂O₂ is 1,453.64 µg/mL. The chemical and morphological analysis ascertained that the surface of the samples treated with microwave assisted H₂O₂ was more ruptured and has a significantly high crystalline index (63.64%) than untreated rice straw sample (52.2%). Microwave assisted H₂O₂ pre-treatment disrupted the silicon waxy structure and broken down allether linkages between lignin and carbohydrates and thus, efficiently remove lignin. The present study proves that microwave assisted H₂O₂ is an effective pretreatment technique for the conversion of rice straw into bioethanol production by enhancing enzymatic saccharification.

Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30°C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems.

Heterogeneous enzymatic reactions are used in many industrial processes including pulp and paper, food, and biofuel production. Industrially-relevant optimization of the enzymes used in these processes requires assaying them with insoluble substrates. However, platforms for high throughput heterogeneous assays do not exist thereby severely increasing the cost and time of enzyme optimization, or leading to the use of assays with soluble substrates for convenient, but non-ideal, optimization. We present an innovative approach to perform heterogeneous reactions in a high throughput fashion using droplet microfluidics. Droplets provide a facile platform for heterogeneous reactions as internal recirculation allows rapid mixing of insoluble substrates with soluble enzymes. Moreover, it is easy to generate hundreds or thousands of picoliter droplets in a small footprint chip allowing many parallel reactions. We validate our approach by screening combinations of cellulases with real-world insoluble substrates, and demonstrate that the chip-based screening is in excellent agreement with the conventional screening methods, while offering advantages of throughput, speed and lower reagent consumption. We believe that our approach, while demonstrated for a biofuel application, provides a generic platform for high throughput monitoring of heterogeneous reactions.

Delignification of paddy straw with the white-rot fungus, Trametes hirsuta under solid state fermentation, for enhanced sugar recovery by enzymatic saccharification was studied. T. hirsuta MTCC136 showed high ligninase and low cellulase activities. Solid state fermentation of paddy straw with T. hirsuta enhanced carbohydrate content by 11.1% within 10 days of incubation. Alkali extracts of Trametes pretreated paddy straw showed high absorbance at 205 nm indicating high lignin break down. The amount of value-added lignin recovered from the Trametes pretreated paddy straw was much higher than controls. Enzymatic hydrolysis of the Trametes pretreated paddy straw yielded much higher sugars than controls and yields increased till 120 h of incubation. Saccharification efficiency of the biologically pretreated paddy straw with Accelerase^® 1500 was 52.69% within 72 h and was higher than controls. Thus, the study brings out the delignification potential of T. hirsuta for pretreatment of lignocellulosic substrate and facilitating efficient enzymatic digestibility of cellulose.

Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this “tethered network” model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.

The high cost of lignocellulolytic enzymes is one of the main barriers towards the development of economically competitive biorefineries. Enzyme engineering can be used to significantly increase the production rate as well as specific activity of enzymes. However, the success of enzyme optimization efforts is currently limited by a lack of robust high-throughput (HTP) cellulase screening platforms for insoluble pretreated lignocellulosic substrates. We have developed a cost-effective microplate based HTP enzyme-screening platform for ionic liquid (IL) pretreated lignocellulose. By performing in-situ biomass regeneration in micro-volumes, we can volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for engineering novel IL-tolerant cellulases. Our platform only requires straightforward liquid-handling steps and allows the integration of biomass regeneration, washing, saccharification, and imaging steps in a single microtiter plate. The proposed method can be used to screen individual cellulases as well as to develop novel cellulase cocktails.

Polysaccharides containing fractions were obtained from the seaweed Enteromorpha compressa by sequential extractions with various solvents. Anion exchange chromatography (AEC) of the oxalate extracted fraction (OSP) yielded rhamnoxylogalactoglucuronan, xyloglucan and glucuronan rich sub-fractions. The heteroglycuronan, which is sulfated, has an apparent molecular mass of 50 ± 20 kDa and is soluble in water. Methylation analysis of the native, desulfated and desulfated reduced heteroglycuronan sulfate suggested that this polymer is branched and contains, inter alia, (1 → 4)- and (1 → 2,4)-linked-rhamnopyranosyl, (1 → 4)-linked xylopyranosyl, and (1 → 4)- and terminally linked glucuronosyl residues. Sulfate groups, when present, are located at C-3 of (1 → 4)-linked rhamnose units and C-2 of (1 → 4)-linked xylose units. Degradation of the xyloglucan rich extract (Ec1OH) by endo-(1 → 4)-β-D-glucanase and analysis of the resulting fragments by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) showed that the E. compressa xyloglucan contains Glc₃Xyl₁, Glc₄, Glc₃Xyl₂(SO₃Na)₂, Glc₄Xyl₂(SO₃Na)₂ and Glc₅Xyl₂(SO₃Na)₂ as major oligomeric building sub-units.