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A representation of a cellulose chain, with reducing and non-reducing ends indicated. The blue brackets with subscript "n" indicate that the degree of polymerization (DP) of the cellulose chain is longer than a DP of 10.
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Lignocellulose has economic potential as a bio-resource for the production of value-added products (VAPs) and biofuels. The commercialization of biofuels and VAPs requires efficient enzyme cocktail activities that can lower their costs. However, the basis of the synergism between enzymes that compose cellulolytic enzyme cocktails for depolymerizing...
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... is sourced from plant material (wood pulp, cotton, or cereals, such as wheat, sugarcane, and rice bagasse), and is a linear polymer that consists of β-Dglucose molecules linked by glycosidic bonds [1,[4][5][6]. The linear structure of a cellulose chain is directional, as it consists of a reducing-end glucose that contains an anomeric carbon (C1) and a non-reducing-end glucose, consisting of hydrogen and a hydroxyl group on the C4 carbon ( Figure 1). Many cellulose chains bundle via hydrogen bonding to constitute cellulose microfibrils, which consist of crystalline regions intersected by amorphous regions. ...
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A systematic evaluation of microorganism’s potential towards biosynthesis of cellulases from inexpensive lignocellulosic feedstock through appropriate kinetic modelling facilitates understanding, optimization and designing of an effective industrial cellulase enzyme production process. The present study aims to optimize a submerged fungal cultivati...
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... Previous studies claimed that crystalline cellulose is recalcitrant to enzymatic activity, and that it generally inhibits endoglucanase activity (van Dyk & Pletschke, 2012, Mafa et al., 2021. In contrast, para-crystalline are easily degraded by endoglucanases. ...
We have identified a HRP enzyme with microcrystalline cellulose activity, which has not yet been explored. The current study investigated the effect of HRP pretreatment on the microcrystalline cellulose substrates, Avicel and filter paper. SEM findings showed that HRP pretreatment catalysed the para-microcrystalline regions of Avicel, cracking and opening the pores on the surface. On filter paper, HRP removed the para-microcrystalline regions exposing fibres. Crystallinity index (CrI) analysis confirmed that HRP increased the CrI of Avicel from 49 % to 54.19 % and filter paper from 42 % to 47 %. The cellulose crystallite sizes increased from 45 to 47 nm at the 002 lattices in Avicel, suggesting a reduction of crystalline cellulose. In addition, endoglucanase displayed 1.15-fold increased activity on HRP-pretreated Avicel, confirming reduced crystalline cellulose. These findings showed that HRP pretreatment changed the structural and chemical properties of Avicel, i.e., loosening crystalline cellulose to make the substrate accessible to enzymes during hydrolysis. Finally, these findings were supported by rooibos microcrystalline cellulose modification post-HRP pretreatment, resulting in a 95 % yield of soluble sugars at 25 mg enzyme cocktail/g biomass.
... Cellulose is characterised as a linear polymer comprising β-D-glucose molecules connected by glycosidic bonds, predominantly found in plant materials, and serves as the principal constituent of lignocellulose. This cellulolytic feedstock is crucial as a resource for generating valueadded products and holds significant potential for biofuel production (McFarlane et al. 2014;Mafa et al. 2021). ...
Brazilian biomes are important sources for environmental microorganisms, including efficient metabolic machineries, like actinomycetes. These bacteria are known for their abilities to produce many bioactive compounds, including enzymes with multiple industrial applications. The present work aimed to evaluate lignocellulolytic abilities of actinomycetes isolated from soil and rhizosphere samples collected at Caatinga, Atlantic and Amazon Forest. Laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and cellulase were evaluated for their efficiency. These enzymes have an essential role in lignin decomposition, through oxidation of phenolic and non-phenolic compounds, as well as enzymatic hydrolysis of vegetal biomass. In this sense, a total of 173 actinomycetes were investigated. Eleven (11) of them were selected by their enzymatic performance. The actinomycete AC166 displayed some activity in all analysed scenarios in terms of Lac, MnP and LiP activity, while AC171 was selected as the most promising strain, showing the following activities: 29.7 U.L⁻¹ for Lac; 2.5 U.L⁻¹ for LiP and 23 U.L⁻¹ for MnP. Cellulolytic activities were evaluated at two pH conditions, 4.8 and 7.4, obtaining the following results: 25 U.L⁻¹ and 71 U.L⁻¹, respectively. Thermostability (4, 30 and 60 o C) and salinity concentrations (0 to 4 M) and pH variation (2.0 to 9.0) stabilities of the obtained LiP and Lac enzymatic extracts were also verified. The actinomycete strain AC171 displayed an adaptable response in distinct pH and salt profiles, indicating that bacterial LiP was some halophilic type. Additionally, the strain AC149 produced an alkali and extreme halophilic lignin peroxidase, which are promising profiles for their future application under lignocellulosic biomass at bioethanol biorefineries.
... Endoglucanase operates by hydrolyzing cellulose chains internally, whereas exoglucanase degrades cellulose from either the reducing or non-reducing end, yielding cellulose oligosaccharides. These oligosaccharides are subsequently hydrolyzed by β-glucosidase to yield glucose [3]. Serving as the rate-limiting enzyme in cellulose degradation, β-glucosidase mitigates feedback inhibition exerted by cellulose oligosaccharides on both endoglucanase and exoglucanase [4], a pivotal aspect ensuring the efficient utilization of cellulose. ...
As a crucial enzyme for cellulose degradation, β-glucosidase finds extensive applications in food, feed, and bioethanol production; however, its potential is often limited by inadequate thermal stability and glucose tolerance. In this study, a functional gene (lq-bg5) for a GH1 family β-glucosidase was obtained from the metagenomic DNA of a hot spring sediment sample and heterologously expressed in E. coli and the recombinant enzyme was purified and characterized. The optimal temperature and pH of LQ-BG5 were 55 °C and 4.6, respectively. The relative residual activity of LQ-BG5 exceeded 90% at 55 °C for 9 h and 60 °C for 6 h and remained above 100% after incubation at pH 5.0–10.0 for 12 h. More importantly, LQ-BG5 demonstrated exceptional glucose tolerance with more than 40% activity remaining even at high glucose concentrations of 3000 mM. Thus, LQ-BG5 represents a thermophilic β-glucosidase exhibiting excellent thermal stability and remarkable glucose tolerance, making it highly promising for lignocellulose development and utilization.
... Previous studies claimed that crystalline cellulose is recalcitrant to enzymatic activity, and that it generally inhibits endoglucanase activity (van Dyk & Pletschke, 2012, Mafa et al., 2021. In contrast, para-crystalline are easily degraded by endoglucanases. ...
We have identified a HRP enzyme with microcrystalline cellulose activity, which has not yet been explored. The current study investigated the effect of HRP pretreatment on the microcrystalline cellulose substrates, Avicel and filter paper. SEM findings showed that HRP pretreatment catalysed the para-microcrystalline regions of Avicel, cracking and opening the pores on the surface. On filter paper, HRP removed the para-microcrystalline regions exposing fibres. Crystallinity index (CrI) analysis confirmed that HRP increased the CrI of Avicel from 49 % to 54.19 % and filter paper from 42 % to 47 %. The cellulose crystallite sizes increased from 45 to 47 nm at the 002 lattices in Avicel, suggesting a reduction of crystalline cellulose. In addition, endoglucanase displayed 1.15-fold increased activity on HRP-pretreated Avicel, confirming reduced crystalline cellulose. These findings showed that HRP pretreatment changed the structural and chemical properties of Avicel, i.e., loosening crystalline cellulose to make the substrate accessible to enzymes during hydrolysis. Finally, these findings were supported by rooibos microcrystalline cellulose modification post-HRP pretreatment, resulting in a 95 % yield of soluble sugars at 25 mg enzyme cocktail/g biomass.
... Various mechanisms have been proposed to explain the enzyme synergy, including (i) the endo/exo effect, where endocellulases create new, free cellodextrin chain ends for cellobiohydrolases, (ii) the exo/exo effect, synergistic action between exocellulases targeting reducing and non-reducing ends, (iii) the endo-BGL effect, synergistic action between endocellulases and β-glucosidases, (iv) enhancement of upstream enzymes by downstream enzymes, such as the waning of cellobiohydrolase inhibition by β-glucosidase, which cleaves its inhibitor cellobiose into glucose, (v) collaborative action of cellulolytic and hemicellulolytic enzymes, enhancing accessibility to each other's respective target substrates, and (vi) physical modification of substrates, such as the loosening of the crystallized region of cellulose by auxiliary proteins and non-hydrolytic enzymes like expansins and LPMOs [35]. ...
Lignocellulosic cellulose serves as a key source for bioethanol production. The efficient conversion of cellulose relies on three major cellulase components. High cost of cellulases and the need for a single microbe that produces all cellulase components in the right proportion and quantities are a few challenges in bioethanol production from cellulose. This investigation is an attempt in developing an enzyme cocktail involving recombinant thermostable cellulases (rMtEgl, rMtCel6A and rMtBgl3c) of the thermophilic mold Myceliophthora thermophila and testing their applicability in the conversion of agro residues to ethanol for the first time. The most effective pretreatment method for paddy straw and sugarcane bagasse was optimized. Pretreatment of sugarcane bagasse with sodium chlorite and acetic acid resulted in a 5.5-fold increase in total reducing sugar liberation, while, in paddy straw, total reducing sugar release increased by 9-fold, when biomass was treated with NaOH and microwaves compared to untreated biomass. The inclusion of recombinant enzymes in the enzyme cocktail supported 80–90% saccharification of pretreated paddy straw and sugarcane bagasse, which is 2-fold higher than that achieved using commercial enzyme mix alone. The ethanol production levels of 55.8 and 37.0 g/L, with the fermentation efficiencies of 80 and 76%, were attained from the pre-treated paddy straw and sugarcane bagasse hydrolysates, respectively. An appropriate blend of each enzyme component and pretreatment method tailored for the specific biomass is crucial for efficient biofuel production.
... Hemicelluloses can bond cellulose microfibrils together, forming a strong load-bearing network. Expansin (EXP) is thought to disrupt the cellulose-hemicellulose association transiently, allowing slippage or movement of cell wall polymers before the association reforms and the integrity of the cell wall network is re-established (Mafa et al. 2021). EXPs are also implicated in other plant developmental processes where cell wall loosening occurs, such as in fruit softening, organ abscission, seed germination, and pollen tube invasion of the grass stigma (Yennawar et al. 2006). ...
... LPMOs are copper-containing AA enzymes that cleave polysaccharides in an oxidative manner (Forsberg et al. 2014). There are two types of cellulose-active LPMOs; C1-hydroxylating LPMOs (EC 1.14.99.54), which produce cellulose fragments that contain a residue of D-glucono-1,5-lactone at the reducing end, which hydrolyses quickly and spontaneously to aldonic acid, and C4 dehydrogenating LPMO (EC 1.14.99.56), which produce cellulose fragments that contain a residue of 4-dehydro-D-glucose at the nonreducing end (Mafa et al. 2021). C1-hydroxylating LPMOs are found in AA9,10 and 14, while C4-dehydrogenating LPMOs are found in AA9 and 10. ...
Plant cell walls are composed of a heterogeneous mixture of polysaccharides that require several different enzymes to degrade. These enzymes are important for a variety of biotechnological processes, from biofuel production to food processing. Several classical mannanolytic enzyme functions of glycoside hydrolases (GH), such as β-mannanase, β-mannosidase and α-galactosidase activities, are helpful for efficient mannan hydrolysis. In this light, we bring three enzymes into the model of mannan degradation that have received little or no attention. By linking their three-dimensional structures and substrate specificities, we have predicted the interactions and cooperativity of these novel enzymes with classical mannanolytic enzymes for efficient mannan hydrolysis. The novel exo-β-1,4-mannobiohydrolases are indispensable for the production of mannobiose from the terminal ends of mannans, this product being the preferred product for short-chain mannooligosaccharides (MOS)-specific β-mannosidases. Second, the side-chain cleaving enzymes, acetyl mannan esterases (AcME), remove acetyl decorations on mannan that would have hindered backbone cleaving enzymes, while the backbone cleaving enzymes liberate MOS, which are preferred substrates of the debranching and sidechain cleaving enzymes. The nonhydrolytic expansins and swollenins disrupt the crystalline regions of the biomass, improving their accessibility for AcME and GH activities. Finally, lytic polysaccharide monooxygenases have also been implicated in promoting the degradation of lignocellulosic biomass or mannan degradation by classical mannanolytic enzymes, possibly by disrupting adsorbed mannan residues. Modelling effective enzymatic mannan degradation has implications for improving the saccharification of biomass for the synthesis of value-added and upcycling of lignocellulosic wastes.
... Plants can use the emissions of carbon dioxide from the biofuel during photosynthesis resulting in an ecofriendly process known as the carbon cycle (I.P.C.C 2007). The biorefinery sector continues to investigate cheaper methods for producing secondary-generation biofuel and value-added products (VAPs) from agricultural residues, which have the potential to produce simple sugars that can be fermented into ethanol (Olsson and Hahn-Hagerdal 1996;Mafa et al. 2021). However, removing lignin from the biomass via ecofriendly means and using enzyme cocktails to degrade lignocellulose to simple sugars has been a challenge, resulting in a higher price for biofuel than petroleum production (Olsson and Hahn-Hagerdal 1996). ...
... Cellulose and hemicellulose are hydrolysed by glycoside hydrolases (GHs) and debranching enzymes to produce sugars that can be fermented to produce ethanol or VAP (Van Dyk and Pletschke 2012; Malgas et al. 2019;Mafa et al. 2021). However, the presence of lignin can lead to non-specific substrate-binding of holocellulolytic enzymes or inhibits the enzymatic degradation of cellulose and hemicellulose (Malhotra and Suman 2021;Kong et al. 2017). ...
... Thoresen et al. (2021) demonstrated that CBHs and endoglucanases do not always display synergy. As a result, some EG and CBH combinations don't produce higher concentration of soluble sugars, the phenomena is called anti-synergy (Thoresen et al. 2021;Mafa et al. 2021). Therefore, the best EGs combination were used to convert the HRP delignified rooibos biomass. ...
The purpose of the study was to pretreat fermented rooibos biomass with partially purified horseradish peroxidase (HRP) for lignin removal and to convert delignified biomass to soluble sugars through saccharification with a formulated holocel-lulolytic enzyme cocktail (HEC). HRP enzyme was extracted from the horseradish root tissue and was partially purified by membrane filters and characterised biochemically. HRP enzyme was used to pretreat the fermented rooibos biomass to remove lignin before hydrolysing it with the HEC. Our findings indicated that HRP is versatile because it displayed activity on guaiacol, 8-aminoquinoline, and decolourised methylene blue dye. HRP had a pH optimum of 4.5 and displayed a mesophilic temperature range. The kinetics studies indicated that HRP displayed a higher affinity towards guaiacol (K m = 0.082 mg/mL) followed by 8-aminoquinoline (K m = 0.221 mg/mL). However, the catalytic efficiency revealed that the enzyme hydrolysed guaiacol (63436.48 s − 1. mg/mL) and 8-aminoquinoline (59189.81 s − 1. mg/mL) efficiently. HRP pretreatment of rooibos biomass significantly removed lignin content and increased pores on the surface as visualised with SEM. FTIR validated the SEM results by showing reductions at 3324.81, 1615.16 and 1018.75 cm − 1 , corresponding to crystalline cellulose , lignin and holocellulose regions, respectively. HRP pretreated biomass had the lowest crystallinity index of 11.2% compared to 20% of the control. HRP delignified rooibos biomass was hydrolysed effectively by the HEC, which released about 10% yield of soluble sugars compared to 6% of control. We conclude that HRP pretreatment significantly modified the structural and chemical properties of the biomass, making it more accessible to hydrolytic enzymes.
... The plant cell wall is composed of a polymeric network of cellulose micro-fibrils cross-linked by hemicellulose and lignin and embedded in pectin (Cosgrove 2000;Mnich et al. 2020;Silva-Sanzana et al. 2020). Cellulose micro-fibrils consist of β-D-1,4-glucan chains, which are the largest component of the cell wall (Mafa et al. 2021). The intra-and intermolecular hydrogen bonds formed between the parallel or overlapping layers of cellulose fibres lead to microcrystalline cellulose (MCF) formation. ...
... Xyloglucan is the major hemicellulose in dicotyledonous plants, while for most grass species arabinoxylans predominate (Mnich et al. 2020). Moreover, cross-linking of xylan to lignin by ferulic acid restricts CWDEs such as xylanases and cellulases from degrading arabinoxylan and MCF, respectively (Mnich et al. 2020;Mafa et al. 2021). ...
... Cellulases produced by pathogenic fungi are part of CWDEs classified under the GHs, which catalyse the hydrolysis of the β-1,4-glycosidic bond in cellulose (Cosgrove 2016;Silva-Sanzana et al. 2020;Mafa et al. 2021). Cellulases can be divided into three groups according to their mode of hydrolysis and substrate specificity, i.e., endoglucanases (EGs), cellobiohydrolase (CBHs), and β-glucosidases (Mafa et al. 2021). ...
Puccinia triticina ( Pt ) is an important pathogen of wheat. While breeding programmes develop resistant wheat cultivars to mitigate the effects of such rust-causing pathogens, the emergence of new rust races with wider virulence mandates the implementation of other control strategies. Our study investigated whether acidic pH conditions affected selected Carbohydrate-Active Enzymes (CAZymes) in Pt- inoculated Thatcher + Lr9 (IR) wheat compared to those found in the Thatcher (IS) wheat. The β-glucosidase and amyloglucosidase activity levels significantly increased in IR compared to the control from 1 to 14 days post-inoculation (dpi). In contrast, activity levels of invertase did not change in the IR wheat relative to the control at 1 and 7 dpi, but were significantly reduced in the IR plants at 14 dpi. The IS had higher activity of all three hexose-producing enzymes under acidic conditions. These enzyme activities could be increased in the IS to produce hexose sugars required by Pt to develop and advance infection. The phenotypic analysis supported this view because leaf rust disease symptoms were only visible in the IS plants. For cell wall loosening-related enzymes, the IR displayed higher activity of exoglucanase, xylanase and peroxidase enzymes compared to IS. The liquid chromatography–mass spectrometry analysis showed IR had higher concentrations of complex oligosaccharides compared to the IS. Thus, we concluded that the higher exoglucanase, xylanase and peroxidase activity could be involved in cell wall loosening under acidic conditions, while oligosaccharides could be building-blocks for synthesizing cell wall barriers that apprehend Pt growth in inoculated Thatcher + Lr9 .
... In general, the cellulose and hemicellulose in pretreated corn kernel fiber need to be further converted into fermentable sugars (pentoses and hexoses) by enzymatic hydrolysis using cellulases and hemicellulases (xylanases). Currently, acid hydrolysis and enzymatic hydrolysis are the most commonly used strategies (Mafa et al., 2021). Enzymatic hydrolysis was considered a more attractive saccharification process due to its milder reaction conditions, lower requirements for equipment, fewer byproducts, and higher saccharification yield, resulting in less environmental pollution compared with acid hydrolysis (Zabed et al., 2017). ...
Due to the rising demand for green energy, bioethanol has attracted increasing attention from academia and industry. Limited by the bottleneck of bioethanol yield in traditional corn starch dry milling processes, an increasing number of studies focus on fully utilizing all corn ingredients, especially kernel fiber, to further improve the bioethanol yield. This mini-review addresses the technological challenges and opportunities on the way to achieving the efficient conversion of corn fiber. Significant advances during the review period include the detailed characterization of different forms of corn kernel fiber and the development of off-line and in-situ conversion strategies. Lessons from cellulosic ethanol technologies offer new ways to utilize corn fiber in traditional processes. However, the commercialization of corn kernel fiber conversion may be hampered by enzyme cost, conversion efficiency, and overall process economics. Thus, future studies should address these technical limitations.
... Hydrolytic bands of cellulases indicated cellulolytic activity in zymogram corresponding to ~ 25 kDa. Cellulase bands in the range of 24.4-185 kDa have been estimated from SDS-PAGE [61][62][63][64]. A similar molecular weight of 25 kDa has been reported in Bacillus licheniformis SVD1 [65], Bacillus subtilis MA139 [66], Penicillium verruculosum [67], and Novosphingobium sp. ...
The characterization of bacteria with hydrolytic potential significantly contributes to the industries. Six cellulose-degrading bacteria were isolated from mixture soil samples collected at Kingfisher Lake and the University of Manitoba campus by Congo red method using carboxymethyl cellulose agar medium and identified as Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Their cellulase production was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, substrate concentration, nitrogen, and carbon sources using the dinitrosalicylic acid and response surface methods. Except for Paenarthrobacter sp. MKAL1, all strains are motile. Only Bacillus sp. MKAL6 was non-salt-tolerant and showed gelatinase activity. Sucrose enhanced higher cellulase activity of 78.87 ± 4.71 to 190.30 ± 6.42 U/mL in these strains at their optimum pH (5-6) and temperature (35-40 °C). The molecular weights of these cellulases were about 25 kDa. These bacterial strains could be promising biocatalysts for converting cellulose into glucose for industrial purposes.
