Search Results (19)

Annually, a considerable amount of agricultural waste is produced leading to serious environmental pollution if not managed effectively. A wide range of bio-decomposers, including fungi, bacteria, and actinomycetes may break down the complex agro-residues in an eco-friendly way through secreting many cellulolytic and amylolytic enzymes. The present study aimed at exploring the ability of Frankia to degrade cellulose and starch and identifying the cellulase and α-amylase genes in Frankia genomes for potential agricultural waste degradation. Frankia alni ACN14a and Frankia casuarinae CcI₃ produced clear zones around growing hyphae on carboxymethyl cellulose (CMC) and starch substrates. The hydrolytic index (HI) ranged from 1 to 2.14 reflecting variation in their degradation efficacy. Quantification of CMCase (carboxymethyl cellulase) production in strain ACN14a presented the maximum activity (0.504 U/mL) under 1% CMC after 16 days whereas strain CcI₃ produced a weak activity after 6 days from incubation. Besides, amylase activity in strain ACN14a reached the highest value (3.215 U/mL) after 4 days of growing with 1% starch, while strain CcI₃ had the superior production (3.04 U/mL) after 12 days from 1% starch condition. Data mining and genome blasting led to the identification of multiple genes related to cellulose and starch degradation. Two endoglucanases (celA1, FRAAL4955 and celA2, FRAAL4956), two glycosyl hydrolase family 16 (FRAAL6120 and FRAAL2663), and one glycosyl hydrolase family 16 (Francci3_3843) were predicted in the two genomes. Likewise, the α-amylase genes (FRAAL5900) from Frankia alni ACN14a and (Francci3_3679) from strain CcI₃ were identified. The gene expression of endo-1, 4-beta-glucanase (celA2, FRAAL4956) revealed the maximum increment in its mRNA abundance under 0.25% CMC exposure and showed a 3.3-fold increase. Frankia capability to degrade cellulose and starch represents a critical process in nutrient cycling and environment protection. Full article

The abscission of fruits has a significant impact on yield, which in turn has a corresponding effect on economic benefits. In order to better understand the molecular mechanism of early coconut fruit abscission, the morphological and structural characteristics, cell wall hydrolysis and oxidase activities, phytohormones, and transcriptomes were analyzed in the abscission zone (AZ) from early-abscised coconut fruits (AFs) and non-abscised coconut fruits (CFs). These results indicated that the weight and water content of AFs are significantly lower than those of CFs, and the color of AFs is a grayish dark red, with an abnormal AZ structure. Cellulase (CEL), polygalacturonase (PG), pectinesterase (PE), and peroxidase (POD) activities were significantly lower than those of CFs. The levels of auxin (IAA), gibberellin (GA), cytokinins (CKs), and brassinosteroid (BR) in AFs were significantly lower than those in CFs. However, the content of abscisic acid (ABA), ethylene (ETH), jasmonic acid (JA), and salicylic acid (SA) in AFs was significantly higher than in CFs. The transcriptome analysis results showed that 3601 DEGs were functionally annotated, with 1813 DEGs upregulated and 1788 DEGs downregulated. Among these DEGs, many genes were enriched in pathways such as plant hormone signal transduction, carbon metabolism, peroxisome, pentose and gluconate interconversion, MAPK signaling pathway—plant, and starch and sucrose metabolism. Regarding cell wall remodeling-related genes (PG, CEL, PE, POD, xyloglucan endoglucosidase/hydrogenase (XTH), expansin (EXP), endoglucanase, chitinase, and beta-galactosidase) and phytohormone-related genes (IAA, GA, CKs, BR, ABA, JA, SA, and ETH) were significantly differentially expressed in the AZ of AFs. Additionally, BHLH, ERF/AP2, WRKY, bZIP, and NAC transcription factors (TFs) were significantly differently expressed, reflecting their crucial role in regulating the abscission process. This study’s results revealed the molecular mechanism of early fruit abscission in coconuts. This provided a new reference point for further research on coconut organ development and abscission. Full article

Thermophilic β-1,4-endoglucanases (Cel5A) have garnered significant interest due to their potential applications in various industries, particularly in biofuel production and biorefineries. However, despite inherent stability, thermophilic Cel5A still face challenges in terms of further enhancing their catalytic efficiency and thermostability. In this study, a novel B-factor analysis method was used to predict beneficial amino acid substitutions within a 4 Å radius of the catalytic site in the tunnel of thermophilic Cel5A from Acidomyces richmondensis (ArCel5A). A combined strategy involving site-saturation mutagenesis and high-throughput screening was employed to identify the variants with the highest endoglucanase activity. Genomic sequencing revealed a mutation at position 299 in the starting strain T. reesei A2H, where the nucleotide sequence changed from TAC to TGC, resulting in a corresponding amino acid substitution from Tyrosine(Y) to Cystine(C). The endoglucanase activity of the mutant ArCel5A reached 3251 IU/mL, representing an 85.2% increase compared to wild-type ArCel5A at the fermentation time of 94 h. Significantly, the ArCel5A-Y299C mutant showed superior thermostability, retaining 93.8% of its initial activity after 30 min at 70 °C, and 91.5% after 10 min at 80 °C. Various computational simulation methods confirmed that the Y299C mutation enhanced the stability of the catalytic pocket, thereby improving the overall stability and catalytic efficiency of ArCel5A. This study offers an effective strategy for mining target sites for rational mutagenesis based on highly conserved sequences, which simultaneously improves both the thermostability and catalytic efficiency of thermophilic Cel5A. Full article

Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process. Full article

Cellulase has been widely used in many industrial fields, such as feed and food industry, because it can hydrolyze cellulose to oligosaccharides with a lower degree of polymerization. Endo-β-1,4-glucanase is a critical speed-limiting cellulase in the saccharification process. In this study, endo-β-1,4-glucanase gene (CelA257) from Myxococcus sp. B6-1 was cloned and expressed in Escherichia coli. CelA257 contained carbohydrate-binding module (CBM) 4-9 and glycosyl hydrolase (GH) family 6 domain that shares 54.7% identity with endoglucanase from Streptomyces halstedii. The recombinant enzyme exhibited optimal activity at pH 6.5 and 50 °C and was stable over a broad pH (6–9.5) range and temperature < 50 °C. CelA257 exhibited broad substrate specificity to barley β-glucan, lichenin, CMC, chitosan, laminarin, avicel, and phosphoric acid swollen cellulose (PASC). CelA257 degraded both cellotetrose (G₄) and cellppentaose (G₅) to cellobiose (G₂) and cellotriose (G₃). Adding CelA257 increased the release of reducing sugars in crop straw powers, including wheat straw (0.18 mg/mL), rape straw (0.42 mg/mL), rice straw (0.16 mg/mL), peanut straw (0.16 mg/mL), and corn straw (0.61 mg/mL). This study provides a potential additive in biomass saccharification applications. Full article

Endoglucanases (EC 3.2.1.4) are important enzymes involved in the hydrolysis of cellulose, acting randomly in the β-1,4-glycosidic bonds present in the amorphous regions of the polysaccharide chain. These biocatalysts have been classified into 14 glycosyl hydrolase (GH) families. The GH7 family is of particular interest since it may act on a broad range of substrates, including cellulose, β-glucan, and xylan, an attractive feature for biotechnological applications, especially in the renewable energy field. In the current work, a gene from the thermophilic fungus Thermothielavioides terrestris, encoding an endoglucanase GH7 (TtCel7B), was cloned in the secretion vector pEXPYR and transformed into the high-protein-producing strain Aspergillus nidulans A773. Purified TtCel7B has a molecular weight of approximately 66 kDa, evidenced by SDS-PAGE. Circular dichroism confirmed the high β-strand content consistent with the canonical GH7 family β-jellyroll fold, also observed in the 3D homology model of TtCel7B. Biochemical characterization assays showed that TtCel7B was active over a wide range of pH values (3.5–7.0) and temperatures (45–70 °C), with the highest activity at pH 4.0 and 65 °C. TtCel7B also was stable over a wide range of pH values (3.5–9.0), maintaining more than 80% of its activity after 24 h. The K_M and Vmax values in low-viscosity carboxymethylcellulose were 9.3 mg mL⁻¹ and 2.5 × 10⁴ U mg⁻¹, respectively. The results obtained in this work provide a basis for the development of applications of recombinant TtCel7B in the renewable energy field. Full article

Cellulases are being widely employed in lignocellulosic biorefineries for the sustainable production of value-added bioproducts. However, the high production cost, sensitivity, and non-reusability of free cellulase enzymes impede their commercial applications. Enzyme immobilization seems to be a potential approach to address the aforesaid complications. The current study aims at the production of recombinant endoglucanase (CelA) originated from the cellulosome of Clostridium thermocellum in Escherichia coli (E. coli), followed by immobilization using modified regenerated cellulose (RC) membranes. The surface modification of RC membranes was performed in two different ways: one to generate the immobilized metal ion affinity membranes RC-EPI-IDA-Co²⁺ (IMAMs) for coordination coupling and another to develop aldehyde functional group membranes RC-EPI-DA-GA (AMs) for covalent bonding. For the preparation of IMAMs, cobalt ions expressed the highest affinity effect compared to other metal ions. Both enzyme-immobilized membranes exhibited better thermal stability and maintained an improved relative activity at higher temperatures (50–90 °C). In the storage analysis, 80% relative activity was retained after 15 days at 4 °C. Furthermore, the IMAM- and AM-immobilized CelA retained 63% and 53% relative activity, respectively, after being reused five times. As to the purification effect during immobilization, IMAMs showed a better purification fold of 3.19 than AMs. The IMAMs also displayed better kinetic parameters, with a higher Vmax of 15.57 U mg⁻¹ and a lower Km of 36.14 mg mL⁻¹, than those of AMs. The IMAMs were regenerated via treatment with stripping buffer and reloaded with enzymes and displayed almost 100% activity, the same as free enzymes, up to 5 cycles of regeneration. Full article

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance. Full article

Cellulase adsorption onto lignin decreases the productivity of enzymatic hydrolysis of lignocellulosic biomass. Here, adsorption of enzymes onto different types of lignin was investigated, and the five major enzymes—cellobiohydrolases (CBHs), endoglucanase (Cel7B), β-glucosidase (Cel3A), xylanase (XYNIV), and mannanase (Man5A)—in a cellulase cocktail obtained from Trichoderma reesei were individually analyzed through SDS-PAGE and zymogram assay. Lignin was isolated from woody (oak and pine lignin) and herbaceous (rice straw and kenaf lignin) plants. The relative adsorption of CBHs compared to the control was in the range of 14.15–18.61%. The carbohydrate binding motif (CBM) of the CBHs contributed to higher adsorption levels in oak and kenaf lignin, compared to those in pine and rice lignin. The adsorption of endoglucanase (Cel7B) by herbaceous plant lignin was two times higher than that of woody lignin, whereas XYNIV showed the opposite pattern. β-glucosidase (Cel3A) displayed the highest and lowest adsorption ratios on rice straw and kenaf lignin, respectively. Mannanase (Man5A) was found to have the lowest adsorption ratio on pine lignin. Our results showed that the hydrophobic properties of CBM and the enzyme structures are key factors in adsorption onto lignin, whereas the properties of specific lignin types indirectly affect adsorption. Full article

In this study, we isolated and identified a thermophilic strain of Aspergillus fumigatus from the “Daqu” samples. Transcriptomic analysis of A. fumigatus identified 239 carbohydrate-active enzymes (CAZy)-encoding genes, including 167 glycoside hydrolase (GH)-encoding genes, 58 glycosyltransferase (GT)-encoding genes, 2 polysaccharide lyase (PLs)-encoding genes and 12 carbohydrate esterase (CEs)-encoding genes, which indicates that the strain has a strong potential for application for enzyme production. Furthermore, we also identified a novel endoglucanase gene (AfCel5A), which was expressed in Pichia pastoris and characterized. The novel endoglucanase AfCel5A exhibited the highest hydrolytic activity against CMC-Na and the optimal activity at 80 °C and pH 4.0 and also showed good stability at pH 3.0–11.0 and below 70 °C. The Km and Vmax values of AfCel5 were 0.16 ± 0.05 mg·mL⁻¹ and 7.23 ± 0.33 mol mg⁻¹·min⁻¹, respectively, using CMC-Na as a substrate. Further, the endoglucanase exhibited a high tolerance toward NaCl as well as glucose. In addition, the finding that the endoglucanase AfCel5A in combination with β-mannanse (ManBK) clearly increased the release of total reducing sugars of glucomannan by up to 74% is significant. Full article

Biomass includes cellulose, hemicelluloses, pectin and lignin; constitutes the components of dietary fibre of plant and alge origins in animals and humans; and can potentially provide inexhaustible basic monomer compounds for developing sustainable biofuels and biomaterials for the world. Development of efficacious cellulases is the key to unlock the biomass polymer and unleash its potential applications in society. Upon reviewing the current literature of cellulase research, two characterized and/or engineered glycosyl hydrolase family-5 (GH5) cellulases have displayed unique properties of processive endoglucanases, including GH5-tCel5A1 that was engineered and was originally identified via targeted genome sequencing of the extremely thermophilic Thermotoga maritima and GH5-p4818Cel5_2A that was screened out of the porcine hindgut microbial metagenomic expression library. Both GH5-tCel5A1 and GH5-p4818Cel5_2A have been characterized as having small molecular weights with an estimated spherical diameter at or < 4.6 nm; being monomodular without a required carbohydrate-binding domain; and acting as processive β-1,4-endoglucanases. These two unique GH5-tCel5A1 and GH5-p4818Cel5_2A processive endocellulases are active in hydrolyzing natural crystalline and pre-treated cellulosic substrates and have multi-functionality towards several hemicelluloses including β-glucans, xylan, xylogulcans, mannans, galactomannans and glucomannans. Therefore, these two multifunctional and monomodular GH5-tCel5A1 and GH5-p4818Cel5_2A endocellulases already have promising structural and functional properties for further optimization and industrial applications. Full article

Pratylenchus loosi is an important root-lesion nematode that causes damage to tea plantations in Iran and all over the world. The present study reports on the characterization and evolution of three ß-1,4-endoglucanase genes: Pl-eng-2, Pl-eng-3 and Pl-eng-4. The gene structure of Pl-eng-2 was fully determined with the predicted signal peptide and devoid of the linker domain and carbohydrate-binding domain, while Pl-eng-3 and Pl-eng-4 were only partially sequenced. The transcription of Pl-eng-2 was localized in the secretory esophageal glands of all life stages, but it was upregulated in male and female stages. The exon/intron structures of Pl-eng-2, Pl-eng-3 and Pl-eng-4 confirmed that they resulted from gene duplication followed by sequence and gene structure diversification with loss of the linker domain and carbohydrate-binding domain during evolution. A phylogenetic analysis further confirmed that nematode endoglucanases resulted from the horizontal gene transfer of a bacterial gene, as Pl-eng-3 showed sister relationships with the CelB cellulase of Bacillus subtilis. Silencing Pl-eng-2 by in vitro RNA interference produced a 60% decrease of the transcript level. The reproductive ability of silenced P. loosi showed a 35% reduction of eggs and larval stages compared to untreated nematodes, suggesting that this gene is involved in the early steps of invasion. Full article

The conventional endo–exo synergism model has extensively been supported in literature, which is based on the perception that endoglucanases (EGs) expose or create accessible sites on the cellulose chain to facilitate the action of processive cellobiohydrolases (CBHs). However, there is a lack of information on why some bacterial and fungal CBHs and EGs do not exhibit synergism. Therefore, the present study evaluated and compared the synergistic relationships between cellulases from different microbial sources and provided insights into how different GH families govern synergism. The results showed that CmixA2 (a mixture of TlCel7A and CtCel5A) displayed the highest effect with BaCel5A (degree of synergy for reducing sugars and glucose of 1.47 and 1.41, respectively) in a protein mass ratio of 75–25%. No synergism was detected between CmixB1/B2 (as well as CmixC1/C2) and any of the EGs, and the combinations did not improve the overall cellulose hydrolysis. These findings further support the hypothesis that “not all endo-to exo-cellulase interactions are synergistic”, and that the extent of synergism is dependent on the composition of cellulase systems from various sources and their compatibility in the cellulase cocktail. This method of screening for maximal compatibility between exo- and endo-cellulases constitutes a critical step towards the design of improved synergistic cellulose-degrading cocktails for industrial-scale biomass degradation. Full article

Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (cel6A) of Thermobifida fusca to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of cel6A.CBC which revealed thermostability of cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass. Full article

The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose^® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg⁻¹, respectively. The K_m and V_max of AcCel12B for CMC were 25.47 mg·mL⁻¹ and 131.75 U·mg⁻¹, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future. Full article