RESUMEN

Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some ß-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na+. The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.

Asunto(s)

Celulasa , Cryptococcus gattii , Escherichia coli , Cryptococcus gattii/genética , Cryptococcus gattii/enzimología , Cryptococcus gattii/química , Celulasa/genética , Celulasa/química , Celulasa/aislamiento & purificación , Celulasa/metabolismo , Celulasa/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/biosíntesis , Expresión Génica , Clonación Molecular , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Concentración de Iones de Hidrógeno , Temperatura

RESUMEN

The pretreatment and saccharification of dewaxed bagasse (DWB) has been investigated under various reaction conditions ranging 2000 to 3200 psi, at 70 ± 1 °C in supercritical carbon dioxide (SCC). This has been in attempt to transform the DWB into fermentable sugar and bioethanol in high yields. The effect of SCC mediated pretreatment and enzymatic hydrolysis on structural and morphological alterations in DWB has been ascertained through diverse analytical methods. The sugar has been released through cellulase (40 FPU/mL) mediated enzymatic hydrolysis of pretreated DWB in sodium acetate buffer (pH 4.7) within 1 h at SCC 2800 psi, 70 ± 1 °C. The released sugar was subsequently fermented in the presence of yeast (Saccharomyces crevices, 135 CFU) at 28 ± 1 °C over 72 h to afford the bioethanol. The SCC mediated process conducted in acetic acid:water media (1:1) at 2800 psi, 70 ± 1 °C over 6 h has afforded the pretreated DWB with maximum yield towards the production of fermentable sugar and bioethanol. The production of fermentable sugar and bioethanol has been electrochemically estimated through cyclic voltammetry (CV) and square wave voltammetry (SWV) over glassy carbon electrode in KOH (0.1 M). The electrochemical methods were found selective and in close agreement for estimation of the yields (%) of fermentable sugars and bioethanol. The yield (%) of fermentable sugar estimated from CV and SWV were 80.10 ± 5.34 and 79.00 ± 5.09 respectively. Whereas the yield (%) of bioethanol estimated from CV and SWV were 81.30 ± 2.78% and 78.6 ± 1.25% respectively. Present investigation delivers a SCC mediated green and sustainable method of pretreatment of DWB to afford the enhanced saccharification, to produce bioethanol in high yields.

Asunto(s)

Biocombustibles , Dióxido de Carbono , Celulosa , Etanol , Fermentación , Etanol/metabolismo , Etanol/química , Celulosa/metabolismo , Celulosa/química , Dióxido de Carbono/metabolismo , Dióxido de Carbono/química , Hidrólisis , Saccharomyces cerevisiae/metabolismo , Celulasa/metabolismo

RESUMEN

Sophorose is the most effective inducer for cellulase production by Trichoderma reesei. Currently, the biosynthesis of sophorose is very inefficient, resulting in that unavailable for cellulase production in industry. In this study, CoGH1A, a multifunctional thermophilic glycoside hydrolase, was employed for sophorose production. Under the optimized conditions, the sophorose yield was 37.86 g/L with a productivity of 9.47 g/L/h which is by far the highest productivity. Meanwhile, the Fe3O4-CS-THP-CoGH1A nanoparticles were constructed to realize the recycling of CoGH1A. After 5 cycles of catalysis, Fe3O4-CS-THP-CoGH1A retained about 83.90 % enzyme activity. Finally, the mixtures of glucose and disaccharides (MGDC) obtained after being catalyzed by CoGH1A was used for cellulase production. As a result, the cellulase productivity achieved 188.38 FPU/L/h in 120 h. These results indicated that sophorose could be efficiently produced from glucose via transglycosylation by CoGH1A, making it possible to be industrially used as the inducer to improving the cellulase productivity.

Asunto(s)

Celulasa , Glucosa , Celulasa/metabolismo , Glucosa/metabolismo , Hypocreales/metabolismo , Glucanos

RESUMEN

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids' potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum.

Asunto(s)

Clostridium butyricum , Plásmidos , Clostridium butyricum/genética , Plásmidos/genética , Humanos , Expresión Génica , Biotecnología/métodos , Glucuronidasa/genética , Glucuronidasa/metabolismo , Celulasa/genética , Celulasa/metabolismo , Genes Reporteros , Microbiología Industrial/métodos , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos

RESUMEN

Apple pomace, an abundant agricultural by-product with low utilization rates, often leads to environmental pollution if not properly managed. This study aimed to enhance the nutritional value of apple pomace by comparing the effects of solid-state fermentation using complex probiotics and cellulase preparation. Additionally, the study investigated the dynamic changes in various components throughout the fermentation process with complex probiotics. The results of single-strain solid-state fermentation tests indicated that Lactiplantibacillus plantarum DPH, Saccharomyces cerevisiae SC9, and Bacillus subtilis C9 were the optimal strains for fermenting the most effective substrate combination, comprising 73 % apple pomace and 20 % millet bran. The strains (complex probiotics) and a cellulase preparation were used for the solid-state fermentation of the apple pomace mixture for nine days, respectively. The contents of acid detergent fiber, neutral detergent fiber, hemicellulose, and insoluble dietary fiber decreased by up to 9.99 %, 9.59 %, 23.21 %, and 14.34 %, respectively. In contrast, the content of soluble dietary fiber significantly increased by up to 29.74 %. Both methods reduced cellulose crystallinity and modified the substrate's surface structure, resulting in a looser arrangement. Fermentation with complex probiotics for three or six days increased the abundance of lactic acid bacteria, which comprised >87 % of the total microbial population. Concurrently, the abundance of detrimental bacteria, such as Salmonella, Acetobacter, Escherichia, and Pantoea, significantly decreased. Furthermore, fermentation with complex probiotics for six or nine days enhanced antioxidant properties, leading to a significant increase in beneficial metabolites, including amino acids, organic acids, gamma-aminobutyric acid, serotonin. In conclusion, complex probiotics can effectively substitute for cellulase preparation in the solid-state fermentation of apple pomace, with a six-day fermentation period yielding optimal results. This study provides valuable insights into enhancing the value of apple pomace in the feed industry and the effective application of agro-industrial by-products.

Asunto(s)

Celulasa , Fermentación , Malus , Probióticos , Malus/microbiología , Probióticos/metabolismo , Celulasa/metabolismo , Fibras de la Dieta/metabolismo , Saccharomyces cerevisiae/metabolismo , Valor Nutritivo , Bacillus subtilis/metabolismo , Lactobacillus plantarum/metabolismo , Microbiología de Alimentos

RESUMEN

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle's terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.

Asunto(s)

Celulosa , Proteínas de Escherichia coli , Escherichia coli , Etanolaminas , Glucosiltransferasas , Celulosa/biosíntesis , Celulosa/metabolismo , Glucosiltransferasas/metabolismo , Glucosiltransferasas/genética , Etanolaminas/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Membrana Celular/metabolismo , Pared Celular/metabolismo , Periplasma/metabolismo , Celulasa/metabolismo , Celulasa/genética

RESUMEN

An innovative binary biol-based deep eutectic solvent (DES), specifically ethylamine hydrochloride-ethylene glycol (EaCl-EG), was developed for efficient pretreatment of eucalyptus biomass. This DES exhibited superior performance in achieving high delignification (85.0%) and xylan removal (80.0%), while preserving a significant amount of cellulose (94.5%) compared to choline chloride-based DES. Notably, the pretreated eucalyptus residues showed a remarkable glucose yield of over 92.5%, representing a substantial enhancement of up to 15 times compared to untreated eucalyptus. Furthermore, the pretreated liquor yielded high-purity lignin with a yield of 97.8%, characterized by well-preserved ß-O-4 structure and nanoscale dimensions. These lignin nanoparticles (LNPs) were subsequently self-assembled into lignin nanobottles (LNBs), adding further value to the pretreatment process. The proposed novel binary EaCl-EG DES presented great potential as an efficient pretreatment solvent for future biomass fractionation processes.

Asunto(s)

Biomasa , Fraccionamiento Químico , Disolventes Eutécticos Profundos , Eucalyptus , Lignina , Eucalyptus/química , Lignina/química , Disolventes Eutécticos Profundos/química , Fraccionamiento Químico/métodos , Hidrólisis , Celulasa/metabolismo , Celulasa/química , Glucosa/química , Solventes/química , Nanopartículas/química

RESUMEN

The mechanical pulp industry is diversifying through the manufacture of high-value paper products, such as microfibrillated cellulose. However, the development of fibre quality is still energy-intensive. Enzymatic hydrolysis is hypothesized to promote fibre cutting, greater fibrillation, and reduce refining energy costs. Despite potential benefits, there is little understanding of the mechanisms behind fibre development during enzymatic hydrolysis of mechanical pulp. This work investigates how incubation pH and temperature during enzymatic hydrolysis impact the refining of mechanical pulp short fibres. Incubation with endoglucanase at pH 5 and 60 °C increased fibre cutting by approximately 20 %. Fibrillation was negatively affected at this condition, resulting in increased slim fines formation with refining. Incubation at pH 8 and 80 °C promoted >15 % reduction in fibre length, despite such conditions being associated with low enzyme activity. The pH variation modified the sedimentation height of the fibres and the conductivity of suspensions, indicating a change in fibre surface charge. Fibre morphology changes were induced by enzyme hydrolysis conducted at conditions representative of the full range of pH and temperature observed in mechanical pulp mills.

Asunto(s)

Celulasa , Celulosa , Temperatura , Hidrólisis , Celulasa/metabolismo , Concentración de Iones de Hidrógeno , Celulosa/química , Celulosa/metabolismo , Papel

RESUMEN

Two multimodular endoglucanases in glycoside hydrolase family 5, ReCel5 and ElCel5, share 73% identity and exhibit similar modular structures: family 1 carbohydrate-binding module (CBM1); catalytic domain; CBMX2; module of unknown function. However, they differed in their biochemical properties and catalytic performance. ReCel5 showed optimal activity at pH 4.0 and 70 °C, maintaining stability at 70 °C (>80% activity). Conversely, ElCel5 is optimal at pH 3.0 and 50 °C (>50% activity at 50 °C). ElCel5 excels in degrading CMC-Na (256 U/mg vs 53 U/mg of ReCel5). Five domain-truncated (TM1-TM5) and four domain-replaced (RM1-RM4) mutants of ReCel5 with the counterparts of ElCel5 were constructed, and their enzymatic properties were compared with those of the wild type. Only RM1, with ElCel5-CBM1, displayed enhanced thermostability and activity. The hydrolysis of pretreated corn stover was reduced in most TM and RM mutants. Molecular dynamics simulations revealed interdomain interactions within the multimodular endoglucanase, potentially affecting its structural stability and complex biological catalytic processes.

Asunto(s)

Celulasa , Hidrólisis , Celulasa/química , Celulasa/metabolismo , Celulasa/genética , Celulosa/metabolismo , Celulosa/química , Dominios Proteicos , Dominio Catalítico , Especificidad por Sustrato , Zea mays/química , Simulación de Dinámica Molecular , Estabilidad de Enzimas

RESUMEN

This study aims to explore the feasibility of introducing, during the manufacture of bakery bread, an enzymatic cocktail coproduced by the fungus Stachybotrys microspora: α-amylases, xylanases and cellulases, using wheat bran as a nutrient source. Among the characteristics of the alveograph (dough tenacity "P" and dough extensibility "L"), the addition of a cocktail of enzymes at a concentration of 2 %, to weak wheat flour, has made it possible to significantly reduce its P/L ratio from 2.45 to 1.41. Furthermore, the use of enzyme cocktails at 2 %, 4 %, and 6 % concentrations increases the brown color of the bread crust. The great reduction in the rate of bread firmness, during storage over 5 days, was obtained in the presence of an enzyme cocktail in comparison with bread control (65.13 N for the control and 22.99 N, 23.24 N, and 18.24 N for bread enriched with enzyme cocktail at 2 %, 4 % and 6 % concentrations, respectively). In conclusion, the enzyme cocktail added can synergistically improve bread dough rheology and bread properties.

Asunto(s)

Pan , Harina , Stachybotrys , alfa-Amilasas , Pan/análisis , alfa-Amilasas/metabolismo , Harina/análisis , Stachybotrys/enzimología , Stachybotrys/química , Reología , Celulasa/metabolismo , Celulasa/química , Triticum/química

RESUMEN

Postharvest rot caused by various fungal pathogens is a damaging disease affecting kiwifruit production and quality, resulting in significant annual economic losses. This study focused on isolating the strain P3-1W, identified as Diaporthe eres, as the causal agent of 'Hongyang' postharvest rot disease in China. The investigation highlighted cell wall degrading enzymes (CWDEs) as crucial pathogenic factors. Specially, the enzymatic activities of cellulase, ß-galactosidase, polygalacturonase, and pectin methylesterases peaked significantly on the second day after infection of D. eres P3-1W. To gain a comprehensive understanding of these CWDEs, the genome of this strain was sequenced using PacBio and Illumina sequencing technologies. The analysis revealed that the genome of D. eres P3-1W spans 58,489,835 bp, with an N50 of 5,939,879 bp and a GC content of 50.7%. A total of 15,407 total protein-coding genes (PCGs) were predicted and functionally annotated. Notably, 857 carbohydrate-active enzymes (CAZymes) were identified in D. eres P3-1W, with 521 CWDEs consisting of 374 glycoside hydrolases (GHs), 108 carbohydrate esterase (CEs) and 91 polysaccharide lyases (PLs). Additionally, 221 auxiliary activities (AAs), 91 glycosyltransferases (GTs), and 108 carbohydrate binding modules (CBMs) were detected. These findings offer valuable insights into the CAZymes of D. eres P3-1W.

Asunto(s)

Actinidia , Ascomicetos , Genoma Fúngico , Enfermedades de las Plantas , Actinidia/microbiología , Enfermedades de las Plantas/microbiología , China , Ascomicetos/genética , Ascomicetos/patogenicidad , Ascomicetos/enzimología , Genoma Fúngico/genética , Poligalacturonasa/genética , Poligalacturonasa/metabolismo , Frutas/microbiología , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Celulasa/genética , Celulasa/metabolismo , Pared Celular/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Nature has developed extremozymes that catalyze complex reaction processes in extreme environmental conditions. Accordingly, a combined approach consisting of extremozyme screening, ancestral sequence resurrection (ASR), and molecular dynamic simulation was utilized to construct a developed endoglucanase. The primary experimental and in-silico data led to the prediction of a hypothetical sequence of endoglucanase (EG5-G131) using Bacillus sp. G131 confirmed by amplification and sequencing. EG5-G131 exhibited noticeable stability in a broad-pH range, several detergents, organic solvents, and temperatures up to 80 °C. The molecular weight, Vmax, and Km of the purified endoglucanase were estimated to be 36 kDa, 4.32 µmol/min, and 23.62 mg/ml, respectively. The calculated thermodynamic parameters for EG5-G131 confirmed its intrinsic thermostability. Computational analysis revealed Glu142 and Glu230 as active-site residues of the enzyme. Furthermore, the enzyme remained bound to cellotetraose at 298 K, 333 K, 343 K, and 353 K for 300 ns, consistent with our experimental data. ASR of EG5-G131 led to the introduction of ancestral ANC204 and ANC205, which show similar thermodynamic characteristics with the last Firmicute common ancestor. Finally, truncating loops from the N-terminal of two sequences created two variants with desirable thermal stability, suggesting the evolutionary deciphering of the functional domain of the GH5 family in Bacillus sp. G131.

Asunto(s)

Bacillus , Celulasa , Estabilidad de Enzimas , Evolución Molecular , Simulación de Dinámica Molecular , Bacillus/enzimología , Bacillus/genética , Celulasa/química , Celulasa/genética , Celulasa/metabolismo , Termodinámica , Concentración de Iones de Hidrógeno , Dominio Catalítico , Secuencia de Aminoácidos , Tetrosas/metabolismo , Tetrosas/química , Temperatura , Filogenia , Cinética , Extremófilos/enzimología , Extremófilos/genética , Celulosa/análogos & derivados

RESUMEN

Cellulases have been widely used in many fields such as animal feed, textile, food, lignocellulose bioconversion, etc. Efficient and low-cost production of cellulases is very important for its industrial application, especially in bioconversion of lignocellulosic biomass. Filamentous fungi are currently widely used in industrial cellulase production due to their ability to secrete large amounts of active free cellulases extracellularly. This review comprehensively summarized the research progress on cellulases from filamentous fungi in recent years, including filamentous fungi used for cellulase production and its modification strategies, enzyme compositions, characterization methods and application of fungal cellulase systems, and the production of fungal cellulase includes production processes, factors affecting cellulase production such as inducers, fermentation medium, process parameters and their control strategies. Also, the future perspectives and research topics in fungal cellulase production are presented in the end of the review. The review helps to deepen the understanding of the current status of fungal cellulases, thereby promoting the production technology progress and industrial application of filamentous fungal cellulase.

Asunto(s)

Celulasa , Fermentación , Hongos , Celulasa/biosíntesis , Celulasa/metabolismo , Hongos/enzimología , Celulasas/metabolismo , Celulasas/biosíntesis , Biomasa , Lignina

RESUMEN

In the context of biorefinery, researchers have been looking for lignocellulosic biomasses and ideal treatments to produce economically viable biofuels. In this scenario, the bamboo culm appears as a plant matrix of great potential, given the high cellulose content of low crystallinity. Thus, the objective and differential of this work was to determine the best conditions for enzymatic hydrolysis of cellulose extracted from bamboo culm and to evaluate its potential application in the production of bioethanol through Separate Hydrolysis and Fermentation (SHF) and Saccharification and Simultaneous Fermentation (SSF) by Saccharomyces cerevisiae modified via CRISPR/Cas9. The average cellulose extraction yield was 41.87 % with an extraction efficiency of 86.76 %. In general, as the hydrolysis time increased, an increase in glucose production was observed in almost all assays, with higher hydrolysis efficiency values at 72 h. The results ranged from 2.09 to 19.8 g/L of glucose obtained with efficiency values of 10.47 to 99 %. The best conditions were found in test 5 (temperature of 36 °C and pH 5.0, with only 10 FPU/g of substrate Cellic Ctec2 Novozymes ® cocktail). It is observed that for all hydrolysis times the independent variables pH and temperature were significant under the hydrolysis efficiency, showing a negative effect, indicating that higher values of the same promote lower values of the response variable. For bioethanol production, a maximum concentration of 7.84 g/L was observed for the SSH process after 4 h of fermentation, while for the SSF process it was 12.6 g/L after 24 h of fermentation, indicating the large potential of the simultaneous process together with the application of bamboo culm biomass for high production of biofuel.

Asunto(s)

Biocombustibles , Sistemas CRISPR-Cas , Celulosa , Etanol , Fermentación , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Hidrólisis , Celulosa/metabolismo , Etanol/metabolismo , Celulasa/metabolismo , Sasa , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Biomasa

RESUMEN

Enzymatic treatment on lignocellulosic biomass has become a trend in preparing nanocellulose (NC), but the process must be optimized to guarantee high production yield and crystallinity. This study offers insights into an innovative protocol using cultivated fungal cellulase and xylanase to improve NC production from raw oil palm leaves (OPL) using five-factor-four-level Taguchi orthogonal design for optimizing parameters, namely substrate and enzyme loading, surfactant concentration, incubation temperature and time. Statistical results revealed the best condition for producing NC (66.06 % crystallinity, 43.59 % yield) required 10 % (w/v) substrate, 1 % (v/v) enzyme, 1.4 % (w/v) Tween-80, with 72-h incubation at 30 °C. Likewise, the highest sugar yield (47.07 %) was obtained using 2.5 % (w/v) substrate, 2.0 % (v/v) enzyme, 2.0 % (w/v) Tween-80, with 72-h incubation at 60 °C. The auxiliary enzymes used in this study, i.e., xylanase, produced higher crystallinity NC, showing widths between 8 and 12 nm and lengths >1 µm and sugars at 47.07 % yield. Thus, our findings proved that optimizing the single-step enzymatic hydrolysis of raw OPL could satisfactorily produce relatively crystalline NC and sugar yield for further transformation into bio-nanocomposites and biofuels. This study presented a simple, innovative protocol for NC synthesis showing characteristics comparable to the traditionally-prepared NC, which is vital for material's commercialization.

Asunto(s)

Celulasa , Celulosa , Hojas de la Planta , Celulosa/química , Celulosa/biosíntesis , Hojas de la Planta/química , Celulasa/química , Celulasa/metabolismo , Azúcares/química , Arecaceae/química , Aceite de Palma/química , Biomasa , Temperatura , Hidrólisis , Endo-1,4-beta Xilanasas/metabolismo , Endo-1,4-beta Xilanasas/química

RESUMEN

In this study, pectin extracted from pomelo peel was investigated using three different combination methods of pulsed electric field (PEF) and cellulase. Three action sequences were performed, including PEF treatment followed by enzymatic hydrolysis, enzymatic hydrolysis followed by PEF treatment, and enzymatic hydrolysis simultaneously treated by PEF. The three corresponding pectins were namely PEP, EPP and SP. The physiochemical, molecular structural and functional properties of the three pectins were determined. The results showed that PEP had excellent physiochemical properties, with the highest yield (12.08 %), total sugar (80.17 %) and total phenol content (38.20 %). The monosaccharide composition and FT-IR analysis indicated that the three pectins were similar. The molecular weights of PEP, EPP and SP were 51.13, 88.51 and 40.00 kDa, respectively. PEP showed the best gel properties, emulsification stability and antioxidant capacity among the three products, due to its high galacturonic acid and total phenol content, appropriate protein and low molecular weight. The mechanism of PEF-assisted cellulase hydrolysis of pomelo peel was also revealed by SEM analysis. These results suggested that PEF pretreatment was the best method, which not only improved the efficiency of enzymatic extraction, but also reduced resource waste and increased financial benefits.

Asunto(s)

Celulasa , Citrus , Peso Molecular , Pectinas , Hidrólisis , Celulasa/metabolismo , Celulasa/química , Pectinas/química , Citrus/química , Antioxidantes/química , Antioxidantes/farmacología , Electricidad , Fenómenos Químicos , Fenoles/química , Frutas/química , Espectroscopía Infrarroja por Transformada de Fourier , Monosacáridos/análisis

RESUMEN

This study evaluates the feasibility of using enzymatic technology to produce novel nanostructures of cellulose nanomaterials, specifically cellulose nanospheres (CNS), through enzymatic hydrolysis with endoglucanase and xylanase of pre-treated cellulose fibers. A statistical experimental design facilitated a comprehensive understanding of the process parameters, which enabled high yields of up to 82.7 %, while maintaining a uniform diameter of 54 nm and slightly improved crystallinity and thermal stability. Atomic force microscopy analyses revealed a distinct CNS formation mechanism, where initial fragmentation of rod-like nanoparticles and subsequent self-assembly of shorter rod-shaped nanoparticles led to CNS formation. Additionally, adjustments in process parameters allowed precise control over the CNS diameter, ranging from 20 to 100 nm, highlighting the potential for customization in high-performance applications. Furthermore, this study demonstrates how the process framework, originally developed for cellulose nanocrystals (CNC) production, was successfully adapted and optimized for CNS production, ensuring scalability and efficiency. In conclusion, this study emphasizes the versatility and efficiency of the enzyme-based platform for producing high-quality CNS, providing valuable insights into energy consumption for large-scale economic and environmental assessments.

Asunto(s)

Celulasa , Celulosa , Nanosferas , Celulosa/química , Hidrólisis , Nanosferas/química , Celulasa/química , Celulasa/metabolismo , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/metabolismo

RESUMEN

The important role of Carbohydrate-binding module (CBM) in the cellulases catalytic activity has been widely studied. CBM3 showed highest affinity for cellulose substrate with 84.69 % adsorption rate among CBM1, CBM2, CBM3, and CBM4 in this study. How CBM affect the catalytic properties of GH5 endoglucanase III from Trichoderma viride (TvEG3) was systematically explored from two perspectives: the deletion of its own CBM(TvEG3dc) and the replacement of high substrate affinity CBM3 (TvEG3dcCBM3). Compared with TvEG3, TvEG3dc lost its binding ability on Avicel and filter paper, but its catalytic activity did not change significantly. The binding ability and catalytic activity of TvEG3dcCBM3 to Avicel increased 348.3 % and 372.51 % than that of TvEG3, respectively. The binding ability and catalytic activity of TvEG3dcCBM3 to filter paper decreased 51.7 % and 33.33 % than that of TvEG3, respectively. Further structural analysis of TvEG3, TvEG3dc, and TvEG3dcCBM3 revealed no changes in the positions and secondary structures of the key amino acids. These results demonstrated that its own CBM1 of TvEG3 did not affect its catalytic activity center, so it had no effect on its catalytic activity. But CBM3 changed the adsorption affinity for different substrates, which resulted in a change in the catalytic activity of the substrate.

Asunto(s)

Celulasa , Celulasa/química , Celulasa/metabolismo , Celulasa/genética , Unión Proteica , Trichoderma/enzimología , Especificidad por Sustrato , Celulosa/metabolismo , Celulosa/química , Catálisis , Secuencia de Aminoácidos , Cinética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética

RESUMEN

Bioeconomy goals for using biomass feedstock for biofuels and bio-based production has arisen the demand for fungal strains and enzymes for biomass processing. Despite well-known Trichoderma and Aspergillus commercial strains, continuous bioprospecting has revealed the fungal biodiversity potential for production of biomass degrading enzymes. The strain Aspergillus fumigatus LMB-35Aa has revealed a great potential as source of lignocellulose-degrading enzymes. Nevertheless, genetic improvement should be considered to increase its biotechnological potential. Molecular manipulation based on homologous direct recombination (HDR) in filamentous fungi poses a challenge since its low recombination rate. Currently, CRISPR/Cas9-mediated mutagenesis can enable precise and efficient editing of filamentous fungi genomes. In this study, a CRISPR/Cas9-mediated gene editing strategy for improving endoglucanase activity of A. fumigatus LMB-35Aa strain was successfully used, which constitutes the first report of heterologous cellulase production in filamentous fungi using this technology. For this, eglA gene from A. niger ATCC 10,864 was integrated into conidial melanin pksP gene locus, which facilitated the selection of edited events discerned by the emergence of albino colonies. Heterologous production of the EglA enzyme in a biofilm fermentation system resulted in a 40% improvement in endoglucanase activity of the mutant strain compared to the wild type.

Asunto(s)

Aspergillus fumigatus , Sistemas CRISPR-Cas , Celulasa , Proteínas Fúngicas , Edición Génica , Aspergillus fumigatus/genética , Aspergillus fumigatus/enzimología , Celulasa/genética , Celulasa/metabolismo , Edición Génica/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Sustitución del Gen , Biopelículas/crecimiento & desarrollo , Fermentación

RESUMEN

In this study, a cellulase-responsive controlled-release formulation (FPR-HMS-HPC) was developed by grafting hydroxypropyl cellulose (HPC) onto fipronil (FPR) loaded hollow mesoporous silica (HMS) nanoparticles via ester linkage. The FPR-HMS-HPC formulation was characterized using scanning and transmission electron microscopies, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The results indicated that FPR-HMS-HPC exhibited a high loading capacity of 10.0 % (w/w) and demonstrated favorable responsiveness to cellulase enzyme. Moreover, its insecticidal efficacy against Reticulitermes flaviceps surpassed that of an equivalent dose of FPR. Toxicology studies showed that the mortality and hatching rates of zebrafish exposed to FPR-HMS-HPC nanoparticles were reduced by >6.5 and 8.0 times, respectively. Thus, HPC-anchored HMS nanoparticles as insecticide delivery systems present a sustainable method for pest control significantly reducing harm to non-target organisms and the environment.

Asunto(s)

Celulasa , Celulosa , Portadores de Fármacos , Nanopartículas , Dióxido de Silicio , Celulosa/análogos & derivados , Celulosa/química , Dióxido de Silicio/química , Animales , Porosidad , Nanopartículas/química , Celulasa/química , Celulasa/metabolismo , Portadores de Fármacos/química , Pez Cebra , Plaguicidas/química , Plaguicidas/farmacología , Insecticidas/química , Insecticidas/farmacología , Pirazoles/química , Pirazoles/farmacología