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1.
Sci Rep ; 13(1): 148, 2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36600032

RESUMO

This research examined the possible pathway of monosaccharide production from the rice straw waste using three integrated enzymatic hydrolysis approaches: boiled hot water pre-treatment with enzyme, alkaline pre-treatment with enzyme, and acid pre-treatment with enzyme, that can be further used as the feedstock for anaerobic digestion. Two cellulase enzymes: SIGMA-ALDRICH laboratory grade cellulase from Aspergillus niger and atres Zymix plus as a commercial cellulase enzyme were applied. It was found that the boiled hot water pre-treatment with the commercial cellulase gave the highest total monosaccharides yields. Glucose was the most significant part (78-86%) of the monosaccharides. For the pre-treatment with dilute acid, glucose was also the main component of monosaccharides; however, for the alkali pre-treatment, xylose was the main monosaccharide. It made up 48-85% of the total monosaccharide compared to glucose that made up 5-49% of total monosaccharide. Boiled rice straw with commercial cellulase enzyme provided the highest glucose yield compared to other experiments. Moreover, the obtained results from GC-MS/MS analysis show that up to 62 species of phenolic compound could be found in enzymatic hydrolysis of the rice straw waste. Aromatic and aliphatic hydrocarbon substances were also detected in the FEEM analysis. From the overall results, the integrated enzymatic hydrolysis with boil hot water pre-treatment was the most efficient method for monosaccharide production from the rice straw waste.


Assuntos
Celulase , Oryza , Monossacarídeos/metabolismo , Oryza/metabolismo , Anaerobiose , Hidrólise , Espectrometria de Massas em Tandem , Celulase/metabolismo , Glucose/metabolismo
2.
Sci Rep ; 13(1): 446, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624114

RESUMO

Application of cost-effective pretreatment of wheat straw is an important stage for massive bioethanol production. A new approach is aimed to enhance the pretreatment of wheat straw by using low-cost ionic liquid [TEA][HSO4] coupled with ultrasound irradiation. The pretreatment was conducted both at room temperature and at 130 °C with a high biomass loading rate of 20% and 20% wt water assisted by ultrasound at 100 W-24 kHz for 15 and 30 min. Wheat straw pretreated at 130 °C for 15 and 30 min had high delignification rates of 67.8% and 74.9%, respectively, and hemicellulose removal rates of 47.0% and 52.2%. Moreover, this pretreatment resulted in producing total reducing sugars of 24.5 and 32.1 mg/mL in enzymatic saccharification, respectively, which corresponds to saccharification yields of 67.7% and 79.8% with commercial cellulase enzyme CelluMax for 72 h. The ethanol generation rates of 38.9 and 42.0 g/L were attained for pretreated samples for 15 and 30 min, equivalent to the yields of 76.1% and 82.2% of the maximum theoretical yield following 48 h of fermentation. This demonstration provided a cheap and promising pretreatment technology in terms of efficiency and shortening the pretreatment time based on applying low-cost ionic liquid and efficient ultrasound pretreatment techniques, which facilitated the feasibility of this approach and could further develop the future of biorefinery.


Assuntos
Celulase , Líquidos Iônicos , Triticum/metabolismo , Análise Custo-Benefício , Fermentação , Hidrólise , Celulase/metabolismo
3.
Appl Environ Microbiol ; 89(1): e0142122, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36602369

RESUMO

The filamentous fungus Trichoderma reesei is one of the most prolific cellulase producers and has been established as a model microorganism for investigating mechanisms modulating eukaryotic gene expression. Identification and functional characterization of transcriptional regulators involved in complex and stringent regulation of cellulase genes are, however, not yet complete. Here, a Zn(II)2Cys6-type transcriptional factor TAM1 that is homologous to Aspergillus nidulans TamA involved in nitrogen metabolism, was found not only to regulate ammonium utilization but also to control cellulase gene expression in T. reesei. Whereas Δtam1 cultivated with peptone as a nitrogen source did not exhibit a growth defect that was observed on ammonium, it was still significantly compromised in cellulase biosynthesis. The absence of TAM1 almost fully abrogated the rapid cellulase gene induction in a resting-cell-inducing system. Overexpression of gdh1 encoding the key ammonium assimilatory enzyme in Δtam1 rescued the growth defect on ammonium but not the defect in cellulase gene expression. Of note, mutation of the Zn(II)2Cys6 DNA-binding motif of TAM1 hardly affected cellulase gene expression, while a truncated ARE1 mutant lacking the C-terminal 12 amino acids that are required for the interaction with TAM1 interfered with cellulase biosynthesis. The defect in cellulase induction of Δtam1 was rescued by overexpression of the key transactivator for cellulase gene, XYR1. Our results thus identify a nitrogen metabolism regulator as a new modulator participating in the regulation of induced cellulase gene expression. IMPORTANCE Transcriptional regulators are able to integrate extracellular nutrient signals and exert a combinatorial control over various metabolic genes. A plethora of such factors therefore constitute a complex regulatory network ensuring rapid and accurate cellular response to acquire and utilize nutrients. Despite the in-depth mechanistic studies of functions of the Zn(II)2Cys6-type transcriptional regulator TamA and its orthologues in nitrogen utilization, their involvement in additional physiological processes remains unknown. In this study, we demonstrated that TAM1 exerts a dual regulatory role in mediating ammonium utilization and induced cellulase production in the well known cellulolytic fungus Trichoderma reesei, suggesting a potentially converged regulatory node between nitrogen utilization and cellulase biosynthesis. This study not only contributes to unveiling the intricate regulatory network underlying cellulase gene expression in cellulolytic fungus but also helps expand our knowledge of fungal strategies to achieve efficient and coordinated nutrient acquisition for rapid propagation.


Assuntos
Celulase , Hypocreales , Trichoderma , Celulase/genética , Celulase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Hypocreales/genética , Expressão Gênica , Trichoderma/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
4.
Bioresour Technol ; 371: 128647, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36681353

RESUMO

Enzyme-mediated hydrolysis of cellulose always starts with an initial rapid phase, which gradually slows down, sometimes resulting in incomplete cellulose hydrolysis even after prolonged incubation. Although mechanisms such as end-product inhibition are known to play a role, the predominant mechanism appears to be reduced cellulose accessibility to the enzymes. When using Simon's stain to quantify accessibility, the accessibility of mechanically disintegrated and phosphoric acid-swollen cellulose substrates decreased as hydrolysis proceeded. In contrast, the poor initial accessibility of Avicel remained low throughout hydrolysis. However, washing the residual cellulose increased cellulose accessibility, likely due to the removal of tightly bound but non-productive enzymes which blocked access to more active enzymes in solution. Atomic force microscopy (AFM) analysis of the initial and residual cellulose collected when the hydrolysis plateaued, showed an increase in the roughness of the cellulose surface, possibly resulting in the tighter binding of less active cellulases.


Assuntos
Celulase , Celulases , Celulose/metabolismo , Celulase/metabolismo , Hidrólise , Celulases/metabolismo , Corantes
5.
Bioresour Technol ; 370: 128510, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36538959

RESUMO

To explore the interaction mechanism of pseudo-lignin (PL) with cellulase and its influence on cellulose hydrolysis, different PLs were extracted from pretreated bamboo holocellulose (HC) using different organic solvents. Meanwhile, the real-time interaction of PL and cellulase was analyzed using surface plasmon resonance (SPR). The results showed that the extraction effect of the tetrahydrofuran and 1, 4-dioxane/water solution on PL was more effective than the ethanol/water solution. The inhibition of PL fraction obtained from HC by acid pretreatment with higher temperature showed less effect on Avicel's enzymatic hydrolysis. SPR analysis revealed that PL formed at higher pretreatment temperature had a lower dissociation rate after adsorption with cellulase. Besides, the binding affinity of PL (160 °C) to cellulase was much greater than that of PL obtained from 180 °C, indicating PL extracted at higher temperature treated biomass is more easily dissociated from cellulase after binding.


Assuntos
Celulase , Celulases , Celulose/metabolismo , Lignina/metabolismo , Ressonância de Plasmônio de Superfície , Hidrólise , Água , Celulase/metabolismo
6.
Bioresour Technol ; 370: 128520, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36565817

RESUMO

To reduce the high cost of (hemi)cellulase production in lignocellulose biorefining, it is important to develop strategies to enhance enzyme productivity from economic and also readily manipulatable carbon sources. In this study, an artificial transcription factor XT was designed by fusing the DNA binding domain of Xyr1 to the transactivation domain of Tmac1. When overexpressed in Trichoderma reesei QM9414 Δxyr1, the XT recombinant strain (OEXT) greatly improved (hemi)cellulase production on repressing glucose compared with QM9414 on Avicel with 1.7- and 8.2-fold increases in pNPCase and xylanase activity, respectively. Both activities were even higher (0.9- and 33.8-fold higher, respectively) than the recombinant strain similarly overexpressing Xyr1. The dramatically enhanced xylanase activities in OEXT resulted from the elevated expression of various hemicellulases in the secretome. Moreover, the enzyme cocktail from OEXT improved the saccharification efficiency toward corn stover by 60% compared with enzymes from QM9414 with equal volume loading.


Assuntos
Celulase , Trichoderma , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Celulase/metabolismo , Glucose/metabolismo , Trichoderma/metabolismo
7.
Sci Rep ; 12(1): 19445, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376415

RESUMO

Trichoderma reesei is a widely used host for producing cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Here, we report a genetic modification strategy for industrial T. reesei that enables enzyme production using simple glucose without inducers, such as cellulose, lactose and sophorose. Previously, the mutated XYR1V821F or XYR1A824V was known to induce xylanase and cellulase using only glucose as a carbon source, but its enzyme composition was biased toward xylanases, and its performance was insufficient to degrade lignocellulose efficiently. Therefore, we examined combinations of mutated XYR1V821F and constitutively expressed CRT1, BGLR, VIB1, ACE2, or ACE3, known as cellulase regulators and essential factors for cellulase expression to the T. reesei E1AB1 strain that has been highly mutagenized for improving enzyme productivity and expressing a ß-glucosidase for high enzyme performance. The results showed that expression of ACE3 to the mutated XYR1V821F expressing strain promoted cellulase expression. Furthermore, co-expression of these two transcription factors also resulted in increased productivity, with enzyme productivity 1.5-fold higher than with the conventional single expression of mutated XYR1V821F. Additionally, that productivity was 5.5-fold higher compared to productivity with an enhanced single expression of ACE3. Moreover, although the DNA-binding domain of ACE3 had been considered essential for inducer-free cellulase production, we found that ACE3 with a partially truncated DNA-binding domain was more effective in cellulase production when co-expressed with a mutated XYR1V821F. This study demonstrates that co-expression of the two transcription factors, the mutated XYR1V821F or XYR1A824V and ACE3, resulted in optimized enzyme composition and increased productivity.


Assuntos
Celulase , Trichoderma , Celulase/metabolismo , Trichoderma/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Glucose/metabolismo , DNA/metabolismo , Regulação Fúngica da Expressão Gênica
8.
Arch Microbiol ; 204(11): 681, 2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36316590

RESUMO

Facing the critical issue of high production costs for cellulase, numerous studies have focused on improving the efficiency of cellulase production by potential cellulolytic microorganisms using agricultural wastes as substrates, extremophilic cellulases, in particular, are crucial in the biorefinery process because they can maintain activity under harsh environmental conditions. This study aims to investigate the ability of a potential carboxymethylcellulose-hydrolyzing bacterial strain H1, isolated from an Algerian saline soil and identified as Bacillus velezensis, to use untreated olive mill wastes as a substrate for the production of an endo-1,4-ß-glucanase. The enzyme was purified 44.9 fold using only two steps: ultrafiltration concentration and ion exchange chromatography, with final recovery of 80%. Its molecular mass was estimated to be 26 kDa by SDS-PAGE. Enzyme identification by LC-MS analysis showed 40% identity with an endo-1,3-1,4-ß-glucanase of GH-16 family. The highest enzymatic activity was significantly measured on barley ß-glucan (604.5 U/mL) followed by lichenan and carboxymethylcellulose as substrates, confirming that the studied enzyme is an endo-1,4-ß-glucanase. Optimal enzymatic activity was at pH 6.0-6.5 and at 60-65 °C. It was fairly thermotolerant, retaining 76.9% of the activity at 70 °C, and halotolerant, retaining 70% of its activity in the presence of 4 M NaCl. The enzyme had a Vmax of 625 U/min/mL and a high affinity with barley ß-glucan resulting a Km of 0.69 mg/mL. It also showed a significant ability to release cello-oligosaccharides. Based on such data, the H1 endo-1,4-ß-glucanase may have significant commercial values for industry, argo-waste treatment, and other biotechnological applications.


Assuntos
Celulase , Olea , beta-Glucanas , Celulase/metabolismo , Carboximetilcelulose Sódica , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Especificidade por Substrato
9.
Microb Cell Fact ; 21(1): 216, 2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253826

RESUMO

BACKGROUND: Cellulose degradation can determine mycelial growth rate and affect yield during the growth of Flammulina filiformis. The degradation of cellulose requires the joint action of a variety of cellulases, and some cellulase-related genes have been detected in mushrooms. However, little is known about the transcriptional regulatory mechanisms of cellulose degradation. RESULTS: In this study, FfMYB15 that may regulate the expression of cellulase gene FfCEL6B in F. filiformis was identified. RNA interference (RNAi) showed that FfCEL6B positively regulated mycelial growth. Gene expression analyses indicated that the expression patterns of FfCEL6B and FfMYB15 in mycelia cultured on the 0.9% cellulose medium for different times were similar with a correlation coefficient of 0.953. Subcellular localization and transcriptional activity analyses implied that FfMYB15 was located in the nucleus and was a transcriptional activator. Electrophoretic mobility shift assay (EMSA) and dual-luciferase assays demonstrated that FfMYB15 could bind and activate FfCEL6B promoter by recognizing MYB cis-acting element. CONCLUSIONS: This study indicated that FfCEL6B played an active role in mycelial growth of F. filiformis and was regulated by FfMYB15.


Assuntos
Celulase , Celulases , Celulase/metabolismo , Celulose/metabolismo , Flammulina , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Microbiologyopen ; 11(5): e1324, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36314761

RESUMO

Microbial cocultures are used as a tool to stimulate natural product biosynthesis. However, studies often empirically combine different organisms without a deeper understanding of the population dynamics. As filamentous organisms offer a vast metabolic diversity, we developed a model filamentous coculture of the cellulolytic fungus Trichoderma reesei RUT-C30 and the noncellulolytic bacterium Streptomyces coelicolor A3(2). The coculture was set up to use α-cellulose as a carbon source. This established a dependency of S. coelicolor on hydrolysate sugars released by T. reesei cellulases. To provide detailed insight into coculture dynamics, we applied high-throughput online monitoring of the respiration rate and fluorescence of the tagged strains. The respiration rate allowed us to distinguish the conditions of successful cellulase formation. Furthermore, to dissect the individual strain contributions, T. reesei and S. coelicolor were tagged with mCherry and mNeonGreen (mNG) fluorescence proteins, respectively. When evaluating varying inoculation ratios, it was observed that both partners outcompete the other when given a high inoculation advantage. Nonetheless, adequate proportions for simultaneous growth of both partners, cellulase, and pigment production could be determined. Finally, population dynamics were also tuned by modulating abiotic factors. Increased osmolality provided a growth advantage to S. coelicolor. In contrast, an increase in shaking frequency had a negative effect on S. coelicolor biomass formation, promoting T. reesei. This comprehensive analysis fills important knowledge gaps in the control of complex cocultures and accelerates the setup of other tailor-made coculture bioprocesses.


Assuntos
Celulase , Trichoderma , Celulase/metabolismo , Técnicas de Cocultura , Celulose/metabolismo , Dinâmica Populacional
11.
Biotechnol Lett ; 44(12): 1465-1475, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36269496

RESUMO

PURPOSE: This study aimed to reveal the roles of the protein kinase A catalytic subunit 1 (pkac1) and carbon catabolite repressor cre1 genes in cellulase production by Trichoderma reesei wild-type strain QM6a. Our strategy might be useful to construct a high-yielding cellulase strain for its wide application. METHODS: This paper describes cellulase activity, plate conidiation, and yellow pigment synthesis assays of QM6a with the disruption of pkac1 and cre1. RESULTS: Deletion of pkac1 (Δpkac1) had no effect on cellulase production or transcript levels of major cellulase genes in the presence of cellulose. Disruption of cre1 (Δcre1) resulted in a remarkable increase in cellulase production and expression of the four major cellulase genes. Double disruption of pkac1 and cre1 significantly improved enzyme activity and protein production. The double disruption also resulted in a significant reduction in yellow pigment production and abrogated conidial production. CONCLUSION: Double deletion of pkac1 and cre1 led to increased hydrolytic enzyme production in T. reesei using cellulose as a carbon source.


Assuntos
Celulase , Trichoderma , Trichoderma/metabolismo , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , Carbono/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
12.
J Phys Chem B ; 126(42): 8472-8485, 2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36251767

RESUMO

Interactions between particles moving on a linear track and their possible blocking by obstacles can lead to crowding, impeding the particles' transport kinetics. When the particles are enzymes processively catalyzing a reaction along a linear polymeric substrate, these crowding and blocking effects may substantially reduce the overall catalytic rate. Cellulose hydrolysis by exocellulases processively moving along cellulose chains assembled into insoluble cellulose particles is an example of such a catalytic transport process. The details of the kinetics of cellulose hydrolysis and the causes of the often observed reduction of hydrolysis rate over time are not yet fully understood. Crowding and blocking of enzyme particles are thought to be one of the important factors affecting the cellulose hydrolysis, but its exact role and mechanism are not clear. Here, we introduce a simple model based on an elementary transport process that incorporates the crowding and blocking effects in a straightforward way. This is achieved by making a distinction between binding and non-binding sites on the chain. The model reproduces a range of experimental results, mainly related to the early phase of cellulose hydrolysis. Our results indicate that the combined effects of clustering of binding sites together with the occupancy pattern of these sites by the enzyme molecules play a decisive role in the overall kinetics of cellulose hydrolysis. It is suggested that periodic desorption and rebinding of enzyme molecules could be a basis of a strategy to partially counter the clustering of and blocking by the binding sites and so enhance the rate of cellulose hydrolysis. The general nature of the model means that it could be applicable also to other transport processes that make a distinction between binding and non-binding sites, where crowding and blocking are expected to be relevant.


Assuntos
Celulase , Celulases , Trichoderma , Celulose/química , Hidrólise , Celulases/química , Cinética , Catálise , Análise por Conglomerados , Celulase/metabolismo
13.
Bioresour Technol ; 365: 128132, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36252752

RESUMO

Sustainable bioproduction usingcarbon neutral feedstocks, especially lignocellulosic biomass, has attracted increasing attention due to concern over climate change and carbon reduction. Consolidated bioprocessing (CBP) of lignocellulosic biomass using recombinantyeast of Saccharomyces cerevisiaeis a promising strategy forlignocellulosic biorefinery. However, the economic viability is restricted by low enzyme secretion levels.For more efficient CBP, MIG1spsc01isolated from the industrial yeast which encodes the glucose repression regulator derivative was overexpressed. Increased extracellular cellobiohydrolase (CBH) activity was observed with unexpectedly decreased cell wall integrity. Further studies revealed that disruption ofCWP2, YGP1, andUTH1,which are functionally related toMIG1spsc01, also enhanced CBH secretion. Subsequently, improved cellulase production was achieved by simultaneous disruption ofYGP1and overexpression ofSED5, which remarkably increased extracellular CBH activity of 2.2-fold over the control strain. These results provide a novel strategy to improve the CBP yeast for bioconversion of carbon neutral biomass.


Assuntos
Celulase , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Biomassa , Carbono/metabolismo , Celulase/metabolismo , Celulose 1,4-beta-Celobiosidase/genética , Celulose 1,4-beta-Celobiosidase/metabolismo , Parede Celular/metabolismo , Fermentação
14.
Int J Mol Sci ; 23(19)2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36233128

RESUMO

Hydrothermal pretreatment (HTP) has long been considered as an efficient and green treatment process on lignocellulosic biomass for bioconversion. However, the variations of cellulose supramolecular structures during HTP as well as their effects on subsequent enzymatic conversion are less understood. In this work, bamboo holocellulose with well-connected cellulose and hemicelluloses polysaccharides were hydrothermally treated under various temperatures. Chemical, morphological, and crystal structural determinations were performed systematically by a series of advanced characterizations. Xylan was degraded to xylooligosaccharides in the hydrolyzates accompanied by the reduced degree of polymerization for cellulose. Cellulose crystallites were found to swell anisotropically, despite the limited decrystallization by HTP. Hydrogen bond linkages between cellulose molecular chains were weakened due to above chemical and crystal variations, which therefore swelled, loosened, and separated the condensed cellulose microfibrils. Samples after HTP present notably increased surface area, favoring the adsorption and subsequent hydrolysis by cellulase enzymes. A satisfying enzymatic conversion yield (>85%) at rather low cellulase enzyme dosage (10 FPU/g glucan) was obtained, which would indicate new understandings on the green and efficient bioconversion process on lignocellulosic biomass.


Assuntos
Celulase , Lignina , Celulase/metabolismo , Celulose/química , Hidrólise , Lignina/metabolismo , Polissacarídeos/metabolismo , Xilanos
15.
J Agric Food Chem ; 70(42): 13574-13582, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36223298

RESUMO

N, N-dimethylformamide is frequently present in industrial wastewater and is environmentally detrimental. The current study aims to assess the utilization and biodegradation of N, N-dimethylformamide-containing wastewater to lessen the associated environmental load. Results show that addition of wastewater containing N, N-dimethylformamide to Trichoderma reesei fermentation media enhances cellulase production and facilitates cellulose hydrolysis. However, N, N-dimethylformamide is a cellulase enhancer that is not degraded during cellulase production in T. reesei fermentation and is retained in the N, N-dimethylformamide-enhanced cellulase solution. Indeed, the cellulosic sugar solution generated via lignocellulose hydrolysis with N, N-dimethylformamide-enhanced cellulase retains N, N-dimethylformamide. We further identified three core enzyme modules─N, N-dimethylformamidase, dimethylamine dehydrogenase, and methylamine dehydrogenase enzyme─which were inserted into Escherichia coli to develop metabolically engineered strains. These strains degraded N, N-dimethylformamide and produced succinate using N, N-dimethylformamide-enhanced cellulosic sugar as the substrate. The platform described here can be applied to effectively convert waste into valuable bioproducts.


Assuntos
Celulase , Trichoderma , Dimetilformamida/metabolismo , Trichoderma/metabolismo , Engenharia Metabólica , Celulase/metabolismo , Celulose/metabolismo , Hidrólise , Fermentação , Carboidratos , Ácido Succínico/metabolismo , Açúcares/metabolismo
16.
Proc Natl Acad Sci U S A ; 119(42): e2117467119, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36215467

RESUMO

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein-carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM-substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose-CBM bond rupture forces exceeding 15 pN.


Assuntos
Celulase , Clostridium thermocellum , Acústica , Proteínas de Bactérias/metabolismo , Carboidratos/química , Celulase/metabolismo , Celulose/metabolismo , Clostridium thermocellum/metabolismo , Análise Espectral , Açúcares
17.
Appl Environ Microbiol ; 88(20): e0107622, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36197104

RESUMO

The Gram-negative bacterium Cytophaga hutchinsonii digests cellulose through a novel cellulose degradation mechanism. It possesses the lately characterized type IX secretion system (T9SS). We recently discovered that N-glycosylation of the C-terminal domain (CTD) of a hypothetical T9SS substrate protein in the periplasmic space of C. hutchinsonii affects protein secretion and localization. In this study, green fluorescent protein (GFP)-CTDCel9A recombinant protein was found with increased molecular weight in the periplasm of C. hutchinsonii. Site-directed mutagenesis studies on the CTD of cellulase Cel9A demonstrated that asparagine residue 900 in the D-X-N-X-S motif is important for the processing of the recombinant protein. We found that the glycosyltransferase-related protein GtrA (CHU_0012) located in the cytoplasm of C. hutchinsonii is essential for outer membrane localization of the recombinant protein. The deletion of gtrA decreased the abundance of the outer membrane proteins and affected cellulose degradation by C. hutchinsonii. This study provided a link between the glycosylation system and cellulose degradation in C. hutchinsonii. IMPORTANCE N-Glycosylation systems are generally limited to some pathogenic bacteria in prokaryotes. The disruption of the N-glycosylation pathway is related to adherence, invasion, colonization, and other phenotypic characteristics. We recently found that the cellulolytic bacterium Cytophaga hutchinsonii also has an N-glycosylation system. The cellulose degradation mechanism of C. hutchinsonii is novel and mysterious; cellulases and other proteins on the cell surface are involved in utilizing cellulose. In this study, we identified an asparagine residue in the C-terminal domain of cellulase Cel9A that is necessary for the processing of the T9SS cargo protein. Moreover, the glycosyltransferase-related protein GtrA is essential for the localization of the GFP-CTDCel9A recombinant protein. Deletion of gtrA affected cellulose degradation and the abundance of outer membrane proteins. This study enriched the understanding of the N-glycosylation system in C. hutchinsonii and provided a link between N-glycosylation and cellulose degradation, which also expanded the role of the N-glycosylation system in bacteria.


Assuntos
Celulase , Celulase/genética , Celulase/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Asparagina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Cytophaga/genética , Cytophaga/metabolismo , Celulose/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Bioresour Technol ; 366: 128176, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36307030

RESUMO

In the present work, the effects of combined enzymatic hydrolysis by cellulase and xylanase (CXEH), fed-batch enzymatic hydrolysis (FBEH) operation and kinetics on production of ferulic acid (FA) and p-coumaric acid (pCA) from pretreated corn straws were investigated. The results showed that CXEH could efficiently increase production of FA and pCA. When performed the FBEH operation by feeding 150 mL enzymatic hydrolysis solution (1.5 % enzyme concentration, 5:4 (v/v) ratio of cellulase to xylanase and 2.0 % substrate loading) to 250 mL batch enzymatic hydrolysis solution at 36 h, the maximum production (2178.58 and 2710.17 mg/L) and production rate (590.95 and 727.89 mg/L.h) of FA and pCA were respectively obtained. Moreover, the disruption of fiber tissues, enhancement of crystallinity and accelerated degradation of hemicelluloses and lignocelluloses caused by CXEH contributed to effectively improving production of FA and pCA in corn straws.


Assuntos
Celulase , Zea mays , Hidrólise , Zea mays/metabolismo , Celulase/metabolismo
19.
Sci Rep ; 12(1): 17219, 2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36241677

RESUMO

The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose-degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification. The PfXyn5 has a molecular mass of ~ 55 kDa and showed broad activity against a range of substrates like xylan, xyloglucan, laminarin and p-nitrophenyl-ß-D-xylopyranoside, with the highest specific activity of 0.7 U/mg against xylan at pH 4.5 and 50 °C. Analysis of the degradation products of xylan and SCB by PfXyn5 showed significant production of xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from two (DP2) to six (DP6), thus, suggesting that the PfXyn5 is an endo-acting enzyme. The enzyme synergistically improved the saccharification of SCB when combined with the crude cellulase cocktail of P. funiculosum with a degree of synergism up to 1.32. The PfXyn5 was further expressed individually and simultaneously with a notable GH16 endoglucanase (PfEgl16) in a catabolite-derepressed strain of P. funiculosum, PfMig188, and the saccharification efficiency of the secretomes from the resulting transformants were investigated on SCB. The secretome of PfMig188 overexpressing Xyn5 or Egl16 increased the saccharification of SCB by 9% or 7%, respectively, over the secretome of PfMig188, while the secretome of dual transformant increased SCB saccharification by ~ 15% at the same minimal protein concentration.


Assuntos
Celulase , Saccharum , Celulase/metabolismo , Celulose/química , Endo-1,4-beta-Xilanases/química , Hidrólise , Saccharum/metabolismo , Açúcares/metabolismo , Talaromyces , Xilanos/metabolismo
20.
Carbohydr Polym ; 297: 120025, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36184173

RESUMO

Pectin is a major component in many agricultural feedstocks. Despite the wide use in industrial production of cellulases and hemicellulases, the fungus Trichoderma reesei lacks a complete enzyme set for pectin degradation. In this study, three representative pectinolytic enzymes were expressed and screened for their abilities to improve the efficiency of T. reesei enzymes on the conversion of different agricultural residues. By replacing 5 % of the T. reesei proteins, endopolygalacturonase and pectin lyase remarkably increased the release of sugars from inferior tobacco leaves. In contrast, pectin methylesterase showed the strongest improving effect (by 31.1 %) on the hydrolysis of beetroot residue. The pectin in beetroot residue was only mildly degraded with the supplementation of pectin methylesterase, which allowed the extraction of pectin keeping the original emulsifying activity with a 51.1 % higher yield. The results provide a basis for precise optimization of lignocellulolytic enzyme systems for targeted valorization of pectin-rich agricultural residues.


Assuntos
Celulase , Celulases , Trichoderma , Biomassa , Celulase/metabolismo , Celulases/metabolismo , Hidrólise , Pectinas/metabolismo , Poligalacturonase/metabolismo , Açúcares/metabolismo
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