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1.
J Chromatogr A ; 1627: 461380, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823093

RESUMO

Microwave-ultrasonic assisted aqueous enzymatic extraction (MUAAEE) was applied to extract tiger nut oil (TNO). The conditions of MUAAEE were optimized by Plackett-Burman design followed Box-Behnken design. An oil recovery of 85.23% was achieved under optimum conditions of a 2% concentration of mixed enzyme including cellulase, pectinase and hemicellulase (1/1/1, w/w/w), particle size <600 µm, microwave power 300 W, ultrasonic power 460 W, radiation temperature 40 °C, time 30 min, enzymolysis temperature 45 °C, pH 4.9, liquid-to-solid ratio 10 mL/g and time 180 min. Oil by MUAAEE revealed the similar fatty acid compositions, triglyceride compositions, thermal behaviour and flavour compared with oil by Soxhlet extraction (SE), while the oil quality of MUAAEE is superior to that of SE. Scanning electron microscopy revealed that structural disruption of tiger nut caused by MUAAEE facilitated the oil extraction. Results suggest that MUAAEE could be an efficient and environment-friendly method for extraction of TNO.


Assuntos
Cyperus/química , Enzimas/metabolismo , Micro-Ondas , Nozes/química , Óleos Vegetais/química , Ultrassom , Varredura Diferencial de Calorimetria , Celulase/metabolismo , Análise Discriminante , Ácidos Graxos/química , Glicosídeo Hidrolases/metabolismo , Poligalacturonase/metabolismo , Análise de Componente Principal , Reprodutibilidade dos Testes , Temperatura , Triglicerídeos/análise , Água/química
2.
Proc Natl Acad Sci U S A ; 117(29): 16776-16781, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636260

RESUMO

A particularly promising approach to deconstructing and fractionating lignocellulosic biomass to produce green renewable fuels and high-value chemicals pretreats the biomass with organic solvents in aqueous solution. Here, neutron scattering and molecular-dynamics simulations reveal the temperature-dependent morphological changes in poplar wood biomass during tetrahydrofuran (THF):water pretreatment and provide a mechanism by which the solvent components drive efficient biomass breakdown. Whereas lignin dissociates over a wide temperature range (>25 °C) cellulose disruption occurs only above 150 °C. Neutron scattering with contrast variation provides direct evidence for the formation of THF-rich nanoclusters (Rg ∼ 0.5 nm) on the nonpolar cellulose surfaces and on hydrophobic lignin, and equivalent water-rich nanoclusters on polar cellulose surfaces. The disassembly of the amphiphilic biomass is thus enabled through the local demixing of highly functional cosolvents, THF and water, which preferentially solvate specific biomass surfaces so as to match the local solute polarity. A multiscale description of the efficiency of THF:water pretreatment is provided: matching polarity at the atomic scale prevents lignin aggregation and disrupts cellulose, leading to improvements in deconstruction at the macroscopic scale.


Assuntos
Biotecnologia/métodos , Lignina/química , Madeira/química , Proteínas de Bactérias/metabolismo , Biomassa , Celulase/metabolismo , Furanos/química , Gluconacetobacter xylinus/enzimologia , Hidrólise , Lignina/metabolismo , Populus/química , Solventes/química , Tensoativos/química
3.
PLoS One ; 15(7): e0236518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702033

RESUMO

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0-9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes.


Assuntos
Celulose/metabolismo , Polissacarídeos/metabolismo , Microbiologia do Solo , Thermoanaerobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Biomassa , Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Filogenia , Especificidade por Substrato , Thermoanaerobacterium/classificação , Thermoanaerobacterium/enzimologia , Thermoanaerobacterium/isolamento & purificação
4.
Food Chem ; 332: 127416, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32619946

RESUMO

Water bamboo shoots quickly deteriorate after harvest as a result of rapid lignification and softening. Nitric oxide (NO) has been used to extend the postharvest life of several other vegetables. Here, we examined the effect of NO on the storage of water bamboo shoots at 4℃ for 28 days. Without NO, fresh weight and firmness decreased quickly, while the cellulose and lignin contents increased sharply during storage. NO treatment delayed softening by maintaining the integrity of the cell wall and inhibiting the degradation of protopectin and the expressions of pectin methylesterase and polygalacturonase. NO treatment also delayed cellulose synthesis by increasing cellulase activity. NO treatment decreased the synthesis of lignin by inhibiting the activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, laccase and peroxidase. These results indicate that NO treatment is effective at suppressing the softening and lignification of water bamboo shoots during postharvest storage.


Assuntos
Armazenamento de Alimentos/métodos , Óxido Nítrico/farmacologia , Poaceae/efeitos dos fármacos , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Celulase/metabolismo , Celulose/metabolismo , Temperatura Baixa , Lignina/metabolismo , Microscopia Eletrônica de Varredura , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Poaceae/metabolismo , Poligalacturonase/metabolismo
5.
Arch Microbiol ; 202(9): 2591-2597, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32607725

RESUMO

To date, the genus Parvularcula consists of 6 species and no potential application of this genus was reported. Current study presents the genome sequence of Parvularcula flava strain NH6-79 T and its cellulolytic enzyme analysis. The assembled draft genome of strain NH6-79 T consists of 9 contigs and 7 scaffolds with 3.68 Mbp in size and GC content of 59.87%. From a total of 3,465 genes predicted, 96 of them are annotated as glycoside hydrolases (GHs). Within these GHs, 20 encoded genes are related to cellulosic biomass degradation, including 12 endoglucanases (5 GH10, 4 GH5, and 3 GH51), 2 exoglucanases (GH9) and 6 ß-glucosidases (GH3). In addition, highest relative enzyme activities (endoglucanase, exoglucanase, and ß-glucosidase) were observed at 27th hour when the strain was cultured in the carboxymethyl cellulose/Avicel®-containing medium for 45 h. The combination of genome analysis with experimental studies indicated the ability of strain NH6-79 T to produce extracellular endoglucanase, exoglucanase, and ß-glucosidase. These findings suggest the potential of Parvularcula flava strain NH6-79 T in cellulose-containing biomass degradation and that the strain could be used in cellulosic biorefining process.


Assuntos
Alphaproteobacteria/enzimologia , Alphaproteobacteria/genética , Genoma Bacteriano/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Composição de Bases , Biomassa , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
6.
Arch Anim Nutr ; 74(4): 309-324, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32441546

RESUMO

The study was conducted to determine effects of a complex of fibre-degrading enzymes (xylanase, cellulase and ß-glucanase) on nutrient digestibility, fibre fermentation and concentrations of short chain fatty acids (SCFA) at different parts of digestive tract in pigs fed different fibre-rich ingredients. A total of 36 barrows fitted with T-cannulas in the distal ileum (initial body weight of 41.1 ± 2.7 kg) were randomly allotted to six dietary treatments with three different high-fibre diets including maize bran (MB), sugar beet pulp (SBP) and soybean hulls (SH) with or without supplementation of fibre-degrading enzymes. Enzyme supplementation improved (p < 0.05) apparent ileal digestibility (AID) of dietary gross energy (GE), crude protein, dry matter (DM), organic matter (OM), total dietary fibre (TDF), neutral detergent fibre (NDF) and apparent total tract digestibility (ATTD) of dietary GE, DM, OM, TDF, insoluble dietary fibre (IDF) when pigs were fed MB, SBP or SH diets. When compared to the SBP and SH diets, the AID of GE, DM, ash, OM and NDF in diet MB was higher (p < 0.05), but the hindgut disappearance and ATTD of nutrients, except for ether extract and crude ash, were lower (p < 0.05). Enzyme supplementation increased acetate and total SCFA concentrations in ileal digesta and faeces of pigs. In conclusion, enzyme addition improved IDF fermentation and SCFA concentration in the whole intestine of pigs, and there was a large variation of digestibility of fibre components among MB, SH and SBP owing to their different fibre composition. Therefore, fibre-degrading enzymes should be applied to fibrous diets to improve efficient production of swine, especially considering low fibre digestibility of fibre-rich ingredients.


Assuntos
Celulase/metabolismo , Digestão , Endo-1,3(4)-beta-Glucanase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Íleo/fisiologia , Nutrientes/fisiologia , Sus scrofa/fisiologia , Ração Animal/análise , Animais , Beta vulgaris/química , Celulase/administração & dosagem , Dieta/veterinária , Suplementos Nutricionais/análise , Endo-1,3(4)-beta-Glucanase/administração & dosagem , Endo-1,4-beta-Xilanases/administração & dosagem , Fermentação , Trato Gastrointestinal , Íleo/efeitos dos fármacos , Masculino , Distribuição Aleatória , Soja/química , Zea mays/química
7.
Chemosphere ; 256: 127126, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32470736

RESUMO

Rapid industrialization and consumption of fossil fuels have led to considerable progress in the production of renewable biofuels like bioethanol. Lignocellulosic biomass such as grasses serves as cheap feedstocks for the production of bioethanol. However, the process involved in lignocellulosic bioethanol production is expensive which restricts its industrial production. The present study thus attempted to investigate a partially consolidated bioprocessing (PCB) approach using two isolated anaerobic thermophiles i.e. Bacillus paranthracis and Bacillus nitratireducens for direct conversion of ultra-sonication assisted sodium hydroxide (UA-NaOH) pretreated Denannath grass to bioethanol in co-culture consortium batch fermentation experiments. The process parameters for the PCB approach were optimized using the Box-Behnken design of Response Surface Methodology (RSM). The parameters that were considered were substrate concentration (5-10 g), incubation time (30-66 h), inoculum volume [1:1 to 3:3 (% v/v) and temperature (50-65 °C). The maximum ethanol concentration of 8.46 mM (0.39 g/L from 7.5 g/L of substrate loading) and ethanol yield (Yp/s) of 0.55 g/g of reducing sugar was obtained at 57.5 °C. In the same conditions the cellulase and xylanase activities were 0.8 U/mL and 11.53 U/mL respectively, while the lactate and acetate concentrations were 0.2 mM (0.009 g/L) and 2.9 mM (0.13 g/L) correspondingly. An increase in the substrate loadings to 250 g/L in a batch fermenter (3 L) resulted in the production of 373.35 mM (17.1 g/L) of ethanol concentration and Yp/s of 0.16 g/g of reducing sugar.


Assuntos
Bacillus/metabolismo , Biocombustíveis/análise , Reatores Biológicos/microbiologia , Etanol/análise , Pennisetum/metabolismo , Anaerobiose , Biomassa , Carboidratos , Celulase/metabolismo , Fermentação , Hidrólise , Hidróxido de Sódio
8.
J Biosci Bioeng ; 130(2): 137-141, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32331776

RESUMO

We had developed a new pretreatment system using cow rumen fluid to improve the methane production from lignocellulosic substrates. However, the pretreatment conditions differ from the in-situ rumen environment, therefore different microbes may be involved in plant cell wall decomposition. In the current study, shotgun metagenomic analysis using MiSeq platform was performed to elucidate the bacteria which produce cellulase and hemicellulase in this pretreatment system. The rumen fluid which contained waste paper pieces (0.1% w/v) were incubated at 37°C during 120 h. The fluid samples were collected from the reactor at each time-point and analyzed for chemical properties. Rumen microbial DNA was extracted from 0-h and 60-h samples and subjected to shotgun-metagenomic analysis. After pretreatment, approximately half of cellulose and hemicellulose contents of the waste paper were decomposed and some volatile fatty acids were accumulated. Clostridia (e.g., Ruminococcus and Clostridium) were the predominant bacteria before and after 60-h pretreatment, and their relative abundance was increased during pretreatment. However, Prevotella and Fibrobacter, one of the most dominant bacteria in-situ rumen fluid, were observed less than 3% before incubation and they were decreased after pretreatment. Genes encoding cellulase and hemicellulase were mainly found in Ruminococcus, Clostridium, and Caldicellulosiruptor. Calicellulosiruptor, which had not been previously identified as the predominant genus in lignocellulose decomposition in in-situ rumen conditions, might be considered as the main fibrolytic bacterium in this system. Thus, this study demonstrated that the composition of fibrolytic bacteria in this system was greatly different from those in the in-situ rumen.


Assuntos
Bactérias/classificação , Bactérias/enzimologia , Microbiologia Industrial , Lignina/metabolismo , Metagenoma , Rúmen/microbiologia , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Biodiversidade , Biomassa , Bovinos , Celulase/genética , Celulase/metabolismo , Ácidos Graxos Voláteis , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Metano/biossíntese
9.
Chemosphere ; 253: 126727, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32289609

RESUMO

The major bottleneck for industrial applications of microbial flocculants is the high production cost. Here, a novel bacterium, Diaphorobacter nitroreducens R9, was isolated that can secret ligninase and cellulase and simultaneously produce bioflocculants (MBF-9) through conversion of ramie biomass. The production of MBF-9 was closely related to the ligninase and cellulase activities of D. nitroreducens. Both ligninase and cellulase showed peak activity at pH 8.5 and 6.0 and retained approximately 80% of cellulase activity and 95% of ligninase activity at pH 8.0. The optimal production conditions with the highest bioflocculant yield (3.86 g/L degumming wastewater) were determined at a fermentation time of 48 h, fermentation temperature of 30 °C, inoculum size of 4.0%, CODCr of ramie degumming wastewater of 1500 mg/L and initial pH of 8.0. In addition, MBF-9 removed 96.2% turbidity, 79.5% chemical oxygen demand (COD), 59.2% lignin, and 63.1% sugar from the pulping wastewater at an MBF-9 dosage of 831.57 mg/L.


Assuntos
Boehmeria/metabolismo , Comamonadaceae/metabolismo , Floculação , Águas Residuárias/microbiologia , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Análise da Demanda Biológica de Oxigênio , Biomassa , Celulase/metabolismo , Comamonadaceae/isolamento & purificação , Fermentação , Concentração de Íons de Hidrogênio , Oxigenases/metabolismo , Temperatura , Poluentes Químicos da Água/metabolismo
10.
World J Microbiol Biotechnol ; 36(3): 51, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157408

RESUMO

Culture-independent molecular-based approaches can be used to identify genes of interest from environmental sources that have desirable properties such as thermo activity. For this study, a putative thermo stable endoglucanase gene was identified from a mixed culture resulting from the inoculation of Brock-CMcellulose (1%) broth with mudspring water from Mt. Makiling, Laguna, Philippines that had been incubated at 90 °C. Genomic DNA was extracted from the cellulose-enriched mixed culture and endo1949 forward and reverse primers were used to amplify the endoglucanase gene, which was cloned into pCR-script plasmid vector. Blastn alignment of the sequenced insert revealed 99.69% similarity to the glycosyl hydrolase, sso1354 (CelA1; Q97YG7) from Saccharolobus solfataricus. The endoglucanase gene (GenBank accession number MK984682) was determined to be 1,021 nucleotide bases in length, corresponding to 333 amino acids with a molecular mass of ~ 37 kDa. The endoglucanase gene was inserted into a pET21 vector and transformed in E. coli BL21 for expression. Partially purified recombinant Mt. Makiling endoglucanase (MM-Engl) showed a specific activity of 187.61 U/mg and demonstrated heat stability up to 80 °C. The thermo-acid stable endoglucanase can be used in a supplementary hydrolysis step to further hydrolyze the lignocellulosic materials that were previously treated under high temperature-dilute acid conditions, thereby enhancing the release of more glucose sugars for bioethanol production.


Assuntos
Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , DNA , Genômica , Água/metabolismo , Sequência de Aminoácidos , Archaea/enzimologia , Archaea/genética , Bactérias/enzimologia , Bactérias/genética , Sequência de Bases , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Filipinas , Proteínas Recombinantes , Alinhamento de Sequência , Sulfolobales/enzimologia , Sulfolobales/genética , Temperatura , Microbiologia da Água
11.
Enzyme Microb Technol ; 134: 109481, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044028

RESUMO

The recombinant endoglucanase gene (EG I) from Trichoderma reesei was successfully expressed in Pichia pastoris for the purpose of producing oligosaccharides from various biomass-derived substrates. Interestingly, the recombinant endoglucanase I (ReEG I) showed the catalytic activity towards both cellulose and xylan hydrolysis, yet it was more efficient with xylans. Among various glucans and xylans substrates (paper pulp, carboxymethylated cellulose, oat spelt xylan, birchwood xylan), birchwood xylan displayed a higher yield of xylooligosaccharides (XOS) (69.5 % after optimization). Eventually, it was observed that ReEG I could simultaneously produce XOS and COS, when the alkali-extracted corncob residues were used as substrate. This is the first report on simultaneous production of XOS and COS by recombinant endoglucanase I from Trichoderma reesei expressed in Pichia pastoris, where a novel application of genetically engineered enzymes is proposed to provide an attractive application for high value utilization of biomass.


Assuntos
Biomassa , Celulase/genética , Oligossacarídeos/biossíntese , Trichoderma/enzimologia , Celulase/metabolismo , Celulose/metabolismo , Hidrólise , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Trichoderma/genética , Xilanos/metabolismo , Zea mays/metabolismo
12.
Appl Microbiol Biotechnol ; 104(5): 2079-2096, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31980921

RESUMO

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the ß-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Enzimas Multifuncionais/metabolismo , Paenibacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Celulase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Hidrólise , Cinética , Manosidases/metabolismo , Simulação de Acoplamento Molecular , Enzimas Multifuncionais/química , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/isolamento & purificação , Mutação , Oligossacarídeos/biossíntese , Paenibacillus/genética , Paenibacillus/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xilosidases/metabolismo
14.
Nucleic Acids Res ; 48(5): 2209-2219, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31943056

RESUMO

Ongoing large-scale genome sequencing projects are forecasting a data deluge that will almost certainly overwhelm current analytical capabilities of evolutionary genomics. In contrast to population genomics, there are no standardized methods in evolutionary genomics for extracting evolutionary and functional (e.g. gene-trait association) signal from genomic data. Here, we examine how current practices of multi-species comparative genomics perform in this aspect and point out that many genomic datasets are under-utilized due to the lack of powerful methodologies. As a result, many current analyses emphasize gene families for which some functional data is already available, resulting in a growing gap between functionally well-characterized genes/organisms and the universe of unknowns. This leaves unknown genes on the 'dark side' of genomes, a problem that will not be mitigated by sequencing more and more genomes, unless we develop tools to infer functional hypotheses for unknown genes in a systematic manner. We provide an inventory of recently developed methods capable of predicting gene-gene and gene-trait associations based on comparative data, then argue that realizing the full potential of whole genome datasets requires the integration of phylogenetic comparative methods into genomics, a rich but underutilized toolbox for looking into the past.


Assuntos
Biologia Computacional/métodos , Epistasia Genética , Genoma , Família Multigênica , Filogenia , Animais , Celulase/classificação , Celulase/genética , Celulase/metabolismo , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Dictyostelium/enzimologia , Dictyostelium/genética , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Dosagem de Genes , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Phascolarctidae/genética , Phascolarctidae/metabolismo , Plantas/classificação , Plantas/genética , Plantas/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(5): 2385-2394, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31953261

RESUMO

Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature's finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), ß-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.


Assuntos
Membrana Celular/metabolismo , Celulossomas/metabolismo , Kluyveromyces/genética , Kluyveromyces/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , Celulossomas/enzimologia , Celulossomas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/genética , Clostridium thermocellum/genética , Etanol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Kluyveromyces/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
16.
Int J Mol Sci ; 21(2)2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31936000

RESUMO

Fermentable sugars are important intermediate products in the conversion of lignocellulosic biomass to biofuels and other value-added bio-products. The main bottlenecks limiting the production of fermentable sugars from lignocellulosic biomass are the high cost and the low saccharification efficiency of degradation enzymes. Herein, we report the secretome of Trichoderma harzianum EM0925 under induction of lignocellulose. Numerously and quantitatively balanced cellulases and hemicellulases, especially high levels of glycosidases, could be secreted by T. harzianum EM0925. Compared with the commercial enzyme preparations, the T. harzianum EM0925 enzyme cocktail presented significantly higher lignocellulolytic enzyme activities and hydrolysis efficiency against lignocellulosic biomass. Moreover, 100% yields of glucose and xylose were obtained simultaneously from ultrafine grinding and alkali pretreated corn stover. These findings demonstrate a natural cellulases and hemicellulases mixture for complete conversion of biomass polysaccharide, suggesting T. harzianum EM0925 enzymes have great potential for industrial applications.


Assuntos
Celulase/metabolismo , Glicosídeo Hidrolases/metabolismo , Lignina/metabolismo , Trichoderma/enzimologia , Biocombustíveis/microbiologia , Fermentação , Glucose/metabolismo , Hidrólise , Trichoderma/metabolismo , Xilose/metabolismo , Zea mays/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-31647988

RESUMO

This review discusses the reaction catalysed, and the structure and function of the cellulase, endo-ß-1,4-glucanase and the hemicellulase enzymes, ß-1,3-glucanase and endo-ß-1,4-mannase that are present in numerous invertebrate groups with a diverse range of feeding specialisations. These range from microbial deposit and filter feeders, micro and macrophagous algal feeders, omnivores to herbivorous leaf litter and wood feeders. Endo-ß-1,4-glucanase from glycosyl hydrolase family 9 (GH9) digests cellulose like ß-1,4-glucans from a range of materials. As it hydrolyses crystalline cellulose very slowly, it is a poor cellulase. Where tested, the enzyme has dual endo-ß-1,4-glucanase and lichenase activity. Its presence does not necessarily indicate the ability of an animal to digest cellulose. It only indicates the ability to digest ß-1,4-glucans and its function, which is discussed in this review, should be considered with reference to the substrates present in the diet. ß-1,3-glucanase (laminarinase) belongs to glycosyl hydrolase family 16 (GH16) and hydrolyses ß-1.3-glucans. These polysaccharides are present in the cell walls of algae, protozoans and yeast, and they also occur as storage polysaccharides within protozoans and algae. Depending on their site of expression, these enzymes may function as a digestive enzyme or may be involved in innate immunity. Enzymes present in the digestive fluids or tissues, would be digestive. Haemolymph GH16 proteins may be involved in innate immunity through the activation of the phenol oxidase system. Insect GH16 proteins expressed within the haemolymph have lost their catalytic residues and function as ß-glucan binding proteins. In contrast, crustacean GH16 proteins expressed within the same tissue, have retained the catalytic residues and thus possibly their ß-1,3-glucanase activity. The potential function of which is discussed. Endo-ß-1,4-mannase from glycosyl hydrolase family 5, subfamily 10 (GH5_10) hydrolyses mannan, glucomannan and galactomannan. These hemicelluloses are present in the cell walls of plants and algae and also function as storage polysaccharides within legume and palm seeds. They are digestive enzymes whose high expression in some species suggests they are a major contributor to hemicellulose digestion. They may also provide the animal with substantial amounts of monosaccharides for energy.


Assuntos
Proteínas de Artrópodes , Celulase , Glicosídeo Hidrolases , Invertebrados , Filogenia , Polissacarídeos/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Celulase/genética , Celulase/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Invertebrados/enzimologia , Invertebrados/genética
18.
Appl Biochem Biotechnol ; 190(1): 113-128, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31301011

RESUMO

Raw domestic wastewater was used as a culture medium for cellulase production in a bubble column reactor (6.2 UFP/mL, 64.6 U/L h) using the strain Trichoderma harzianum TRIC03-LPBII. Cellulases presented optimum pH and temperature between 4 and 5 and 50 and 70 °C, respectively. Enzymatic extract was concentrated through ultrafiltration and then a cellulolytic formulation was prepared with the addition of sorbitol (50% w/v) and benzoic acid (0.05% w/v). High cellulase stability of around 100% was reached after 30 days at 4 °C. The concentrated extract was also dried in a spray-dryer with the addition of maltodextrin at 20% (w/v), resulting in powder enzymatic formulation with 85% stability after 60 days. With these characteristics, the liquid and powder cellulase products have potential to be used in different industrial applications.


Assuntos
Reatores Biológicos , Celulase/metabolismo , Pós , Águas Residuárias/química , Meios de Cultura , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Temperatura , Trichoderma/enzimologia , Trichoderma/metabolismo
19.
Appl Biochem Biotechnol ; 190(2): 448-463, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31378843

RESUMO

Cellulases, as environmentally appropriate catalysts specifically acting on cellulosic substrates, are important for the industrial conversion of lignocellulose and modification of cellulose products. After decades of research, a fundamental understanding of cellulase-mediated hydrolysis of cellulose is that its ability to processively act as a key for the complete enzymatic hydrolysis of natural crystalline cellulose. Two types of processive cellulases are known: exoglucanases and processive endoglucanases. Exoglucanases are typical processive enzymes, and they have been studied in detail so that their modes of action and mechanisms are reasonably well characterized. Conversely, endoglucanases are less well characterized because of the non-universality and structural diversity. However, processive endoglucanases have certain characteristics that exoglucanases lack such as hydrolysis product diversity and independent hydrolyze natural crystalline cellulose. Therefore, besides the conversion of cellulose to monosaccharide, they might be useful for modification of fibrous substrates and preparation of cellulose oligosaccharides. Herein, we review in detail the sources, hydrolysis products, application, and possible hydrolysis mechanisms of processive endoglucanases.


Assuntos
Celulase/metabolismo , Bactérias/enzimologia , Catálise , Celulases/metabolismo , Celulose/metabolismo , Fungos/enzimologia
20.
Artigo em Inglês | MEDLINE | ID: mdl-31751767

RESUMO

Millipedes represent a model for the study of organic matter transformation, animal-microbial interactions, and compartmentalisation of digestion. The activity of saccharidases (amylase, laminarinase, cellulase, xylanase, chitinase, maltase, cellobiase, and trehalase) and protease were measured in the midgut and hindgut contents and walls of the millipedes Archispirostreptus gigas and Epibolus pulchripes. Assays done at pH 4 and 7 confirmed activities of all enzymes except xylanase. Hydrolysing of starch and laminarin prevailed. The hindgut of E. pulchripes was shorter, less differentiated. Micro-apocrine secretion was observed only in the midgut of A. gigas. Merocrine secretion was present in midgut and hindgut of E. pulchripes, and in the pyloric valve and anterior hindgut of A. gigas. Alpha-polysaccharidases were mostly active in the midgut content and walls, with higher activity at pH 4. The low activity of amylase (A. gigas) and laminarinase (E. pulchripes) in midgut tissue may indicate their synthesis in salivary glands. Cellulases were found in midgut. Chitinases, found in midgut content and tissue (E. pulchripes) or concentrated in the midgut wall (A. gigas), were more active at an acidic pH. Polysaccharidases were low in hindguts. Protease shows midgut origin and alkaline activity extending to the hindgut in E. pulchripes, whereas in A. gigas it is of salivary gland origin and acid activity restricted to the midgut. Some disaccharidases, with more alkaline activity, showed less apparent midgut-hindgut differences. It may indicate an axial separating of the primary and secondary digestion along the intestinal pH gradient or the presence of enzymes of hindgut parasites.


Assuntos
Artrópodes/enzimologia , Quitinases/metabolismo , Animais , Artrópodes/classificação , Celulase/metabolismo , Quitinases/fisiologia , Trato Gastrointestinal/enzimologia , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Polissacarídeos/metabolismo , Especificidade por Substrato
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