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1.
J Food Sci ; 85(2): 324-331, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31968392

RESUMO

Anthocyanins in wine principally depend on grape skin extractable anthocyanin content, that is, the amount of anthocyanins present in grape skin that are released to wine during the maceration stage. This amount of extractable anthocyanins is closely linked to the cell wall degradation of skin cells. Indeed, among other methodologies, the maceration in presence of different enzymes can be used to increase cell wall degradation, and therefore, the amount of anthocyanins extracted from grape skins to wine. Vitis vinifera L. cv. Tempranillo and Syrah red grapes have been identified as samples with low anthocyanin extraction potential by near infrared hyperspectral imaging. Grape skins have been macerated in the presence of cellulase, glucosidase, and pectinase. Then, color of the supernatants and phenolic compounds extracted from grape skins (total phenols, total flavanols, and total and individual anthocyanins) has been determined. Cellulase and glucosidase have shown a positive effect in the extraction of phenolic compounds from these grapes. Macerations carried out in the presence of cellulase have produced supernatants with a more intense color (lower lightness and higher chroma values), and a higher extraction of flavanols and anthocyanins than the respective control essays. However, pectinase treatments have produced the opposite effect, which could be partially explained by an eventual interaction between the cell wall polysaccharides liberated by pectinase and the phenolic compounds extracted. Synergy effects do not appear between cellulase and glucosidase. Moreover, the negative effect of the addition of pectinase might be due to the interactions between the cell wall material liberated by pectinase and the phenolic compounds extracted. PRACTICAL APPLICATION: In the present study, grape samples with a low anthocyanin extraction potential have been identified, and these samples have been macerated in the presence of different enzymes. The applied enzymes were three of the most common enzymes that are applied in the wine industry. Individual enzymes and mixtures have been applied to Syrah and Tempranillo grape skin samples and the results have been compared to control macerations. Knowledge in this topic will help the production of quality wines.


Assuntos
Antocianinas/análise , Antocianinas/isolamento & purificação , Fenóis/análise , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Vitis/química , Biocatálise , Celulase/química , Cor , Frutas/química , Glucosidases/química , Fenóis/química , Poligalacturonase/química , Vinho/análise
3.
Carbohydr Polym ; 230: 115661, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887893

RESUMO

This study indicated tailoring efficient polymer-enzyme bioconjugates with superb stability and activity for practical utilization of cellulase enzyme in hydrolyzing lignocellulosic biomass. To this goal, a dual crosslinking (DC) strategy was presented to synthesize novel 3D networks of carboxymethyl cellulose grafted copolymers of 2-acrylamido-2methyl propane sulfonate and acrylamide (CMC-g-poly(AMPS-co-AAm)) hydrogels. Graphene oxide (GO) nano-sheets were utilized as nano-filler and physical cross-linker making H-bondings between polymeric chains to prepare GO@CMC-g-poly(AMPS-co-AAm) networks. The GO content effects on the performance of as-synthesized architectures in conjugation to a model enzyme (PersiCel1) were examined. PersiCel1 immobilization on the GO reinforced hydrogels resulted in noticeable retaining near 60 % of its maximum activity at 90 °C, along with the remarkable enhancement of its specific activity and storage stability. Compared with the free PersiCel1, the immobilized enzyme on the GO containing hydrogels showed 154.8 % increase in conversion of alkalin-treated sugar beet pulp, while the PersiCel1/neat-Hydrogel indicated an increment of 66.7 %, under the same conditions.


Assuntos
Celulase/química , Enzimas Imobilizadas/química , Lignina/química , Acrilamida/química , Acrilamidas/química , Alcanossulfonatos/química , Carboximetilcelulose Sódica/química , Grafite/química , Hidrogéis/química , Hidrólise
4.
J Biosci Bioeng ; 129(1): 104-109, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31400993

RESUMO

Succinic acid, an important intermediate in the manufacture of plastics and other commodity and specialty chemicals, is currently made primarily from petroleum. We attempted to biosynthesize succinic acid through microbial fermentation of cellulosic sugars derived from the bagasse of sweet sorghum, a renewable feedstock that can grow in a wide range of climates around the world. We investigated pretreating sweet sorghum bagasse (SSB) with concentrated phosphoric acid at mild conditions (40-85°C) at various residence times and biomass concentrations. We then subjected the pretreated SSB to enzymatic hydrolysis with a commercial cellulase to release glucose. The highest glucose yield was obtained when SSB was pretreated at 50°C for 43 min at 130 g/L biomass concentration on dry basis. Fermentation was carried out with Actinobacillus succinogenes 130Z, which readily converted 29.2 g/L of cellulosic glucose to 17.8 g/L of succinic acid in a 3.5-L bioreactor sparged with CO2 at a rate of 0.5 vvm, thus reducing the carbon footprint of the process. Overall, we demonstrated, for the first time, the use of SSB for production of succinic acid using practices that lower energy use, future equipment cost, waste generation, and carbon footprint.


Assuntos
Actinobacillus/metabolismo , Celulose/metabolismo , Sorghum/microbiologia , Ácido Succínico/metabolismo , Actinobacillus/crescimento & desenvolvimento , Biocatálise , Biomassa , Reatores Biológicos/microbiologia , Celulase/química , Celulose/química , Fermentação , Hidrólise , Sorghum/química , Ácido Succínico/química
5.
ChemSusChem ; 13(4): 663-667, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-31802645

RESUMO

The solid-solid (immobilized cellulase-insoluble cellulose) phase cellulose hydrolysis reaction is significant in cellulosic biomass conversion processes but hindered because of its low efficiency. Herein, a smart temperature-pH dual-responsive material (D-N-N material) was prepared to be used as a carrier for cellulase recovery. This D-N-N material could undergo reversible and switchable transitions between solution, hydrogel, and solid phases. The following results were demonstrated: 1) the hydrolytic degree of this strategy could be as high as that of free cellulase in buffer solution; 2) the cellulase could be encapsulated into the D-N-N hydrogel without significant leaching and most of the cellulase activity was retained after recycling for at least 10 batches; and 3) more than 95 % of the glucose inside the hydrogel could be extracted during the hydrogel-solid transition within 1 h, which can assist in the high-efficiency separation of cellulase from glucose. The results suggested that this strategy provides a feasible platform for efficient cellulose hydrolysis and could be applied to other bio-derived reactions.


Assuntos
Celulase/química , Celulose/química , Enzimas Imobilizadas/química , Hidrogéis/química , Polímeros/química , Acrilamida/química , Celulase/metabolismo , Celulose/metabolismo , Enzimas Imobilizadas/metabolismo , Glucose/química , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Transição de Fase , Temperatura
6.
Mater Sci Eng C Mater Biol Appl ; 106: 110169, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753391

RESUMO

Bacteria mediated synthesis of magnetic nanoparticles (MNPs) for biotechnological applications is an important area of nanotechnology. This study demonstrates the use of iron tolerant bacterium for synthesis of MNPs for cellulase immobilization and photocatalytic activity. The enrichment, isolation, screening and molecular identification led to the selection of Pseudomonas stutzeri KDP_M2 with high degree of iron tolerance. The synthesis parameters such as 1 mM ferric quinate, pH 9 and 96 h static incubation were found optimum for maximum yield of 210 mg/L. The characterization using various techniques indicated that MNPs were Hematite (Fe2O3) with particle size between 10 and 20 nm. Further, vibrating sample magnetometer and thermogravimetric analyses demonstrated the superparamagnetic nature with high thermal stability. The MNPs were found an excellent support for immobilization of industrially important cellulase with 96.5% binding efficiency. The immobilization which was confirmed by Fourier transform infrared spectroscopy indicated that immobilization did not reduce the cellulase activity, rather enhanced the thermal stability and operational temperature range of cellulase. The immobilized cellulase showed maximum cellulolytic activity at pH 4.6 and retained 80% activity upto 3rd cycle of reuse, therefore, can be utilized repeatedly at acidic conditions.The monitoring the photocatalytic activity showed rapid degradation of methyl violet and methylene blue within initial 10 min. of reaction.


Assuntos
Celulase/metabolismo , Ferro/farmacologia , Nanopartículas de Magnetita/química , Pseudomonas stutzeri/efeitos dos fármacos , Pseudomonas stutzeri/metabolismo , Celulase/química , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura
7.
Biochim Biophys Acta Gen Subj ; 1864(1): 129434, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525408

RESUMO

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts ß-lactoglobulin, which has mainly ß-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original ß-sheet structure. Here we extend these studies to the large ß-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.


Assuntos
Celulase/química , Desdobramento de Proteína/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Calorimetria , Celulase/efeitos dos fármacos , Dicroísmo Circular , Cinética , Polietilenoglicóis/farmacologia , Conformação Proteica/efeitos dos fármacos , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Sordariales/enzimologia , Tensoativos/química , Difração de Raios X
8.
Carbohydr Polym ; 229: 115464, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826395

RESUMO

It is important to make full utilization of industrial biomass residues. Pulp was prepared from licorice residues by soda-anthraquinone pulping followed by peroxyacetic acid bleaching. Cellulose nanofibril was obtained by enzymatic pretreatment followed by homogenization of the pulp (ETCNF). The effects of enzymatic pretreatment on ETCNF were investigated. Chitosan nanofibril (CHN) and lignin nanoparticles (LNPs) were prepared and used for ETCNF composites, respectively. The results showed that ETCNF exhibited clear nanofibrillar structure and a highly relative colloidal stability, and a much higher crystallinity index and thermal stability compared to TEMPO-medicated oxidized one; the cellulose composite films incorporated with CHN or LNPs exhibited good thermal stability and hydrophobicity. Compared with ETCNF film, ETCNF@LNPs-5.0% film showed higher UV-blocking ability and thermal stability, but reduced light transmittance, while ETCNF@CHN-5.0% film showed improved mechanical properties and similar light transmittance. This study would expend licorice residues as potential materials for CNF and its applications.


Assuntos
Celulose/química , Glycyrrhiza/química , Nanofibras/química , Nanopartículas/química , Celulase/química , Celulose/isolamento & purificação , Hidrólise , Lignina/química , Lignina/isolamento & purificação
9.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140248, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31279935

RESUMO

Fungi cellulases are used to degrade cellulose-containing biomass for bioethanol production. Industrial cellulases such as Cel7A from Trichoderma reesei (TrCel7A) are critical in this process. Thus, the understanding of structure and dynamics is crucial for engineering variants with improved cellulolytic activity. This cellulase consists of two domains connected by a flexible and highly glycosylated linker. However, the linker flexibility has hindered the determination of Cel7A complete structure. Herein, based on atomic and sparse data, we applied integrative modelling to build a model of the complete enzyme structure. Next, through simulations, we studied the glycosylation effects on the structure and dynamics of a solubilized TrCel7A. Essential dynamics analysis showed that O-glycosylation in the linker led to the stabilization of protein overall dynamics. O-linked glycans seem to restrict protein dihedral angles distribution in this region, selecting more elongated conformations. Besides the reduced flexibility, functional interdomain motions occurred in a more concerted way in the glycosylated system. In contrast, in the absence of glycosylation, we observed vast conformational plasticity with the functional domains frequently collapsing. We report here evidence that targeting Cel7A linker flexibility by point mutations including modification of glycosylation sites could be a promising design strategy to improve cellulase activity.


Assuntos
Celulase/química , Modelos Moleculares , Trichoderma/enzimologia , Proteínas Fúngicas/química , Glicosilação , Conformação Proteica
10.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1138-1147, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793907

RESUMO

The catalytic domain (residues 128-449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.


Assuntos
Proteínas de Bactérias/química , Celobiose/metabolismo , Celulase/química , Celulose 1,4-beta-Celobiosidase/química , Celulose/metabolismo , Neocallimastigales/enzimologia , Trioses/metabolismo , beta-Glucosidase/química , Sítios de Ligação , Cristalografia por Raios X/métodos , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
11.
Environ Sci Pollut Res Int ; 26(35): 35648-35656, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31792789

RESUMO

Lignocellulosic materials are mainly consisted of lignin, cellulose, and hemicellulose. Lignin is recognized as the main obstacle for the enzymatic saccharification of cellulose towards the fermentable sugars' production. Hence, the removal of lignin from the lignocellulosic feedstock is beneficial for reducing the recalcitrance of lignocellulose for enzymatic attack. For this purpose, various different alkaline pretreatments were examined in order to study their effect on the enzymatic saccharification of wheat straw, as a typical lignocellulosic material. Results revealed that the alkaline pretreatments promoted delignification reactions. Regarding the removal of lignin, the most efficient pretreatments were alkaline treatment with hydrogen peroxide 10% and NaOH 2% autoclave with delignification efficiencies of 89.60% and 84.86% respectively. X-ray diffraction analysis was performed to enlighten the structural changes of raw and pretreated materials. The higher the delignification of the raw material, the higher the conversion of cellulose during enzymatic saccharification. In all cases after enzymatic saccharification, the cellulosic conversion was much higher (32-77%) than the untreated wheat straw (8.6%). After undergoing alkaline peroxide 10% pretreatment and cellulase treatment, 99% of the initial raw straw was eventually solubilized. Thus, wheat straw could be considered as an ideal material for the production of glucose with proper pretreatments and effective enzymatic hydrolysis.


Assuntos
Celulase/química , Celulose/química , Lignina/química , Polissacarídeos/química , Triticum/química , Hidrólise
12.
ACS Appl Mater Interfaces ; 11(47): 44913-44921, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31670943

RESUMO

Exploring a suitable immobilization strategy to improve catalytic efficiency and reusability of cellulase is of great importance to lowering the cost and promoting the industrialization of cellulose-derived bioethanol. In this work, a layered structure with a thin PEG hydrogel as the inner layer and sodium polyacrylate (PAANa) brush as the outer layer was fabricated on low density polyethylene (LDPE) film by visible-light-induced graft polymerization. Two enzymes, ß-glucosidase (BG) and cellulase, were separately coimmobilized onto this hierarchical film. As supplementary to cellulase for improving catalytic efficiency, BG was in situ entrapped into the inner PEG hydrogel layer during the graft polymerization from the LDPE surface. After graft polymerization of sodium acrylate on the PEG hydrogel layer was reinitiated, cellulase was covalently attached on the outer PAANa brush layer. Owing to the mild reaction condition (visible-light irradiation and room temperature), the immobilized BG could retain a high activity after the graft polymerization. The immobilization did not alter the optimal pH and temperature of BG or the optimal temperature of cellulase. However, the optimal pH of cellulase shifts to 5.0 after immobilization. Compared with the original activity of single cellulase system and isolated BG/cellulase immobilization system, the dual-enzyme system exhibited 82% and 20% increase in catalytic activity, respectively. The dual-enzyme system could maintain 93% of carboxymethylcellulose sodium salt (CMC) activity after repeating 10 cycles of hydrolysis and 89% of filter paper activity after 6 cycles relative to original activity, exhibiting excellent reusability. This layer coimmobilization system of BG and cellulase on the polymer film displays tremendous potential for practical application in a biorefinery.


Assuntos
Celulase/química , Polímeros/química , beta-Glucosidase/química , Biocatálise , Carboximetilcelulose Sódica/química , Celulose/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Hidrogéis/química , Concentração de Íons de Hidrogênio , Luz , Polietilenoglicóis/química , Polimerização/efeitos da radiação , Propriedades de Superfície , Temperatura
13.
Colloids Surf B Biointerfaces ; 184: 110568, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31627101

RESUMO

In this paper, the dispersion performance of biomacromolecule hydrolyzed cellulase from Trichoderma reesei on copper phthalocyanine (CuPc) pigment was first studied. The effect of hydrolysis time, cellulase concentration and environmental pH on the dispersion performance was investigated by particle size distribution and suspension transmittance measurement. The hydrolysis degree of cellulase was determined by FTIR, XRD, UV-vis and fluorescence spectra, potential and particle size analysis, respectively. Subsequently, the hydrolyzed cellulase was combined with sodium dodecyl sulfate (SDS) for acquiring better CuPc suspension based on their synergetic effects on dispersion. The optimal mass ratio of hydrolyzed cellulase to SDS was found to be 1:9. The resulting CuPc dispersion by this hydrolyzed cellulase/SDS composite was characterized by FTIR, TG, TEM, XRD analysis, respectively. This study demonstrated that there were strong interactions between hydrolyzed cellulase and SDS to result in synergistic dispersing effect on CuPc for better stability.


Assuntos
Celulase/química , Corantes/química , Indóis/química , Compostos Organometálicos/química , Dodecilsulfato de Sódio/química , Concentração de Íons de Hidrogênio , Hidrólise , Substâncias Macromoleculares/química , Tamanho da Partícula , Pigmentos Biológicos , Propriedades de Superfície
14.
Enzyme Microb Technol ; 131: 109389, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615669

RESUMO

Cross-linked enzyme aggregate (CLEA) is a technology to overcome the limitation of enzymes for its application in chemical industries. The inability of repeated use of enzymes, stability and ease of separation from reaction mixture limits its applications. Here, magnetic combi-CLEA has been synthesised by adding amino-functionalized magnetic nanoparticles into pectinase ultra-clear (containing pectinases, xylanases and cellulases). Enzymes were precipitated on the surface of amino-functionalized magnetic nanoparticles with ethanol and cross-linked using glutaraldehyde. The structural characterization of magnetic combi-CLEA was studied by Scanning Electron Microscopy. Thermal stability was performed at 70 °C for pectinase and 80 °C for xylanase and cellulase respectively. Half-life (t1/2) of the xylanase, cellulase and pectinase in free form remarkably enhance from 84.51, 29.36, and 25.29 min respectively to 533.07, 187.29 and 147.44 min in magnetic-combi CLEA respectively. Magnetic combi-CLEA can be efficiently reused till 12th cycle after which pectinase, xylanase and cellulase retain 86.45%, 90.3% and 88.62% activity respectively. Using this CLEA preparation bioethanol concentration increases to 1.82-fold as compared to free enzyme, when simultaneous saccharification and fermentation was performed using wheat straw as the substrate. Magnetic combi-CLEA can be used for a variety of industrial applications like food processing, textile industry and bioethanol production.


Assuntos
Celulase/isolamento & purificação , Celulase/metabolismo , Enzimas Imobilizadas/metabolismo , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Magnetismo , Nanopartículas Metálicas , Biotransformação , Celulase/química , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Temperatura Alta , Microscopia Eletrônica de Varredura , Temperatura , Triticum/metabolismo
15.
Biochimie ; 165: 275-284, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31472178

RESUMO

Glycoside hydrolase (GH) family 45 is one of the smallest and poorly studied endoglucanase family with a broad biotechnological application ranging from treatment of textiles to conversion of complex cell wall polysaccharides into simple oligo- and monosaccharides. In a present study, GH45 cellulase from Neurospora crassa OR74A (NcCel45A) was characterized both biochemically and structurally. HPLC analysis of the hydrolytic products confirmed the endo-ß(1,4) mode of action of the enzyme. Moreover, such pattern revealed that NcCel45A cannot hydrolyze efficiently oligosaccharides with a degree of polymerization smaller than six. The crystal structure of NcCel45A catalytic domain in the apo-form was determined at 1.9 Šresolution and the structure of the enzyme bound to cellobiose was solved and refined to 1.8 Šresolution. Comparative structural analyses and molecular dynamics simulations show that the enzyme dynamics is affected by substrate binding. Taken together, MD simulations and statistical coupling analysis revealed previously unknown correlation of a loop 6 with the breakdown of cellulose substrates by GH45.


Assuntos
Celulase/química , Celulose/metabolismo , Neurospora crassa/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Hidrólise , Simulação de Dinâmica Molecular , Conformação Proteica , Especificidade por Substrato
16.
Int J Biol Macromol ; 141: 484-492, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31479677

RESUMO

Sugarcane bagasse (SCB) was pretreated by sodium hydroxide (SH), alkaline ethanol (AE) and alkaline hydrogen peroxide (AHP), and the solid residue with similar lignin content after different pretreatment was selected. Enzymatic saccharification with different substrates was performed at 2%, 10%, and 20% (w/v) solid loading. After that, the lignin from different substrate was extracted and its structure was characterized. Furthermore, the adsorption capability of isolated lignin to cellulase and its effect on enzymatic hydrolysis were studied. The results showed that, as the substrate content increased from 2% (w/v) to 20% (w/v), the glucose yield after digestion of SH, AE, and AHP pretreated SCB reduced by 41.8%, 35.4%, and 28.7%, respectively. The inhibitory effect of different prepared lignin on the digestibility of Avicel was as follows, SH pretreated lignin (SHL) > cellulase enzymatic hydrolysis lignin (CEL) > AE pretreated lignin (AEL) > AHP pretreated lignin (AHPL), which exhibited positive correlation with its non-productive adsorption capability to cellulase. Lignin samples with low negative zeta potential, high hydrophobicity and high ration of syringyl (S) to guaiacyl (G) were unfavorable for the enzymatic saccharification of cellulose.


Assuntos
Celulase/química , Celulose/química , Lignina/química , Saccharum/química , Hidróxido de Sódio/química , Etanol/química , Hidrólise
17.
Protein J ; 38(6): 640-648, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31549278

RESUMO

Cellulase from Trichoderma reesei was immobilized by covalent or non-covalent binding onto magnetic hierarchical porous carbon (MHPC) nanomaterials. The immobilization yield and the enzyme activity were higher when covalent immobilization approach was followed. The covalent immobilization approach leads to higher immobilization yield (up to 96%) and enzyme activity (up to 1.35 U mg-1) compared to the non-covalent cellulase binding. The overall results showed that the thermal, storage and operational stability of the immobilized cellulase was considerably improved compared to the free enzyme. The immobilized cellulose catalyzed the hydrolysis of microcrystalline cellulose up to 6 consecutive successive reaction cycles, with a total operation time of 144 h at 50 °C. The half-life time of the immobilized enzyme in deep eutectic solvents-based media was up to threefold higher compared to the soluble enzyme. The increased pH and temperature tolerance of the immobilized cellulase, as well as the increased operational stability in aqueous and deep eutectic solvents-based media indicate that the use of MHPCs as immobilization nanosupport could expand the catalytic performance of cellulolytic enzymes in various reaction conditions.


Assuntos
Celulase/química , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Carbono/química , Estabilidade Enzimática , Cinética , Fenômenos Magnéticos , Nanopartículas/química , Porosidade , Trichoderma/metabolismo
18.
J Biotechnol ; 306: 118-124, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31550489

RESUMO

Using multi-step error prone PCR (ep-PCR) of the gene encoding endoglucanase Cel12A (27 kDa) from Thermotoga neapolitana, mutants were obtained with many fold increase in the enzyme activity. The best mutant (C6, N47S/E57 K/ V88A/S157 P/K165 H) obtained after four rounds of ep-PCR showed 2.7-, 5- and 4.8-fold increase in activity against CMC, RAC and Avicel, respectively, compared with the wild type enzyme. The other characteristics of the mutated enzyme with respect to stability, optimum working pH and temperature were comparable to the wild type enzyme.C6 mutant showed higher binding efficiency towards the rice straw (∼50%) than the wild type (∼41%). The structural information obtained from the protein docking of the wild type Cel12A and its mutant showed that E57 K improved the binding affinity between enzyme and ligand by producing conformational changes in the catalytic cavity. The other mutations can facilitate the enzyme-substrate binding interactions to enhance catalytic activity although they are not directly involved in catalysis. The wild type and mutant enzyme produce cellobiose as the major products for both soluble and insoluble substrates, suggesting that this enzyme should be a cellobiohydrolase instead of endoglucanase as previously reported.


Assuntos
Celulase/genética , Celulase/metabolismo , Thermotoga neapolitana/enzimologia , Catálise , Celulase/química , Celulose/metabolismo , Evolução Molecular Direcionada , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Mutação , Reação em Cadeia da Polimerase/métodos , Relação Estrutura-Atividade , Temperatura , Thermotoga neapolitana/genética , Thermotoga neapolitana/metabolismo
19.
Chem Biodivers ; 16(11): e1900369, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31529789

RESUMO

Lenzites betulina has been recognized as a rich source of chemical components, including polysaccharides, sterides and sugar alcohols. In this study, cellulase-ultrasonic synergistic extraction method was applied to extract polysaccharides from L. betulina, and the response surface methodology was used to optimize the extraction conditions. The eight basic factors affecting extraction yield were evaluated by Plackett-Burman design (PBD). Then, the four important factors significantly affecting the yield of polysaccharides from L. betulina, including enzymolysis temperature, enzymolysis time, ultrasound time and ultrasound temperature, were optimized by Box-Behnken design (BBD). Maximum extraction yield of L. betulina polysaccharides was 13.64±0.09 % at a cellulase dosage of 0.8 %, enzymolysis temperature of 60 °C, enzymolysis time of 180 min, pH of 4.5, liquid-solid ratio of 45 ml/g, ultrasound power of 300 W, ultrasound time of 20 min and ultrasound temperature of 45 °C. Subsequently, the characteristic structure of crude polysaccharides was determined by FT-IR. Results indicated that cellulase-ultrasonic synergistic treatment is suitable for L. betulina polysaccharides extraction, and it has good prospect for development and utilization.


Assuntos
Celulase/metabolismo , Polyporaceae/química , Polissacarídeos/isolamento & purificação , Ultrassom , Celulase/química , Polissacarídeos/química , Polissacarídeos/metabolismo , Temperatura
20.
J Oleo Sci ; 68(9): 881-891, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31413240

RESUMO

Enzyme-assisted aqueous extraction of rice germ oil (RGO) was performed in this study. The physicochemical properties, fatty acid composition, bioactive substances and antioxidant activity of RGO were analyzed. An enzyme composed of alcalase and cellulase (1:1, w/w) was found to be the most effective in the extraction yield of oil. The optimal oil yield of 22.27% was achieved under the conditions of an enzyme concentration of 2% (w/w), incubation time of 5 h, incubation temperature of 50°C, water to seed ratio of 5:1, and pH 6.0. The predominant fatty acids of RGO were oleic acid (39.60%), linoleic acid (34.20%) and palmitic acid (20.10%). The total saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) composition of RGO were 22.50%, 39.60% and 36.00%, respectively. RGO yielded a high content of γ-oryzanol (530 mg/100 g oil), tocotrienol (62.96 mg/100 g oil), tocopherol (23.24 mg/100 g oil) and a significant amount of phytosterol (372.14 mg/100 g oil). It exhibited notable antioxidant activities with IC50 values of 32.37 and 41.13 mg/mL, according to the DPPH radical scavenging assay and ß-carotene/linoleic acid bleaching test, respectively.


Assuntos
Depuradores de Radicais Livres/química , Oryza/química , Óleos Vegetais/química , Sementes/química , Celulase/química , Ácidos Graxos/análise , Depuradores de Radicais Livres/análise , Depuradores de Radicais Livres/isolamento & purificação , Fitosteróis/análise , Óleos Vegetais/análise , Óleos Vegetais/isolamento & purificação , Extração em Fase Sólida/métodos , Subtilisinas/química , Tocoferóis/análise
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