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1.
Food Chem ; 298: 124999, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31261010

RESUMO

Glycoside hydrolase family 8 (GH8) includes endoglucanases, lichenases, chitosanases and xylanases, which are essential for polysaccharides breakdown. In this work, we studied a thermally stable GH8 from the cellulose synthase complex of Enterobacter sp. R1, for deconstruction of ß-glucans. The biochemical characterization of the recombinant GH8ErCel showed high specificity towards barley ß-glucan and lichenan and lower activity on carboxymethylcellulose and swollen cellulose, yielding different length oligosaccharides. By molecular modeling, six conserved subsites for glucose binding and some possible determinants for its lack of xylanase and chitosanase activity were identified. GH8ErCel was active at a broad range of pH and temperature and presented remarkable stability at 60 °C. Additionally, it hydrolyzed ß-glucan from oat and wheat brans mainly to tri- and tetraoligosaccharides. Therefore, GH8ErCel may be a good candidate for enzymatic deconstruction of ß-glucans at high temperature in food and feed industries, including the production of prebiotics and functional foods.


Assuntos
Celulase/química , Celulase/metabolismo , Celulose/metabolismo , Enterobacter/enzimologia , beta-Glucanas/metabolismo , Argentina , Carboximetilcelulose Sódica/metabolismo , Celulase/genética , Enterobacter/genética , Enterobacter/isolamento & purificação , Estabilidade Enzimática , Glucanos/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Microbiologia do Solo , Especificidade por Substrato , Temperatura Ambiente , beta-Glucanas/química
2.
J Sci Food Agric ; 99(13): 5784-5791, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31162677

RESUMO

BACKGROUND: The use of byproducts such as rejected plantain with final disposition problems and conversion processes with 'green' technologies are important research topics. Bioethanol production from crops with a high content of fermentable sugars is an alternative to that from traditional crops (corn and sugar cane). The aim of this work was to study the use of whole (peel and pulp) unripe plantain (WP) for bioethanol production. RESULTS: Lab-scale liquefaction and saccharification of both materials released mainly three carbohydrates, glucose (9.02 mg g-1 ), maltose (0.45 mg g-1 ) and xylose (0.25 mg g-1 ). The WP saccharification required the use of pectinase and cellulase because of the high amounts of pectin and cellulose associated with the peel. Fermentation for 11 h produced similar ethanol concentration for both samples, but at the end of fermentation (32 h), the ethanol production was higher in the WP (58.6 mL L-1 ) compared with the plantain pulp (PP) (45.5 mL L-1 ). The theoretical ethanol yield was lower with WP (67%) than with PP (90%). CONCLUSION: WP can be an alternative raw material for bioethanol production. © 2019 Society of Chemical Industry.


Assuntos
Biocombustíveis/análise , Etanol/metabolismo , Microbiologia Industrial/métodos , Musa/química , Saccharomyces cerevisiae/metabolismo , Resíduos/análise , Biocatálise , Celulase/química , Etanol/análise , Fermentação , Frutas/química , Frutas/microbiologia , Musa/microbiologia , Poligalacturonase/química
3.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 605-615, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205022

RESUMO

The discovery of new glycoside hydrolases that can be utilized in the chemoenzymatic synthesis of carbohydrates has emerged as a promising approach for various biotechnological processes. In this study, recombinant Ps_Cel5A from Pseudomonas stutzeri A1501, a novel member of the GH5_5 subfamily, was expressed, purified and crystallized. Preliminary experiments confirmed the ability of Ps_Cel5A to catalyze transglycosylation with cellotriose as a substrate. The crystal structure revealed several structural determinants in and around the positive subsites, providing a molecular basis for a better understanding of the mechanisms that promote and favour synthesis rather than hydrolysis. In the positive subsites, two nonconserved positively charged residues (Arg178 and Lys216) were found to interact with cellobiose. This adaptation has also been reported for transglycosylating ß-mannanases of the GH5_7 subfamily.


Assuntos
Proteínas de Bactérias/química , Celulase/química , Celulose/química , Pseudomonas stutzeri/enzimologia , Trioses/química , Celulose/metabolismo , Cristalização , Cristalografia por Raios X/métodos , Escherichia coli , Glicosilação , Especificidade por Substrato , Trioses/metabolismo
4.
Chem Commun (Camb) ; 55(57): 8219-8222, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31210215

RESUMO

Here we reported a new strategy to construct synthetic metabolons using dCas9-guided assembly. Three orthogonal dCas9 proteins were exploited to guide the independent and site-specific assembly of their fusion partners onto a single DNA scaffold. This new platform was applied towards the construction of a two-component cellulosome. Because of the superior binding affinity, the resulting structures exhibited both improved assembly and reducing sugar production. Conditional enzyme assembly was made possible by utilizing toehold-gated sgRNA (thgRNA), which blocks cellulosome formation until the spacer region is unblocked by a RNA trigger. This platform is highly modular owing to the ease of target synthesis, combinations of possible Cas9-fusion arrangements, and expansion to other metabolic pathways.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , RNA Guia/metabolismo , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Celulase/química , Celulase/genética , Celulase/metabolismo , Celulossomas/química , Celulossomas/metabolismo , DNA/química , DNA/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Guia/genética
5.
J Basic Microbiol ; 59(7): 667-679, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31087565

RESUMO

A psychrotolerant yeast strain Mrakia robertii A2-3 isolated from cryoconites of Hamtah glacier, Himalaya, India was investigated for the production of cold-tolerant endoglucanase. Optimum endoglucanase production was found at 15°C with an initial pH of 5.5, and potent inducers were 1% wt/vol of xylose and KNO3 and 0.1% wt/vol of NaCl. Under optimum conditions, the enzyme production was 1.81-fold higher than the unoptimized conditions. Crude enzyme was partially purified by ammonium sulfate precipitation followed by dialysis. The enzyme was purified to 2.53-fold and the yield was 6.03% with specific activity of 17.38 U/mg and molecular weight ~57 kDa. The Km and Vmax values of the partially purified enzyme were found to be 1.57 mg/ml and 142.85 U/mg, respectively. The characterization study revealed that the best temperature was 15°C for activity and stability. Furthermore, the enzyme showed the highest activity at pH 11.0 and was stable at pH 6.0. Fe2+ , Mn2+ , Na2+ , Cu2+ , Co2+ , Ca2+ proved to be activators of endoglucanase. Ethylenediamine tetraacetic acid showed very low effect on the enzyme activity whereas it was active with Tween-80 and sodium deoxycholate. The present study successfully produced a cold-active endoglucanase with novel properties making it promising as a biocatalyst for industrial processes.


Assuntos
Basidiomycota/enzimologia , Celulase/fisiologia , Temperatura Baixa , Camada de Gelo/microbiologia , Basidiomycota/classificação , Basidiomycota/fisiologia , Carboximetilcelulose Sódica/metabolismo , Celulase/química , Celulase/isolamento & purificação , Celulase/metabolismo , DNA Fúngico/genética , DNA Ribossômico/genética , Detergentes , Ativadores de Enzimas , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Índia , Cinética , Peso Molecular , Filogenia , Análise de Sequência de DNA
6.
J Agric Food Chem ; 67(22): 6248-6256, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31090409

RESUMO

A lignin amphoteric surfactant and betaine could enhance the enzymatic hydrolysis of lignocellulose and recover cellulase. The effects of lignosulfonate quaternary ammonium salt (SLQA) and dodecyl dimethyl betaine (BS12) on enzymatic hydrolysis digestibility, ethanol yield, yeast cell viability, and other properties of high-solid enzymatic hydrolysis and fermentation of a corncob residue were studied in this research. The results suggested that SLQA and 1 g/L BS12 effectively improved the ethanol yield through enhancing enzymatic hydrolysis. SLQA had no significant effect on the yeast cell membrane and glucose fermentation. However, 5 g/L BS12 reduced the ethanol yield as a result of the fact that 5 g/L BS12 damaged the yeast cell membrane and inhibited the conversion of glucose to ethanol. Our research also suggested that 1 g/L BS12 enhanced the ethanol yield of corncob residue fermentation, which was attributed to the fact that lignin in the corncob adsorbed BS12 and decreased its concentration in solution to a safe level for the yeast.


Assuntos
Biotecnologia/métodos , Celulose/metabolismo , Etanol/química , Etanol/metabolismo , Lignina/metabolismo , Resíduos/análise , Leveduras/metabolismo , Zea mays/microbiologia , Biocatálise , Biotecnologia/instrumentação , Celulase/química , Fermentação , Glucose/metabolismo , Hidrólise , Lignina/química , Tensoativos/química , Zea mays/metabolismo
7.
Food Chem ; 290: 47-55, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31000055

RESUMO

The immobilization of cellulase on amine-functionalized Fe3O4 magnetic nanoparticles (MNPs), via metal affinity immobilization, as a nano-biocatalyst was investigated. Copper was chosen as ligand and loaded onto MNPs in a buffering environment without adding any intermediates. Immobilization conditions were optimized by a 23 full factorial design method. Under optimized working conditions (Cu/MNPs = 1, E/MNPs = 0.11, pH = 6), the relative enzyme activity and the amount of enzyme immobilization were 91% and 164 (mg enzyme/g MNPs), respectively. The immobilized cellulase (tested by carboxymethyl cellulose hydrolysis at 1% concentration) was found to be more stable than the free enzyme. Also, the immobilized enzyme still retained 73% of its initial activity after five cycles of usage. Furthermore, the free and immobilized cellulases retained 70 and 84% of their initial activity after eight days storage at 4 °C, respectively. Immobilization of enzymes, using this method, could be a good and economic option for various industries.


Assuntos
Biocatálise , Celulase/química , Celulase/metabolismo , Cobre/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Nanopartículas de Magnetita/química , Carboximetilcelulose Sódica/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Ligantes , Temperatura Ambiente
8.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31032817

RESUMO

(1) Background: Processivity is common among enzymes and mechanochemical motors that synthesize, degrade, modify or move along polymeric substrates, such as DNA, RNA, polysaccharides or proteins. Processive enzymes can make multiple rounds of modification without releasing the substrate/partner, making their operation extremely effective and economical. The molecular mechanism of processivity is rather well understood in cases when the enzyme structurally confines the substrate, such as the DNA replication factor PCNA, and also when ATP energy is used to confine the succession of molecular events, such as with mechanochemical motors. Processivity may also result from the kinetic bias of binding imposed by spatial confinement of two binding elements connected by an intrinsically disordered (ID) linker. (2) Method: By statistical physical modeling, we show that this arrangement results in processive systems, in which the linker ensures an optimized effective concentration around novel binding site(s), favoring rebinding over full release of the polymeric partner. (3) Results: By analyzing 12 such proteins, such as cellulase, and RNAse-H, we illustrate that in these proteins linker length and flexibility, and the kinetic parameters of binding elements, are fine-tuned for optimizing processivity. We also report a conservation of structural disorder, special amino acid composition of linkers, and the correlation of their length with step size. (4) Conclusion: These observations suggest a unique type of entropic chain function of ID proteins, that may impart functional advantages on diverse enzymes in a variety of biological contexts.


Assuntos
Enzimas/química , Enzimas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Celulase/química , Celulase/metabolismo , Fenômenos Químicos , Sequência Conservada , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
9.
Int J Mol Sci ; 20(7)2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30935060

RESUMO

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15⁻21% increase in specific activity against carboxymethylcellulose and ß-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52⁻58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15⁻22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.


Assuntos
Celulase/química , Dissulfetos/química , Proteínas Fúngicas/química , Penicillium/enzimologia , Termotolerância , Celulase/genética , Celulase/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Simulação de Dinâmica Molecular , Penicillium/genética , Penicillium/metabolismo , Engenharia de Proteínas/métodos , Especificidade por Substrato
10.
Acta Crystallogr D Struct Biol ; 75(Pt 4): 426-436, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30988259

RESUMO

Although endogenous animal cellulases have great potential for industrial applications such as bioethanol production, few investigations have focused on these enzymes. In this study, the glycoside hydrolase family 45 (GH45) subfamily B endoglucanase EG27II from the snail Ampullaria crossean was expressed using a Pichia pastoris expression system and the crystal structure of the apo form was determined at 1.00 Šresolution; this is the highest resolution structure of an animal endoglucanase. The results showed that EG27II has a double-ψ six-stranded ß-barrel and that the structure of EG27II more closely resembles those of subfamily C enzymes than those of subfamily A enzymes. The structure of EG27II complexed with cellobiose was also determined under cryoconditions and at room temperature at three pH values, pH 4.0, 5.5 and 8.0, and no structural changes were found to be associated with the change in pH. The structural comparison and catalytic activity measurements showed that Asp137 and Asn112 function as the catalytic acid and base, respectively, and that Asp27 is also an important residue for catalysis. These high-resolution structures of EG27II provide a large amount of information for structure-based enzyme modification and cell-surface engineering, which will advance biofuel production using animal-derived cellulases.


Assuntos
Celulase/química , Celulase/metabolismo , Caramujos/enzimologia , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X/métodos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica
11.
Phys Rev Lett ; 122(9): 098102, 2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30932525

RESUMO

The microscopic kinetics of enzymes at the single-molecule level often deviate considerably from those expected from bulk biochemical experiments. Here, we propose a coarse-grained-model approach to bridge this gap, focusing on the unexpectedly slow bulk hydrolysis of crystalline cellulose by cellulase, which constitutes a major obstacle to mass production of biofuels and biochemicals. Building on our previous success in tracking the movements of single molecules of cellulase on crystalline cellulose, we develop a mathematical description of the collective motion and function of enzyme molecules hydrolyzing the surface of cellulose. Model simulations robustly explained the experimental findings at both the microscopic and macroscopic levels and revealed a hitherto-unknown mechanism causing a considerable slowdown of the reaction, which we call the crowding-out effect. The size of the cellulase molecule impacted significantly on the collective dynamics, whereas the rate of molecular motion on the surface did not.


Assuntos
Celulase/química , Modelos Químicos , Celulose/química , Celulose 1,4-beta-Celobiosidase/química , Hidrólise , Cinética , Trichoderma/enzimologia
12.
J Basic Microbiol ; 59(7): 692-700, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30980726

RESUMO

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.


Assuntos
Celulase/isolamento & purificação , Celulase/metabolismo , Haliclona/microbiologia , Haloferax/enzimologia , Animais , Organismos Aquáticos/enzimologia , Organismos Aquáticos/microbiologia , Carboximetilcelulose Sódica/metabolismo , Celulase/química , Celulase/fisiologia , Estabilidade Enzimática , Haliclona/classificação , Haloferax/classificação , Haloferax/fisiologia , Concentração de Íons de Hidrogênio , Índia , Microbiota/genética , Microbiota/fisiologia , Peso Molecular , Filogenia , Salinidade , Especificidade por Substrato , Temperatura Ambiente
13.
Carbohydr Polym ; 214: 1-7, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30925976

RESUMO

In this work, cellulase, low-concentration cold alkali and cellulase combined with cold alkali were used to pretreat unbleached bagasse pulp from which cellulose nanofibrils (CNFs), about 30 nm in diameter, were successfully prepared through ultrafine grinding and high-pressure homogenization. X-ray diffraction analysis showed that cellulase pretreatment increased the crystallinity of CNFs. After low-concentration cold alkali pretreatment, the crystallinity of CNFs significantly reduced and the crystal structure of the cellulose changed from type I to type II. Thermogravimetric analysis showed that CNFs prepared by cellulase combined with cold alkali treatment produced more regenerated cellulose and had lower thermal stability. The use of cellulase and low-concentration cold alkali pretreatments combined with ultrafine grinding and high-pressure homogenization is an environment-friendly method for preparing CNFs. The use of low-concentration cold alkali reduces the consumption of alkali and clean water.


Assuntos
Celulase/química , Celulose/química , Química Verde/métodos , Nanofibras/química , Cristalização , Estrutura Molecular , Tamanho da Partícula , Saccharum/química , Hidróxido de Sódio/química , Temperatura Ambiente
14.
Carbohydr Polym ; 211: 349-359, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30824099

RESUMO

One of the major challenges in biofuels production from lignocellulosic biomass is the generation of high glucose titers from cellulose in the enzymatic hydrolysis stage of pretreated biomass to guarantee a cost-effective process. Therefore, the enzymatic saccharification on cellulose at high solid loading is an alternative. In this work, the agave bagasse was hydrothermally pretreated and optimized at 194 °C/30 min, obtaining a pretreated solid rich in cellulose content (>46.46%), and subjected to enzymatic hydrolysis at high solid levels. A horizontal bioreactor was designed for enzyme saccharification at high solid loadings [25% (w/v)]. The bioreactor improved mixing efficiency, with cellulose conversions up to 98% (195.6 g/L at 72 h). Moreover, mathematical modeling of cellulase deactivation demonstrated that cellulases lose most of their initial activity in the first hours of the reaction. Also, cellulose was characterized by X-ray diffraction, and the pretreated solids were visualized using scanning electron microscopy.


Assuntos
Agave , Celulase/química , Celulose/química , Modelos Teóricos , Reatores Biológicos , Temperatura Alta , Hidrólise
15.
Carbohydr Polym ; 211: 57-68, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30824104

RESUMO

Plant biomass is a low-cost and abundant source of carbohydrates for production of fuels, "green" chemicals and materials. Currently, biochemical conversion of the biomass into sugars via enzymatic hydrolysis is the most viable technology. Here, the role of carbohydrate binding modules (CBMs) in the disruption of insoluble polysaccharide structures and their capacity to enhance cellulase-promoted lignocellulosic biomass hydrolysis was investigated. We show that CBM addition promotes generation of additional reducing ends in the insoluble substrate by cellulases. On the contrary, bovine serum albumin (BSA), widely used in prevention of a non-specific protein binding, causes an increase in soluble reducing-end production, when applied jointly with cellulases. We demonstrate that binding of CBMs to cellulose is non-homogeneous, irreversible and leads to its amorphisation. Our results also reveal effects of CBM-promoted amorphogenesis on cellulose hydrolysis by cellulases.


Assuntos
Carboidratos/química , Celulase/química , Celulose/química , Proteínas Fúngicas/química , Adsorção , Hidrólise , Ligação Proteica , Soroalbumina Bovina/química
16.
J Biotechnol ; 296: 42-52, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30885654

RESUMO

The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-ß-galactanase and an α-l-arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (kcat values of ˜177 and ˜133 s-1, respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases - endo-1,4-ß-xylanase (XynA), endo-1,4-ß-mannanase (ManB/Man5A) and ß-glucosidase (GghA) - to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases.


Assuntos
Celulase/química , Glicosídeo Hidrolases/química , Lignina/química , Polissacarídeos/química , Biocombustíveis , Biomassa , Celulase/genética , Estabilidade Enzimática/genética , Escherichia coli/genética , Fermentação , Glicosídeo Hidrolases/genética , Hidrólise/efeitos dos fármacos , Polissacarídeos/genética , Temperatura Ambiente , Thermotoga neapolitana/enzimologia , Thermotoga neapolitana/genética
17.
Int J Biol Macromol ; 131: 564-571, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30831164

RESUMO

In this work, cellulose was extracted from potato pulp, a low-efficiently utilized by-product from potato starch industry, by a combined alkali and sodium sulfite treatment. Its physicochemical properties were characterized and compared with cellulose from corn stalk. The yield and purity of cellulose from potato pulp were determined to be 24.74% and 81.34% respectively. Cellulose from potato pulp had more loosened and porous structure, lower crystallinity index and larger specific surface area (SSA) compared with cellulose from corn stalk. It also provided the highest accessibility to cellulase (5.7 mg protein/g glucan) and had the highest enzymatic digestibility (with glucose yield of 88.46%). Maximum adsorption capacity (Wmax) and equilibrium constant (K) were obtained by fitting the adsorption data with the Langmuir adsorption isotherm, suggesting that cellulose from potato pulp had the highest cellulase adsorption efficiency. The data further indicated that the presence of non-cellulosic components, especially for pectin, appeared to have a considerable effect on the hydrolysis by cellulase due to non-productive adsorption. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue Wuxi 214122, Jiangsu Province, People's Republic of China.


Assuntos
Celulase/química , Celulose/química , Solanum tuberosum/química , Hidrólise , Cinética , Peso Molecular , Solanum tuberosum/enzimologia , Análise Espectral
18.
Appl Biochem Biotechnol ; 189(1): 65-75, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30863987

RESUMO

Corn stover silage (CSS) is regarded as a promising feedstock for bioethanol production. The two-step pretreatment using a sequential non-ionic surfactant and ferric nitrate pretreatment was investigated for improving the enzymatic hydrolysis of CSS. The first-step pretreatment using non-ionic surfactant (Tween-80, 2.0 wt.%) at 60 °C for 60 min achieved 30.48% the removal of lignin. Compared with the raw material, the cellulose content of first-step treated CSS increased by 15.86%. The second step using ferric nitrate resulted in 94.56% hemicellulose removal and achieved 72.53% cellulose purity at 130 °C for 30 min, while the yields of furfural and HMF were only 0.36 and 0.32 g/100 g dry material, respectively. The maximum enzymatic digestibility of the two-step treated CSS was 90.98% with a low cellulose dosage (15 FPU/g-glucan), which was approximately 32.07% higher than that of the first-step pretreatment only with Tween-80.


Assuntos
Celulase/química , Celulose/química , Compostos Férricos/química , Nitratos/química , Silagem , Açúcares/isolamento & purificação , Tensoativos/química , Zea mays/química
19.
Int J Biol Macromol ; 131: 676-681, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30904528

RESUMO

Natural lignocellulose is used as raw material to produce chemicals through biological transformation. The accessibility of cellulase to substrate was also one of the limiting factors of industrial production. Polyethylene glycol (PEG) can be used as additive in enzymatic hydrolysis of lignocellulose. In this study, enzymatic activity on simultaneous or non-simultaneous addition of PEG 4000 was investigated, and the partly delignified rice straw, the rice straw and filter paper were used as substrates, respectively. Enzyme activity was characterized by reducing sugar concentration in supernatant which was quantified through 3,5-dinitrosalicylic acid (DNS) method. Addition of PEG has been proven to facilitate enzymatic hydrolysis of lignocellulosic materials. Furthermore, PEG had the positive effect on hydrolytic enzyme activity of pure cellulose materials without lignin. Changes in lignocellulose materials have been observed by inverted microscope and Scanning electron microscope (SEM), and no chemical changes were shown by Fourier transform infrared spectroscopy (FTIR). The promotion of PEG on enzymatic hydrolysis of pure cellulose materials may be due to its loose physical structure and similar phenomenon in natural lignin materials. PEG loosens the physical structure of lignocellulose, thus facilitating enzymatic hydrolysis. This may be a new idea to optimize the lignocellulosic enzymatic hydrolysis process.


Assuntos
Celulase/química , Lignina/química , Polietilenoglicóis/química , Carboidratos/química , Hidrólise , Estrutura Molecular , Especificidade por Substrato
20.
J Biotechnol ; 295: 55-62, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30853632

RESUMO

Endoglucanase, an important component of cellulases, is used as additives in ensiling of forage crops. However, its detailed role is unclear in ensilages. In the present study, two endoglucanases Cel5 and Cel9 produced by strain Paenibacillus panacisoli SDMCC050309, previously isolated from ensiled corn stover, were identified in the cultures by microcrystalline cellulose absorption coupled with zymogram analysis. After heterologously expressed in Escherichia coli DE3 and purified, these two proteins were biochemically characterized. Cel5 was 61 kDa and showed maximal activity at pH 7.0 and 45 °C, while the maximum activity was at pH 8.0 and 65 °C for Cel9 with 97 kDa in size. Both of them could degrade carboxymethyl cellulose into cellooligosaccharides, in which cellobiose and cellotriose could be used as substrates for the growth of homofermentative strains Lactobacillus plantarum CGMCC6888 and L. farciminis CCTCC AB2016237, but not for the heterofermentative strains L. brevis SDMCC050297 and L. parafarraginis SDMCC050300. Therefore, we concluded that the added endoglucanase contributed to enhance the growth of homofermentative lactic acid bacteria for high level of lactic acid production in ensilages.


Assuntos
Celulase/química , Celulase/metabolismo , Lactobacillus/metabolismo , Silagem/microbiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulase/genética , Celulose/metabolismo , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Oligossacarídeos/metabolismo , Paenibacillus/enzimologia , Paenibacillus/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
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