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1.
Pestic Biochem Physiol ; 158: 128-134, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378348

RESUMO

Indoleacetic acid (IAA)-carbendazim was synthesized to assess whether this conjugate could retain the fungicidal activity of carbendazim and gain root-inducing properties upon the addition of an indoleacetic acid group. An indoor virulence test demonstrated that the conjugate retained the fungicidal activity of carbendazim towards Cylindrocladium parasiticum. The conjugate was detected in roots after soaking Ricinus communis L. leaves into a solution of the IAA-carbendazim, which confirmed its phloem mobility. The activities of the cellulase, polygalacturonase and xylanase produced by Cylindrocladium parasiticum treated with different concentrations of the conjugate were determined, and the peak activities appeared at 72 h or 96 h. More importantly, the conjugate showed the ability to promote root growth. These results revealed that indoleacetic acid-carbendazim may be useful in preventing Cylindrocladium parasiticum and other diseases.


Assuntos
Ascomicetos/efeitos dos fármacos , Benzimidazóis/farmacologia , Carbamatos/farmacologia , Fungicidas Industriais/farmacologia , Ácidos Indolacéticos/farmacologia , Celulase/metabolismo , Floema/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Poligalacturonase/metabolismo , Ricinus/efeitos dos fármacos
2.
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
3.
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
4.
J Microbiol Biotechnol ; 29(6): 905-912, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154746

RESUMO

Bioethanol has attracted much attention in recent decades as a sustainable and environmentally friendly alternative energy source. In this study, we compared the production of bioethanol by Candida molischiana and Saccharomyces cerevisiae at different initial concentrations of cellobiose and glucose. The results showed that C. molischiana can utilize both glucose and cellobiose, whereas S. cerevisiae can only utilize glucose. The ethanol yields were 43-51% from different initial concentrations of carbon source. In addition, different concentrations of microcrystalline cellulose (Avicel) were directly converted to ethanol by a combination of Trichoderma reesei and two yeasts. Cellulose was first hydrolyzed by a fully enzymatic saccharification process using T. reesei cellulases, and the reducing sugars and glucose produced during the process were further used as carbon source for bioethanol production by C. molischiana or S. cerevisiae. Sequential culture of T. reesei and two yeasts revealed that C. molischiana was more efficient for bioconversion of sugars to ethanol than S. cerevisiae. When 20 g/l Avicel was used as a carbon source, the maximum reducing sugar, glucose, and ethanol yields were 42%, 26%, and 20%, respectively. The maximum concentrations of reducing sugar, glucose, and ethanol were 10.9, 8.57, and 5.95 g/l, respectively, at 120 h by the combination of T. reesei and C. molischiana from 50 g/l Avicel.


Assuntos
Biocombustíveis , Candida/metabolismo , Celobiose/metabolismo , Celulose/metabolismo , Etanol/metabolismo , Glucose/metabolismo , Saccharomyces cerevisiae/metabolismo , Candida/crescimento & desenvolvimento , Celobiose/química , Celulase/metabolismo , Celulose/química , Proteínas Fúngicas/metabolismo , Glucose/química , Hidrólise , Saccharomyces cerevisiae/crescimento & desenvolvimento , Açúcares/metabolismo , Trichoderma/crescimento & desenvolvimento , Trichoderma/metabolismo
5.
Mol Plant Microbe Interact ; 32(10): 1402-1414, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31216219

RESUMO

Xylella fastidiosa is a gram-negative bacterium that causes Pierce's disease (PD) in grapevine. X. fastidiosa is xylem-limited and interfaces primarily with pit membranes (PMs) that separate xylem vessels from one another and from adjacent xylem parenchyma cells. PMs are composed of both pectic and cellulosic substrates, and dissolution of PMs is facilitated by X. fastidiosa cell wall-degrading enzymes. A polygalacturonase, which hydrolyzes the pectin component of PMs, is required for both movement and pathogenicity in grapevines. Here, we demonstrate that two X. fastidiosa ß-1,4-endoglucanases (EGases), EngXCA1 and EngXCA2, also play a role in how X. fastidiosa interfaces with grapevine PMs. The loss of EngXCA1 and EngXCA2 in tandem reduces both X. fastidiosa virulence and population size and slows the rate of PD symptom development and progression. Moreover, we demonstrate that single and double EGases mutants alter the rate of PD progression differently in two grapevine cultivars, Cabernet Sauvignon and Chardonnay, and that Chardonnay is significantly more susceptible to PD than Cabernet Sauvignon. Interestingly, we determined that there are quantitative differences in the amount of fucosylated xyloglucans that make up the surface of PMs in these cultivars. Fucosylated xyloglucans are targets of the X. fastidiosa EGases, and xyloglucan abundance could impact PM dissolution and affect PD symptom development. Taken together, these results indicate that X. fastidiosa EGases and the PM carbohydrate composition of different grape cultivars are important factors that influence PD symptom development and progression.


Assuntos
Celulase , Vitis , Xylella , Celulase/metabolismo , Doenças das Plantas/microbiologia , Especificidade da Espécie , Vitis/classificação , Vitis/microbiologia , Xylella/enzimologia
6.
Food Chem ; 292: 81-89, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054696

RESUMO

How to effectively increase or decrease the ability of A. oryzae to produce enzymes was the key to improve the quality of soy sauce. However, multi-core property of A. oryzae resulted in genetic instability of the new strain. Here, A. oryzae 3.042-3 which can stably produce mononuclear spores was constructed based on A. oryzae 3.042. A. oryzae 3.042-3-c obtained by transformation of the fragment of cis-CreA into A. oryzae 3.042-3 exhibited genetic stability. The fragment containing the cis-acting and the promoter CreA from A. oryzae was connected to chromosome VII in A. oryzae 3.042-3-c. Compared with A. oryzae 3.042-3, the cellulase activity of A. oryzae 3.042-3-c was reduced by 50.5% and the pectinase activity was decreased by 10.0%. At the end of the soy sauce fermentation, the salt-free solid content of A. oryzae 3.042-3-c was higher 58.9% than that of A. oryzae 3.042-3. The kinds and contents of the flavor components of the soy sauce from the fermentation by A. oryzae 3.042-3-c were higher than those of the A. oryzae 3.042 and A. oryzae 3.042-3, especially in alcohols and esters. HEMF was only found in the soy sauce from A. oryzae 3.042-3-c. The results indicated that the new strain A. oryzae 3.042-3-c could improve the quality of soy sauce from the low-salt solid fermentation by decreasing enzyme activity of cellulase and pectinase.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Alimentos de Soja/análise , Aspergillus oryzae/genética , Celulase/genética , Celulase/metabolismo , Cromossomos Fúngicos , Qualidade dos Alimentos , Proteínas Fúngicas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alimentos de Soja/microbiologia
7.
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
8.
Bioresour Technol ; 287: 121417, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31076294

RESUMO

Herbel-tolerant strains exhibit considerable environmental and commercial values not only due to their harmless treatment of herbal-extraction residues (HERs) but also because of their use in preparing high-quality cellulase cocktails. In this study, three typical HERs were evaluated for enzymatic in situ saccharification performance. A HERs-tolerance fungus, identified as Penicillium oxalicum G2, can grow in 1.5% (w/v) Radix isatidis residues (RIR), thereby exhibiting the highest FPase (2.2 U/mL), carboxymethyl cellulase (13.3 U/mL), and ß-glucosidase (4.6 U/mL) activities. The most effective production of cellulase cocktail was achieved via orthogonal experiment in a system with pH 6.0, 30 °C, and 96 h. Cellulase cocktail from P. oxalicum G2 can directly saccharify the extraction RIR, thereby achieving a maximum reducing sugar yield of 7.2 mg/mL, which is 1.7-fold higher than those of commercial cellulases. Results illustrate the potential of P. oxalicum G2 for enzymatic in situ saccharification.


Assuntos
Celulase/metabolismo , Hidrólise , Penicillium/enzimologia , beta-Glucosidase/metabolismo
9.
J Food Sci ; 84(6): 1382-1389, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31141617

RESUMO

Evolution of Rhizopus oryzae and Trichoderma reesei biomass in rice bran, their enzyme activity, and the profile of phenolic compounds released from the lignocellulosic matrices were determined and correlated by principal component analysis (PCA). PCA analysis confirms that cultivation of rice bran affected the release of methanol-soluble phenolic compounds (MSPC), ethanol-soluble phenolic compounds (ESPC), and bound phenolic compounds (BPC) positively, due to their enzymatic activity. The release of MSPC was influenced by the activity of cellulase and endoglucanase, which increased 110.6% and 136.3%, respectively, for Rhizopus oryzae and Trichoderma reesei. Gallic acid was the main component in the MSPC and ESPC compound fractions. Ferulic and syringic acids were found in its bound (BPC) form in the biomass. This study showed that bioactive compounds be released from lignocellulosic materials by fungus action and this process can be conducted to obtain specific phenolic compounds. PRACTICAL APPLICATION: Due the demand by natural compounds with biological activity, such as phenolic compounds, it is interesting to purpose alternatives to enhance their yield, like for instance, by fungal fermentation of lignocellulosic material. Therefore, understanding the relations among different phenolic compounds released and the production of fungal hydrolases during growth of Rhizopus oryzae and Trichoderma reesei in solid state cultivation using rice bran as a substrate is fundamental to control the process. This knowledge gets viable scale up to apply the phenolic compounds as preservative in food chain, because this becomes possible directing the process to obtain specific bioactive compounds in less time of cultivation and with low cost.


Assuntos
Celulase/metabolismo , Oryza/química , Fenóis/análise , Rhizopus/enzimologia , Trichoderma/enzimologia , Biomassa , Celulose/metabolismo , Ácidos Cumáricos/análise , Meios de Cultura , Fermentação , Ácido Gálico/análogos & derivados , Ácido Gálico/análise , Rhizopus/crescimento & desenvolvimento , Trichoderma/crescimento & desenvolvimento
10.
Curr Microbiol ; 76(7): 879-887, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089795

RESUMO

The goal of this study was to elucidate the role of the outer membrane protein A (ompA) gene of Xanthomonas axonopodis pv. glycines in bacterial pustule pathogenesis of soybean. An ompA mutant of X. axonopodis pv. glycines KU-P-SW005 was shown to significantly decrease cellulase, pectate lyase, and polysaccharide production. The production of these proteins in the ompA mutant was approximately five times lower than that of the wildtype. The ompA mutant also exhibited modified biofilm development. More importantly, the mutant reduced disease severity to the soybean. Ten days after inoculation, the virulence rating of the susceptible soybean cv. SJ4 inoculated with the ompA mutant was 11.23%, compared with 87.98% for the complemented ompA mutant. Production of cellulase, pectate lyase, polysaccharide was restored, biofilm, and pustule numbers were restored in the complemented ompA mutant that did not differ from the wild type. Taken together, these data suggest that OmpA-mediated invasion plays an important role in protein secretion during pathogenesis to soybean.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Doenças das Plantas/microbiologia , Soja/microbiologia , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidade , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Celulase/metabolismo , Teste de Complementação Genética , Mutação , Folhas de Planta/microbiologia , Polissacarídeo-Liase/metabolismo , Polissacarídeos Bacterianos/metabolismo , Virulência/genética
11.
Microb Cell Fact ; 18(1): 81, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31077201

RESUMO

BACKGROUND: Cellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion. The high cost of cellulase is still a significant challenge to commercial biofuel production. Improving cellulase production in T. reesei for application in the cellulosic biorefinery setting is an urgent priority. RESULTS: Trichoderma reesei hyper-cellulolytic mutant SS-II derived from the T. reesei NG14 strain exhibited faster growth rate and more efficient lignocellulosic biomass degradation than those of RUT-C30, another hyper-cellulolytic strain derived from NG14. To identify any genetic changes that occurred in SS-II, we sequenced its genome using Illumina MiSeq. In total, 184 single nucleotide polymorphisms and 40 insertions and deletions were identified. SS-II sequencing revealed 107 novel mutations and a full-length wild-type carbon catabolite repressor 1 gene (cre1). To combine the mutations of RUT-C30 and SS-II, the sequence of one confirmed beneficial mutation in RUT-C30, cre196, was introduced in SS-II to replace full-length cre1, forming the mutant SS-II-cre196. The total cellulase production of SS-II-cre196 was decreased owing to the limited growth of SS-II-cre196. In contrast, 57 genes mutated only in SS-II were selected and knocked out in RUT-C30. Of these, 31 were involved in T. reesei growth or cellulase production. Cellulase activity was significantly increased in five deletion strains compared with that in two starter strains, RUT-C30 and SS-II. Cellulase production of T. reesei Δ108642 and Δ56839 was significantly increased by 83.7% and 70.1%, respectively, compared with that of RUT-C30. The amount of glucose released from pretreated corn stover hydrolyzed by the crude enzyme from Δ108642 increased by 11.9%. CONCLUSIONS: The positive attribute confirmed in one cellulase hyper-producing strain does not always work efficiently in another cellulase hyper-producing strain, owing to the differences in genetic background. Genome re-sequencing revealed novel mutations that might affect cellulase production and other pathways indirectly related to cellulase formation. Our strategy of combining the mutations of two strains successfully identified a number of interesting phenotypes associated with cellulase production. These findings will contribute to the creation of a gene library that can be used to investigate the involvement of various genes in the regulation of cellulase production.


Assuntos
Celulase , Genômica/métodos , Trichoderma , Biomassa , Celulase/genética , Celulase/metabolismo , Glucose/metabolismo , Mutação , Trichoderma/genética , Trichoderma/crescimento & desenvolvimento , Trichoderma/metabolismo
12.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067833

RESUMO

In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-ß-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters Km and Vmax were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0-8.0) and was highly thermostable at 55 °C for 72 h. Low Cu2+ concentrations (0.1-1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse "in natura", sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.


Assuntos
Aspergillus fumigatus/enzimologia , Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Lignina/metabolismo , Pichia/metabolismo , Aspergillus fumigatus/genética , Biomassa , Celulase/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Hidrólise , Pichia/genética , Pichia/crescimento & desenvolvimento , Termotolerância
13.
J Dairy Sci ; 102(7): 6235-6241, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31079907

RESUMO

The purpose of this study was to optimize the conditions of a previously proposed enzymatic method used to estimate in situ undigested neutral detergent fiber (uNDF). We used a multi-step enzymatic approach, in which samples were first solubilized in NaOH solutions as a preincubation (PreInc) phase. After rinsing, samples were incubated (24 h at 39°C) in a buffered solution (pH 6) containing hemicellulase, cellulase, and Viscozyme L enzymes (Sigma-Aldrich s.r.l., Milan, Italy), followed by incubation (24 h at 39°C) in a buffered solution (pH 5) containing xylanase. Two sets of experiments were performed: a calibration trial (that tested different PreInc conditions on 9 selected forages) and a validation trial (that verified the results by testing multiple samples of 6 different forage types and a group of fibrous by-products). In the calibration trial, samples (300 mg in Ankom F57 filter bags; Ankom Technology Corp., Fairport, NY) were preincubated at 39°C in a 0.1 M NaOH solution for 90, 180, or 240 min, or in 0.2, 0.5, 1.0, or 2.0 M NaOH solution for 90 min. The results indicated that the best PreInc method, in terms of intra-laboratory repeatability and estimation of reference in situ values, was 90 min in a 0.2 M NaOH solution. Thus, we used this PreInc condition to determine enzymatic uNDF of 257 samples in the validation trial. Although the selected method generally had good accuracy in predicting in situ uNDF, inconsistencies were noted for certain forage types. Overall, when enzymatic uNDF was used to predict the in situ uNDF of all samples, the regression was satisfactory (intercept = 7.098, slope = 0.920, R2 = 0.73). The regression models developed for alfalfa hays, corn silages, and small grain silages had also acceptable regression performances and mean square error of prediction (MSEP) values, and the main sources of MSEP variation were error due to incomplete (co)variation and random error. Even when R2 values were >0.70, the MSEP value of the regression model for grass hays was 149.55, and that for nonforage fibrous feeds was 155.16. Although enzymatic uNDF partially overestimated the in situ uNDF, particularly in grass silages, the proposed procedure seems to be promising for accurately predicting in situ uNDF, because it generally had good repeatability and provided satisfactory estimates of in situ uNDF.


Assuntos
Ração Animal/análise , Bovinos/metabolismo , Fibras na Dieta/análise , Fibras na Dieta/metabolismo , Digestão , Animais , Celulase/metabolismo , Feminino , Glicosídeo Hidrolases/metabolismo , Itália , Medicago sativa/metabolismo , Complexos Multienzimáticos/metabolismo , Reprodutibilidade dos Testes , Rúmen , Silagem/análise , Zea mays/metabolismo
14.
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
15.
Food Chem ; 289: 232-239, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955607

RESUMO

A novel method for producing germinated brown rice (GBR) was developed by cyclic cellulase conditioning and germination treatment. Changes in the γ-aminobutyric acid (GABA) content and the cooking and taste qualities of GBR were analysed. The results indicated GABA accumulation reached its highest level under 50 mg/ml cellulase solution treatment for 90 min followed by 32 h germination at 30 °C. The treatment conditions had no significant influence on the cooking or taste qualities of GBR. Correlation analysis showed the taste value of cooked GBR significantly correlated with hardness, springiness, chewiness, and stickiness. The cyclic cellulase conditioning treatment could contribute to improved cooking and taste qualities of GBR and higher GABA content of GBR compared to the soaking treatment. The cooking and taste qualities of GBR were improved by the degradation of the crude fibre of the cortex and the increase in GBR water absorption rate under cyclic cellulase conditioning.


Assuntos
Celulase/metabolismo , Paladar/fisiologia , Ácido gama-Aminobutírico/análise , Cromatografia Líquida de Alta Pressão , Culinária , Germinação , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Temperatura Ambiente , Água/química
16.
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
17.
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
18.
Food Chem ; 289: 453-460, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955636

RESUMO

Carrot residues were upgraded as pectin-enriched fractions (PEFs) useful for functional food formulation due to co-extracted antioxidants (α- and ß-carotenes, lutein, α-tocopherol), and gelling effect. High power ultrasound (US)-enzyme assisted extraction was applied for efficiency and sustainability. Carrot powder (CP) in citrate-buffer (pH 5.20) was submitted to US-pretreatment (12.27 W/cm2: 20 kHz, 80% amplitude, 20 min) and a subsequent digestion (5 h-40 °C) without or with hemicellulase or cellulase. US-hemicellulase led to the highest PEF yield (27.1%), and extracted almost the whole pectin content of CP. US-pretreatment increased the extraction yield of all PEFs, but the existence of an additional positive effect of the following step depended on the enzyme used. PEFs contained 40-47% of UA with low DM (24-49.9%), and co-extracted antioxidants. US decreased the antioxidant contents, DM, and molecular weight, but allowed obtaining calcium crosslinked true gels, also with higher elastic modulus than non-US-extracted PEFs, being promising as food additives.


Assuntos
Antioxidantes/isolamento & purificação , Daucus carota/química , Pectinas/isolamento & purificação , Carotenoides/isolamento & purificação , Celulase/metabolismo , Manipulação de Alimentos/métodos , Luteína/isolamento & purificação , Raízes de Plantas/química , Ultrassom , alfa-Tocoferol/isolamento & purificação
19.
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
20.
Appl Biochem Biotechnol ; 189(1): 249-261, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30972706

RESUMO

To date, bioethanol is not economically competitive. One strategy to overcome this limitation is co-producing ethanol and high value-added products as an integrated process. The results of this study demonstrated that flavonoids could be extracted from rice straw, and the flavonoids apigenin and kaempferol were detected by HPLC. Compared with untreated straw, ball-milling slightly increased the total amount of flavonoids and antioxidant activity measured by ABTS, DPPH, and FRAP assays. The saccharification step in the bioconversion of straw strongly affected the extraction of flavonoids from straw. The residue obtained after saccharification of ball-milled straw for glucose production was more suitable for flavonoid extraction than untreated and ball-milled straw. The yield of flavonoids from the residue was 1.51-fold higher than that from untreated straw. The antioxidant activity of flavonoids derived from the residue was similar to that of flavonoid-rich biomasses such as rice bran and wheat bran. More importantly, saccharification may significantly affect the conditions of flavonoid extraction. In this respect, treatment with cellulase may reduce the extraction time from 2.0 to 0.5 h and the extraction temperature from 80 to 30 °C. Therefore, saccharification in the bioconversion of straw may be considered as an enzyme pretreatment step for the efficient extraction of flavonoids from straw, serving as a sustainable process for straw utilization.


Assuntos
Apigenina/isolamento & purificação , Celulose/metabolismo , Quempferóis/isolamento & purificação , Oryza/metabolismo , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Celulase/metabolismo , Etanol/metabolismo , Fermentação , Temperatura Alta
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