Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 54
Filter
1.
Journal of Southern Medical University ; (12): 257-264, 2023.
Article in Chinese | WPRIM | ID: wpr-971523

ABSTRACT

OBJECTIVE@#To investigate the inhibitory effects of levofloxacin (LEV) combined with cellulase against bacille CalmetteGuerin (BCG) biofilms in vitro.@*METHODS@#The mature growth cycle of BCG biofilms was determined using the XTT method and crystal violet staining. BCG planktonic bacteria and BCG biofilms were treated with different concentrations of LEV and cellulose alone or jointly, and the changes in biofilm biomass were quantified with crystal violet staining. The mature BCG biofilm was then treated with cellulase alone for 24 h, and after staining with SYTO 9 and Calcofluor White Stain, the number of viable bacteria and the change in cellulose content in the biofilm were observed with confocal laser scanning microscopy. The structural changes of the treated biofilm were observed under scanning electron microscopy.@*RESULTS@#The MIC, MBC and MBEC values of LEV determined by broth microdilution method were 4 μg/mL, 8 μg/mL and 1024 μg/mL, respectively. The combined treatment with 1/4×MIC LEV and 2.56, 5.12 or 10.24 U/mL cellulase resulted in a significant reduction in biofilm biomass (P < 0.001). Cellulase treatments at the concentrations of 10.24, 5.12 and 2.56 U/mL all produced significant dispersion effects on mature BCG biofilms (P < 0.001).@*CONCLUSION@#LEV combined with cellulose can effectively eradicate BCG biofilm infections, suggesting the potential of glycoside hydrolase therapy for improving the efficacy of antibiotics against biofilmassociated infections caused by Mycobacterium tuberculosis.


Subject(s)
Levofloxacin/pharmacology , Gentian Violet/pharmacology , BCG Vaccine/pharmacology , Anti-Bacterial Agents/pharmacology , Biofilms , Cellulases/pharmacology , Microbial Sensitivity Tests
2.
Chinese Journal of Biotechnology ; (12): 4927-4938, 2023.
Article in Chinese | WPRIM | ID: wpr-1008069

ABSTRACT

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, β-glucan metabolic process, cellulase activity, endo-1,4-β-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.


Subject(s)
Animals , Cattle , Neocallimastigales/metabolism , Anaerobiosis , Rumen/microbiology , Propionates/metabolism , Isobutyrates/metabolism , Cellulose/metabolism , Fungi , Starch/metabolism , Glucose/metabolism , Acetates , Sucrose/metabolism , Cellulases , Cellulase
3.
Chinese Journal of Biotechnology ; (12): 4593-4607, 2023.
Article in Chinese | WPRIM | ID: wpr-1008044

ABSTRACT

The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.


Subject(s)
Aspergillus niger/genetics , Xylose/metabolism , Molecular Docking Simulation , Xylosidases/genetics , Cellulases , Tea , Hydrogen-Ion Concentration , Substrate Specificity
4.
China Journal of Chinese Materia Medica ; (24): 2309-2314, 2022.
Article in Chinese | WPRIM | ID: wpr-928109

ABSTRACT

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.


Subject(s)
Agaricales , Ascomycota , Basidiomycota , Catechol Oxidase , Cellulases , Gastrodia
5.
Electron. j. biotechnol ; 51: 79-87, May. 2021. tab, ilus, graf
Article in English | LILACS | ID: biblio-1343441

ABSTRACT

BACKGROUND: At present, cellulases are the most important enzymes worldwide, and their demand has been increasing in the industrial sector owing to their notable hydrolysis capability. RESULTS: In the present study, contrary to conventional techniques, three physical parameters were statistically optimized for the production of cellulase by thermophilic fungi by using response surface methodology (RSM). Among all the tested thermophilic strains, the best cellulase producing fungus was identified as Talaromyces thermophilus ­ both morphologically and molecularly through 5.8S/ITS rDNA sequencing. The central composite design (CCD) was used to evaluate the interactive effect of the significant factors. The CCD was applied by considering incubation period, pH, and temperature as the model factors for the present investigation. A second-order quadratic model and response surface method revealed that the independent variables including pH 6, temperature 50 C, and incubation period 72 h significantly influenced the production of cellulases. The analysis of variance (ANOVA) indicated that the established model was significant (P 0.05) and showed the high adequacy of the model. The actual and predicted values of CMCase and FPase activity showed good agreement with each other and also confirmed the validity of the designed model. CONCLUSIONS: We believe the present findings to be the first report on cellulase production by exploiting Kans grass (Saccharum spontaneum) as a substrate through response surface methodology by using thermophilic fungus, Talaromyces thermophilus.


Subject(s)
Talaromyces/metabolism , Cellulases/biosynthesis , Analysis of Variance , Saccharum , Fermentation , Hot Temperature , Hydrogen-Ion Concentration
6.
Rev. argent. microbiol ; 52(4): 61-70, dic. 2020. graf
Article in English | LILACS | ID: biblio-1340921

ABSTRACT

Resumen La demanda de xilanasas fúngicas en los procesos biotecnológicos industriales muestra un claro aumento en todo el mundo, por lo que hay un interés en ajustar las condiciones de producción de xilanasas microbianas. En este estudio se optimizó la capacidad del hongo Fusarium solani para producir xilanasas extracelulares con escasa actividad celulolítica mediante el diseño de Box-Wilson. Se determinaron las mejores condiciones de cultivo para obtener una preparación enzimática cruda con una actividad xilanolítica significativa y poca actividad celulolítica. En la mayoría de los tratamientos, la actividad xilanolítica fue mayor que la actividad celulolítica. Se observó un efecto negativo sobre la producción de endoxilanasas, p-xilosidasasy endocelulasascon el aumento de la concentración dexilano. El aumento del tiempo de incubación afectó adversamente la producción de endocelulasas y p-xilosidasas. De acuerdo con el modelo matemático y las pruebas experimentales, es posible producir endoxilanasas con una actividad endocelulasa mínima aumentando el tiempo de incubación y la concentración de sulfato de amonio. Las condiciones de cultivo óptimas para producir una mayor cantidad de endoxilanasas (10,65 U/mg) y mínima cantidad de endocelulasas fueron 2,5% (p/v) de xilano y 5, 2 y 0,4 g/l de extracto de levadura, sulfato de amonio y urea, respectivamente, con 120 h de incubación.


Resumen La demanda de xilanasas fúngicas en los procesos biotecnológicos industriales muestra un claro aumento en todo el mundo, por lo que hay un interés en ajustar las condicionesde producción de xilanasas microbianas. En este estudio se optimizó la capacidad del hongo Fusarium solani para producir xilanasas extracelulares con escasa actividad celulolítica medi-ante el dise˜no de Box-Wilson. Se determinaron las mejores condiciones de cultivo para obteneruna preparación enzimática cruda con una actividad xilanolítica significativa y poca actividad celulolítica. En la mayoría de los tratamientos, la actividad xilanolítica fue mayor que laactividad celulolítica. Se observó un efecto negativo sobre la producción de endoxilanasas, xylanolytic activity and little cellulolytic activity. In most treatments, the xylanolytic activity was higher than the cellulolytic activity. A negative effect on the production of endoxylanases, p-xylosidases and endocellulases was observed with the increasing of xylan concentration. Increasing the incubation time adversely affected the production of endocellulases and p-xylosidases. According to the mathematical model and experimental tests, it is possible to produce endoxylanases with minimal endocellulase activity increasing incubation time and the concentration of ammonium sulfate. The optimal culture conditions to produce a greater amount of endoxylanases (10.65 U/mg) and low endocellulases from F. solani were: 2.5% (w/v) xylan, 5.0, 2.0 and 0.4g/l, of yeast extract, ammonium sulfate and urea, respectively, with 120 h of incubation.


Subject(s)
Cellulases , Endo-1,4-beta Xylanases/biosynthesis , Fermentation , Research Design , Industrial Microbiology , Fusarium , Hydrogen-Ion Concentration
7.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 1069-1074, May-June, 2020. tab
Article in Portuguese | LILACS, VETINDEX | ID: biblio-1129781

ABSTRACT

The objective was to evaluate the digestive tract characteristics, metabolizability and nutrient retention of broilers fed diets supplemented with enzyme complex (EC). To evaluate the characteristics of the digestive tract 600 female Cobb 500 birds were used, distributed in a completely randomized design, with 5 inclusion levels of the EC (0; 100, 200, 300 and 400 g/ton) and 6 replicates of 20 birds each. To evaluate the metabolizability and the retention of nutrients 200 female Cobb 500 birds at 15 days of age were used, distributed in a completely randomized design with 5 levels of supplementation of the EC and 4 replicates of 10 birds each. No significant effects (P>0.05) were observed for the supplementation of the EC in the intestinal pH, digestive organ weight, intestinal length and metabolizable coefficients of dry matter and crude protein. The metabolizable coefficient of ethereal extract was influenced in a quadratic decreasing form (P<0.01). The metabolizable coefficients of calcium (Ca) and phosphorus (P) were influenced in a quadratic increase (P<0.01), resulting in increased Ca retention in 21.39% and P in 9.56%. Supplementation of the EC in broiler diets improves the metabolizability and retention of P and Ca, without affecting the other parameters evaluated.(AU)


Subject(s)
Animals , Nutrients/administration & dosage , Chickens/metabolism , Gastrointestinal Tract/metabolism , Enzymes/administration & dosage , Peptide Hydrolases , Dietary Supplements/analysis , Cellulases
8.
Biosci. j. (Online) ; 36(3): 924-931, 01-05-2020. tab
Article in English | LILACS | ID: biblio-1146988

ABSTRACT

Fungi are capable of sensing light from ultraviolet to far-red and they use light as a source of information about the environment anticipating stress and adverse conditions. Lentinus crinitus is a lignin-degrading fungus which produces laccase and other enzymes of biotechnological interest. The effect of blue light on fungal enzymatic activity has been studied; however, it has not been found studies on the effect of the blue light on carbohydrate-active enzymes and on mycelial biomass production of L. crinitus. We aimed to investigate carbohydrate-active enzymes activity and mycelial biomass production of L. crinitus cultivated under continuous illumination with blue light. L. crinitus was cultivated in malt extract medium in the dark, without agitation, and under continuous illumination with blue light-emitting diodes. The blue light reduced the total cellulase, pectinase and xylanase activities but increased the endoglucanase activity. Blue light reduced the mycelial growth of L. crinitus in 26% and the enzymatic activity-to-mycelial biomass ratio (U mg-1 dry basis) increased in 10% total cellulase, 33% endoglucanase, and 16% pectinase activities. Also, it is suggested that L. crinitus has a photosensory system and it could lead to new process of obtaining enzymes of biotechnological interest.


Fungos são capazes de sentir a luz com comprimentos de onda que variam do ultravioleta ao infravermelho e usam a luz como fonte de informação sobre o ambiente, antecipando condições adversas e de estresse. Lentinus crinitus é um fungo ligninolítico que produz lacase e outras enzimas de interesse biotecnológico. O efeito da luz azul na atividade enzimática de fungos já foi estudado, contudo, ainda não há estudos sobre o efeito da luz azul na produção de enzimas ativas sobre carboidratos (CAZymes, carbohydrate-active enzymes) e de biomassa micelial de L. crinitus. O objetivo deste estudo foi investigar a avitivade de CAZymes e a produção de biomassa micelial de L. crinitus cultivado sob iluminação continua com luz azul. L. crinitus foi cultivado em meio extrato de malte, sem agitação, na ausência de luz e sob luz continua fornecida por diodos emissores de luz azul. A luz azul reduziu a atividade de cellulase total, pectinase e xilanase, mas aumentou a atividade de endoglucanase. A luz azul reduziu o crescimento micelial de L. crinitus em 26% e aumentou a razão atividade enzimática/biomassa micelial (U mg-1 em base seca) de cellulase total em 10%, endoglucanase em 33% e pectinase em 16%. Além disso, sugere-se que L. crinitus possua um sistema fotossensorial que poderia ser explorado para a otimização de bioprocessos que visam a obtenção de enzimas de interesse biotecnológico.


Subject(s)
Polygalacturonase , Lentinula , Cellulases , Light
9.
Braz. arch. biol. technol ; 63: e20170710, 2020. tab, graf
Article in English | LILACS | ID: biblio-1132254

ABSTRACT

Abstract (1) Background: The aim of this study was to evaluate the production and partial characterization of xylanase and avicelase by a newly isolated Penicillium sp. in solid-state fermentation, using soybean hulls as substrate. (2) Methods: Temperature, time, number of spores, and substrate moisture on xylanase and avicelase bioproduction were evaluated, maximizing activity with 30°C, 1x106 spores/g substrate, 14 and 7 days of fermentation with 70 and 76% substrate moisture contents, for xylanase and avicelase, respectively. (3) Results: Different solvents, temperatures, and agitation in the enzymatic extraction were evaluated, obtaining higher activities, 430.77 and 26.77 U/g for xylanase and avicelase using 30 min extraction and 0.05 M citrate buffer solution (pH 4.5 ), respectively at 60°C and 175 rpm and 50°C and 125 rpm. The optimum pH and temperature for enzymatic activity determination were 5.3 and 50°C. Enzyme extract stability was evaluated, obtaining higher stability with pH between 4.5 and 5.5, higher temperature of up to 40°C. The kinetic thermal denaturation (Kd), half-life time, D-value, and Z-value were similar for both enzymes. The xylanase Ed value (89.1 kJ/mol) was slightly lower than the avicelase one (96.7 kJ/mol), indicating higher thermostability for avicelase. (4) Conclusion: In this way, the production of cellulases using alternative substrates is a way to reduce production costs, since they represent about 10% of the world demand of enzymes, with application in animal feed processing, food production and breweries, textile processing, detergent and laundry production, pulp manufacturing and the production of biofuels.


Subject(s)
Penicillium/isolation & purification , Penicillium/enzymology , Glycine max/microbiology , Xylosidases/biosynthesis , Cellulases/biosynthesis , Temperature , Time Factors , Substrates for Biological Treatment
10.
Electron. j. biotechnol ; 41: 60-71, sept. 2019. graf, tab, ilus
Article in English | LILACS | ID: biblio-1087169

ABSTRACT

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.


Subject(s)
Sordariales/enzymology , Glucan 1,3-beta-Glucosidase/chemistry , Temperature , Enzyme Stability , Cellulases , Glucan 1,3-beta-Glucosidase/isolation & purification , Electrophoresis, Polyacrylamide Gel , Tandem Mass Spectrometry , Enzyme Assays , Hydrogen-Ion Concentration
11.
Electron. j. biotechnol ; 41: 1-8, sept. 2019. tab, ilus, graf
Article in English | LILACS | ID: biblio-1053552

ABSTRACT

Background: The bioethanol produced from biomass is a promising alternative fuel. The lignocellulose from marginal areas or wasteland could be a promising raw material for bioethanol production because it is present in large quantities, is cheap, renewable and has favorable environmental properties. Despite these advantages, lignocellulosic biomass is much more difficult to process than cereal grains, due to the need for intensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Therefore, there is a need to develop an efficient and cost-effective method for the degradation and fermentation of lignocellulosic biomass to ethanol. Results: The usefulness of lignocellulosic biomass from wasteland for the production of bioethanol using pretreatment with the aid of ionic liquids of 1-ethyl-3-methylimidazolium acetate and 1-ethyl-3-methylimidazolium chloride was evaluated in this study. The pretreatment process, enzymatic hydrolysis and alcoholic fermentation lasted a total of 10 d. The largest amounts of bioethanol were obtained from biomass originating from agricultural wasteland, in which the dominant plant was fireweed (Chamaenerion angustifolium) and from the field where the common broom (Cytisus scoparius) was the dominant. Conclusions: The plants such as fireweed, common broom, hay and goldenrod may be useful for the production of liquid biofuels and it would be necessary in the further stage of research to establish and optimize the conditions for the technology of ethyl alcohol producing from these plant species. Enzymatic hydrolysis of biomass from agricultural wastelands results in a large increase in fermentable sugars, comparable to the enzymatic hydrolysis of rye, wheat, rice or maize straw.


Subject(s)
Soil/chemistry , Biomass , Ethanol/metabolism , Biodegradation, Environmental , Cellulases/analysis , Enzymes/metabolism , Ionic Liquids , Biofuels , Hydrolysis , Lignin/analysis
12.
Rio de Janeiro; s.n; 2019. xiv, 152 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-1049943

ABSTRACT

Celulases fúngicas têm sido usadas para degradar a biomassa lignocelulósica para a produção de bioetanol. Celulases industriais como Cel7A de Trichoderma reesei (TrCel7A) são críticas neste processo. A compreensão da estrutura e dinâmica é crucial para a reengenharia da atividade celulolítica. Esta enzima é formada por dois domínios ligados por um linker flexível e altamente glicosilado. No entanto, a flexibilidade do linker tem dificultado a determinação da estrutura completa da Cel7A. Assim, na ausência de dados experimentais de alta resolução, aplicamos a modelagem integrativa para construir um modelo da enzima completa. Em seguida, estudamos os efeitos da glicosilação na estrutura e dinâmica da apo TrCel7A por meio de simulações. A análise da dinâmica essencial mostrou que a O-glicosilação no linker levou à estabilização da dinâmica global da proteína. Os glicanos O-ligados parecem restringir a distribuição dos ângulos diedros desta região, selecionando conformações mais alongadas. Além da flexibilidade reduzida, os movimentos interdomínios funcionais foram preservados no sistema glicosilado. Em contraste, observamos grande plasticidade conformacional na ausência de glicosilação, mas os domínios funcionais frequentemente colapsaram. Nós relatamos aqui evidências de que a flexibilidade dirigida no linker de Cel7A por mutações pontuais, incluindo modificações de sítios de glicosilação, poderia ser uma estratégia promissora para melhorar a atividade da celulase. (AU)


Subject(s)
Humans , Trichoderma , Glycosylation , Mutagenesis, Insertional , Cellulases
13.
Braz. j. microbiol ; 49(4): 879-884, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-1039268

ABSTRACT

ABSTRACT The multi-enzyme complex (crude extract) of white rot fungi Pleurotus ostreatus, Pleurotus eryngii, Trametes versicolor, Pycnosporus sanguineus and Phanerochaete chrysosporium were characterized, evaluated in the hydrolysis of pretreated pulps of sorghum straw and compared efficiency with commercial enzyme. Most fungi complexes had better hydrolysis rates compared with purified commercial enzyme.


Subject(s)
Fungal Proteins/chemistry , Sorghum/chemistry , Cellulases/chemistry , Fungi/enzymology , Lignin/chemistry , Fungal Proteins/metabolism , Plant Stems/microbiology , Plant Stems/chemistry , Sorghum/microbiology , Cellulases/metabolism , Biocatalysis , Fungi/chemistry , Hydrolysis , Lignin/metabolism
14.
Rev. argent. microbiol ; 50(3): 234-243, set. 2018. ilus, tab
Article in English | LILACS | ID: biblio-977237

ABSTRACT

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57 -#9702;C and pH 5.5 and the other, a temperature of 61 -#9702;C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24 h until 144 h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.


El objetivo de este estudio fue aislar, seleccionary caracterizar bacterias con actividad celulolítica a partir de 2 diferentes pilas de compostaje de residuos de café, una con temperatura interna de 57°C y pH 5,5; la otra con temperatura interna de 61 °C y pH 9,3. Se utilizaron medios de cultivo con carboximetilcelulosa y celulosa cristalina como únicas fuentes de carbono. La actividad enzimàtica fue evaluada por formación de halos de hidrólisis, producción de azúcares reductores y zimogramas. De 20 cepas aisladas, 3 presentaron mayor actividad enzimàtica y fueron identificadas como Bacillus subtilis sobre la base de sus características morfológicas, fisiológicas y bioquímicas y del análisis de las secuencias de la región 16S del ADNr. Los extractos enzimáticos de las 3 cepas seleccionadas presentaron actividad de exocelulasa y de endocelulasa, con máximos de 0,254 y 0,519 U/ml, respectivamente; la actividad de estas enzimas se mantuvo incluso a pH ácido (4,8) o básico (9,3) y a temperaturas de hasta 60 °C. Las actividades enzimáticas halladas en este estudio se ubican dentro de las más altas reportadas para celulasas producidas por bacterias del género Bacillus. En los zimogramas se demostró actividad de endocelulasa desde las 24h hasta las 144h de incubación. Asimismo, se reporta por primera vez el efecto del pH sobre la actividad de endocelulasa observado por zimogramas. Los resultados de este estudio abren la posibilidad de hacer uso de estas enzimas en la obtención de sustratos fermentables para la producción de energía a partir de los residuos generados en grandes cantidades por la agroindustria del café.


Subject(s)
Bacillus subtilis , Coffee , Cellulases , Bacillus subtilis/isolation & purification , Bacillus subtilis/enzymology , Composting , Cellulose , Cellulases/metabolism
15.
Electron. j. biotechnol ; 34: 29-36, july. 2018. ilus, tab, graf
Article in English | LILACS | ID: biblio-1045993

ABSTRACT

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.


Subject(s)
Bacillus/enzymology , Cellulases/biosynthesis , Temperature , Enzyme Stability , Gene Expression , Cell Wall/enzymology , Polymerase Chain Reaction , Cloning, Molecular , Cellulases/isolation & purification , Cellulases/metabolism , Escherichia coli/metabolism , Plant Cells/enzymology , Hydrogen-Ion Concentration , Hydrolysis
16.
Mycobiology ; : 370-378, 2017.
Article in English | WPRIM | ID: wpr-729651

ABSTRACT

Cylindrocarpon destructans is an ascomycete soil-borne pathogen that causes ginseng root rot. To identify effective biocontrol agents, we isolated several bacteria from ginseng cultivation soil and evaluated their antifungal activity. Among the isolated bacteria, one isolate (named JH7) was selected for its high antibiotic activity and was further examined for antagonism against fungal pathogens. Strain JH7 was identified as a Chromobacterium sp. using phylogenetic analysis based on 16S rRNA gene sequences. This strain was shown to produce antimicrobial molecules, including chitinases and proteases, but not cellulases. Additionally, the ability of JH7 to produce siderophore and solubilize insoluble phosphate supports its antagonistic and beneficial traits for plant growth. The JH7 strain suppressed the conidiation, conidial germination, and chlamydospore formation of C. destructans. Furthermore, the JH7 strain inhibited other plant pathogenic fungi. Thus, it provides a basis for developing a biocontrol agent for ginseng cultivation.


Subject(s)
Ascomycota , Bacteria , Cellulases , Chromobacterium , Fungi , Genes, rRNA , Germination , Panax , Peptide Hydrolases , Plants , Soil
17.
Electron. j. biotechnol ; 19(6): 56-62, Nov. 2016. ilus
Article in English | LILACS | ID: biblio-840314

ABSTRACT

Background: Endoglucanase, one of three type cellulases, can randomly cleave internal p-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene (JqCel5A) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance.


Subject(s)
Actinomycetales/enzymology , Cellulases/chemistry , Cellulases/isolation & purification , Cellulases/genetics , Hydrogen-Ion Concentration , Mutagenicity Tests , Temperature
18.
Electron. j. biotechnol ; 19(6): 79-83, Nov. 2016. ilus
Article in English | LILACS | ID: biblio-840317

ABSTRACT

Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus. Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5-pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5-pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C-40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co2+ and Fe2+, but decreased by Cd2+, Zn2+, Li+, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS. Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.


Subject(s)
Cellulases/metabolism , Citrobacter/enzymology , Escherichia coli/enzymology , Myxococcus xanthus/enzymology , Cold Temperature , Genetic Vectors , Recombinant Proteins
19.
Rev. argent. microbiol ; 48(3): 191-195, set. 2016. graf
Article in English | LILACS | ID: biblio-1290558

ABSTRACT

Se estudió la producción de enzimas hidrolíticas (celulasas, laminarinasas y xilanasas) en cultivos de Lentinula edodes en pulpa de café estéril. Se tomaron muestras de sustrato colonizado por el micelio después de 7, 14, 21, 28 y 35 días de incubación a 25°C (W1 a W5) y durante el período de fructificación en diferentes etapas: formación de primordios (PF), primera cosecha (H) y una semana después de la primera cosecha (PH). La actividad enzimática fue menor al inicio del crecimiento micelial y mostró mayores niveles en la formación y el desarrollo de basidiomas. Durante la etapa reproductiva del hongo, las muestras se sometieron a un tratamiento de remojo. Sin embargo, no fue posible relacionar este tratamiento con el aumento de la producción de enzimas. Los niveles de actividad enzimática sugieren que la secreción de las enzimas estudiadas no influye en la capacidad de adaptación de las cepas al sustrato


Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate


Subject(s)
Shiitake Mushrooms/growth & development , Shiitake Mushrooms/enzymology , Enzymes/analysis , Cellulases/isolation & purification
20.
Braz. j. microbiol ; 47(1): 110-119, Jan.-Mar. 2016. tab, graf
Article in English | LILACS | ID: lil-775128

ABSTRACT

Abstract The bioconversion of cellulosic wastes into high-value bio-products by saccharification and fermentation processes is an important step that can reduce the environmental pollution caused by agricultural wastes. In this study, enzymatic saccharification of treated and untreated date palm cellulosic wastes by the cellulases from Geobacillus stearothermophilus was optimized. The alkaline pre-treatment of the date palm wastes was found to be effective in increasing the saccharification percentage. The maximum rate of saccharification was found at a substrate concentration of 4% and enzyme concentration of 30 FPU/g of substrate. The optimum pH and temperature for the bioconversions were 5.0 and 50 °C, respectively, after 24 h of incubation, with a yield of 31.56 mg/mL of glucose at a saccharification degree of 71.03%. The saccharification was increased to 94.88% by removal of the hydrolysate after 24 h by using a two-step hydrolysis. Significant lactic acid production (27.8 mg/mL) was obtained by separate saccharification and fermentation after 72 h of incubation. The results indicate that production of fermentable sugar and lactic acid is feasible and may reduce environmental pollution by using date palm wastes as a cheap substrate.


Subject(s)
Cellulases/metabolism , Cellulose/metabolism , Geobacillus stearothermophilus/enzymology , Glucose/metabolism , Industrial Waste , Lactic Acid/metabolism , Phoeniceae/metabolism , Alkalies , Biotransformation , Fermentation , Hydrogen-Ion Concentration , Phoeniceae/drug effects , Temperature
SELECTION OF CITATIONS
SEARCH DETAIL