Crude Extracellular Extract Research Articles

Evaluation of the Antibacterial Activity of Crude Extracts Obtained From Cultivation of Native Endophytic Fungi Belonging to a Tropical Montane Rainforest in Colombia.

Bioactive secondary metabolite production from endophytic fungi has gained a recurring research focus in recent decades as these microorganisms represent an unexplored biological niche for their diverse biotechnological potential. Despite this focus, studies involving tropical endophytes remain scarce, particularly those isolated from medicinal plants of these ecosystems. In addition, the state of the art of the pharmaceutical industry has experienced stagnation in the past 30years, which has pushed pathogenic infections to get one step ahead, resulting in the development of resistance to existing treatments. Here, five fungal endophytes were isolated from the medicinal plant Otoba gracilipes (Myristicaceae), which corresponded to the genera Xylaria and Diaporthe, and screened to demonstrate the promissory potential of these microorganisms for producing bioactive secondary metabolites with broad-spectrum antibacterial activities. Thus, the evaluation of crude organic extracts obtained from the mycelia and exhaust medium allowed the elucidation of Xylaria sp. and Diaporthe endophytica potential toward providing crude extracellular extracts with promising bioactivities against reference strains of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923), according to the determined half-maximum inhibitory concentration (IC50) with values down to 3.91 and 10.50mg/ml against each pathogen, respectively. Follow-up studies provided insights into the polarity nature of bioactive compounds in the crude extracts through bioactivity guided fractionation using a polymeric resin absorbent alternative extraction procedure. In addition, evaluation of the co-culturing methods demonstrated how this strategy can enhance endophytes biosynthetic capacity and improve their antibacterial potential with a 10-fold decrease in the IC50 values against both pathogens compared to the obtained values in the preliminary evaluations of Xylaria sp. and D. endophytica crude extracts. These results support the potential of Colombian native biodiversity to provide new approaches concerning the global emergence of antibiotics resistance and future production of undiscovered compounds different from the currently used antibiotics classes and simultaneously call for the value of preserving native habitats due to their promising ecosystemic applications in the biotechnological and pharmaceutical industries.

Phytoremediation of synthetic textile dyes: biosorption and enzymatic degradation involved in efficient dye decolorization by Eichhornia crassipes (Mart.) Solms and Pistia stratiotes L.

The effectiveness of four aquatic floating plants: Eichhornia crassipes, Pistia stratiotes, Lemna minor, Salvinia sp., and a submerged plant Hydrilla sp. on decolorization and detoxification of five structurally different textile dyes: CI Direct Blue 201 (DB 201), Cibacron Blue FR, Cibanone Gold Yellow RK, Vat Green FFB, and Moxilon Blue GRL were studied. The E. crassipes and P. stratiotes showed complete decolorization of all the dyes tested, while Salvinia sp. (79-86%), L. minor (16-24%), and Hydrilla sp. (6-13%) were recorded as the least tolerance for all the dyes even after 14 days of incubation. Therefore, E. crassipes and P. stratiotes were selected for further studies using DB 201 as the model dye. E. crassipes and P. stratiotes showed complete decolorization of DB 201 at 48 and 84 h of incubation, respectively, and decolorization was well effective in the pH range 6-9. The crude extract of intracellular enzymes obtained from the roots of E. crassipes (46%) and P. stratiotes (20%) showed significant involvement on decolorization of DB 201, compared with the activity of crude extracellular extract and isolated endophytic bacteria and fungi (p ≤ 0.05). Further, 18 and 22% of biosorption of DB 201 dye were recorded by E. crassipes and P. stratiotes, respectively, suggesting that decolorization mechanisms of DB 201 dye by E. crassipes and P. stratiotes were based on biosorption and intracellular enzyme activities. The FTIR spectra and seed germination assay confirmed biodegradation and detoxification of DB 201 dye by E. crassipes and P. stratiotes plants along with complete color removal. Thus, present study confers the potential applicability of E. crassipes and P. stratiotes plants for textile dye removal and release to the environment without further treatment.

Exploring the Extracellular Macromolecular Composition of Crude Extracts of Penicillium rubens Strain 212 for Elucidation Its Mode of Action as a Biocontrol Agent.

Penicillium rubens strain 212 (PO212) acts as an inducer of systemic resistance in tomato plants. The effect of crude extracellular extracts of PO212 on the soil-borne pathogen Fusarium oxysporum f. sp. lycopersici has been evaluated. Evidence of the involvement of soluble, thermo-labile, and proteinase-inactivated macromolecules present in PO212 crude extracts in the control of Fusarium vascular disease in tomato plants was found. Proteomic techniques and the availability of the access to the PO212 genome database have allowed the identification of glycosyl hydrolases, oxidases, and peptidases in these extracellular extracts. Furthermore, a bioassay-guided fractionation of PO212 crude extracellular extracts using an integrated membrane/solid phase extraction process was set up. This method enabled the separation of a PO212 crude extracellular extract of seven days of growth into four fractions of different molecular sizes and polarities: high molecular mass protein fraction >5 kDa, middle molecular mass protein fraction 5–1 kDa, low molecular mass metabolite fraction, and nutrients from culture medium (mainly glucose and minerals). The high and middle molecular mass protein fractions retained disease control activity in a way similar to that of the control extracts. Proteomic techniques have allowed the identification of nine putatively secreted proteins in the high molecular mass protein fraction matching those identified in the total crude extracts. Therefore, these enzymes are considered to be potentially responsible of the crude extracellular extract-induced resistance in tomato plants against F. oxysporum f. sp. lycopersici. Further studies are required to establish which of the identified proteins participate in the PO212’s action mode as a biocontrol agent.

Aspergillus flavipes is a novel efficient biocontrol agent of Phytophthora parasitica

Phytophthora spp are one of the most devastating pathogens to a wide range of crop plants. Chemical fungicides are commonly used for controlling Phytophthora diseases, however, due to environmental concerns and development of fungicide resistant Phytophthora strains, alternative low-risk method such as biological controls are the prospective approaches. In an in vitro bioactivity-driven screen of rhizosphere-inhabiting fungi, Aspergillus flavipes was identified as a strong inhibitor for growth of various species of Phytophthora. As well as, the crude extracellular extract of broth cultures of A. flavipes displayed a significant growth inhibition of various Phytophthora spp, with EC50 values ranged from 0.018−0.02 units. Bioactivity-driven fractionation analyses revealed that the antiphytophthoral activity was mostly reported in the ethyl acetate fraction. In addition to mycelial growth inhibition, the ethyl acetate fraction inhibited the zoospore germination and sporangial production in P. parasitica. Microscopic analyses of P. parasitica hyphae treated with A. flavipes ethyl acetate extract, revealed severe growth abnormalities such as irregular condensed hyphal aggregations, shorter hypha and thicker cells, in addition to the strongly inhibition of P. parasitica infection and pathogenicity to Nicotiana benthamiana. From the direct co-culturing, P. parasitica and its metabolites had no effect on over elicitation of the active metabolites of A. flavipes. The putative antiphytophthoral compounds from A. flavipes were chemically verified as 3-hydroxy-2′,4,4′,6′-tetramethoxychalocone, 7,3,4,5′-tetramethoxyflavanone, isovitexin and amodiaquine at concentrations 35.8, 9.1, 26.4 and 11.4%, respectively. The cultures of A. flavipes and its active extracts did not display any phytotoxicity to N. benthamiana and S. lycopersicum suggesting their potentially for controlling the diseases of Phytophthora spp.

Purification and characterization of xylanases from Trichoderma inhamatum

Normal 0 21 false false false PT-BR JA X-NONE Background : Two xylanases, Xyl I and Xyl II, were purified from the crude extracellular extract of a Trichoderma inhamatum strain cultivated in liquid medium with oat spelts xylan. Results : The molecular masses of the purified enzymes estimated by SDS-PAGE and gel filtration were, respectively, 19 and 14 kDa for Xyl I and 21 and 14.6 kDa for Xyl II. The enzymes are glycoproteins with optimum activity at 50oC in pH 5.0 - 5.5 for Xyl I and 5.5 for Xyl II. The xylanases were very stable at 40oC and in the pH ranges from 4.5 - 6.5 for Xyl I and 4.0 - 8.0 for Xyl II. The ion Hg 2+ and the detergent SDS strongly reduced the activity while 1,4-dithiothreitol stimulated both enzymes. The xylanases showed specificity for xylan, K m and V max of 14.5, 1.6 mg ● mL -1 and 2,680.2 and 462.2 U ● mg of protein -1 (Xyl I) and 10.7, 4.0 mg ● mL -1 and 4,553.7 and 1,972.7 U ● mg of protein -1 (Xyl II) on oat spelts and birchwood xylan, respectively. The hydrolysis of oat spelts xylan released xylobiose, xylotriose, xylotetrose and larger xylooligosaccharides. Conclusions : The enzymes present potential for application in industrial processes that require activity in acid conditions, wide-ranging pH stability, such as for animal feed, or juice and wine industries. /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Times New Roman,serif; mso-ansi-language:PT-BR; mso-fareast-language:PT-BR;} Background: Results: Conclusions:

Biodegradation and extracellular enzymatic activities of Pseudomonas aeruginosa strain GF31 on β-cypermethrin.

Pseudomonas aeruginosa strain GF31, isolated from a contaminated soil, can effectively degrade β-cypermethrin (β-CP), as well as fenpropathrin, fenvalerate, and cyhalothrin. The highest level of degradation (81.2%) was achieved with the addition of peptone. Surprisingly, the enzyme responsible for degradation was mainly localized to the extracellular areas of the bacteria, in contrast to the other known pyrethroid-degrading enzymes, which are intracellular. Although intact bacterial cells function at about 30°C for biodegradation, similar to other degrading strains, the crude extracellular extract of strain GF31 remained biologically active at 60°C. Moreover, the extract fraction showed good storage stability, maintaining >50% of its initial activity following storage at 25°C for at least 20days. Significant differences in the characteristics of the crude GF31 extracellular extract compared with the known pyrethroid-degrading enzymes indicate the presence of a novel pyrethroid-degrading enzyme. Furthermore, the identification of 3-phenoxybenzoic acid and 2,2-dimethylcyclopropanecarboxylate from the degradation products suggests the possibility that β-CP degradation by both the strain and the crude extracellular fraction is achieved through a hydrolysis pathway. Further degradation of these two metabolites may lead to the development of an efficient method for the mineralization of these types of pollutants.

Preliminary studies on the biobleaching of loblolly pine Kraft pulp with Trametes trogii crude extracts

Trametes trogii (BAFC 463), Coriolus versicolor var. antarcticus (BAFC 266) and Trametes villosa (BAFC 2755) are Argentinean white-rot fungi that proved to be efficient ligninolytic enzyme producers in previous studies. The aim of the present work was investigating the ability of their crude extracellular extracts for the biobleaching of loblolly pine Kraft pulp. The production of laccase, manganese peroxidase, endoxylanase and endoglucanase was also evaluated. Out of the three fungi studied, the best biobleaching results were obtained with culture supernatants from T. trogii grown for 21 days in a synthetic medium with glucose and asparagine as carbon and nitrogen sources, respectively, with the addition of 1 mM copper sulfate. Such medium rendered high level of ligninolytic enzymes: 0.25 U ml −1 of manganese peroxidase and 44 U ml −1 of laccase. A strong brightness increase was attained after the enzymatic treatment followed by H 2O 2. Bleaching up to 82% ISO brightness (compared with 37% in the peroxide-bleached control) was obtained. Moreover, after the enzymatic treatment, hydrogen peroxide requirements decreased (as reflected by the higher residual peroxide), and the pulp luminance reached a value of 92.5. Addition of known laccase mediators such as HBT, ABTS, and HBA did not improve the process. The supernatant did not significantly lose its bleaching capacity even after successive recovery and reapplication to unbleached pulp. Correspondingly, laccase and manganese peroxidase activities in the recovered supernatant, exhibited near 100% of their initial values.

Optimization of lignocellulolytic enzyme production by the white-rot fungus Trametes trogii in solid-state fermentation using response surface methodology

The white-rot basidiomicete Trametes trogii (MYA 28-11) is an outstanding producer of laccase. A Doehlert experimental design was applied to optimize its lignocellulolytic enzyme production in solid-state fermentation. The impact on enzyme production of three quantitative variables, namely pH, copper and nitrogen concentrations, was investigated by using a wood-based solid medium supplemented with malt extract. Optimization was aimed at simultaneously minimizing cellulase activity and maximizing ligninolytic enzyme production. Such conditions were: pH 4.5, peptone 12.5 g l −1, and CuSO 4 11 mM. Highest activities achieved were: laccase 901 and Mn-peroxidase 20 U g −1. There was not a direct correlation between the levels of enzymatic activities and the extent of losses of wood weight or components. The factorial approach also allowed us to quantify the enzyme activity in any part of the experimental domain, consequently, it was also possible to determine a culture medium to obtain crude extracellular extracts with high laccase (510 U g −1) and endoxylanase (780 U g −1) yields. These enzymes have gained renewed interest mainly due to their applications in paper industries for pulp treatment, improving the effectiveness of conventional bleaching. Considering the results obtained, T. trogii could be an attractive source of both enzymes.

Mapping substrate selectivity of lipases from thermophilic fungi

New methods were adapted to screen, fast and easily, the lipase specificity (topo- or enantio-selectivity) on crude extracellular extracts from thermophilic fungi. Substrate acyl chain length specificity was tested using p-nitrophenyl esters and vinyl esters by the detection of released p-nitrophenolate anions in the first case and protonation of p-nitrophenolate anions (color diminution) in the second case. Enantioselectivity was tested using either the direct reaction rates on individual enantiomers of glycidyl butyrate or on competition between these enantiomers and resorufin esters (-butyrate or -acetate). Among a library of 44 thermophilic fungi, 10 strains were pre-selected (based on their capabilities to produce constitutively extracellular lipases) for further lipase specificity studies. The above methods were applied to lipases from these pre-selected fungi and also to other several lipases preparations from bacterial, fungal and mammalian origin. Remarkably, the method on competition allowed the accurate determination of the enantiomeric ratio ( E), since experimental data fitted correctly with the E determined by classical chemical methods. Consequently, these methods can be applicable for screening selectivity in a high number of lipases or esterases from wild isolates or variants generated by directed evolution, using directly in the test, the substrate (i.e. esters) that will be worked out in a given process.

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme.

The ability of a Brazilian strain of Pleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase), P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorization in vivo was tested using three dyes--Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability of P. pulmonarius to decolorize industrial dyes.

A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces.

Two distinct pathogenic mechanisms, adhesion to polymer surfaces and subsequent accumulation of sessile bacterial cells, are considered important pathogenic steps in foreign body infections caused by Staphylococcus epidermidis. By using mitomycin mutagenesis, we have recently generated a mutant, strain M7, from S. epidermidis RP62A which is unaffected in adhesion but deficient in accumulation on glass or polystyrene surfaces and lacks a 115-kDa extracellular protein (designated the 140-kDa antigen; F. Schumacher-Perdreau, C. Heilmann, G. Peters, F. Götz, and G. Pulverer, FEMS Microbiol. Lett. 117:71-78, 1994). To evaluate the role of this protein in accumulation, we harvested extracellular proteins from S. epidermidis RP62A grown on dialysis membranes placed over chemically defined medium, purified the protein by using ion-exchange chromatography, determined its N-terminal amino acid sequence, and raised antiserum in rabbits. The antibody recognized only a single band in a Western immunoblot of the crude extracellular extract. With the microtiter biofilm test, antiserum at a dilution of < or =1:1,000 blocked accumulation of RP62A up to 98% whereas preimmune serum did not. The 140-kDa antigen was found only in extracellular products from bacteria grown under sessile conditions. Of 58 coagulase-negative clinical isolates, 32 strains were 140-kDa antigen positive and produced significantly larger amounts of biofilm than the 26 strains that were 140-kDa antigen negative. The 140-kDa protein appears to be biochemically and functionally unrelated to any previously described factors associated with biofilm formation. Thus, the 140-kDa antigen, referred to as accumulation-associated protein, may be a factor essential in S. epidermidis accumulation and, due to its immunogenicity, may allow the development of novel immunotherapeutic strategies for prevention of foreign body infection.

Detection of polygalacturonases and pectin esterases in lactic acid bacteria

Of 80 strains of lactic acid bacteria tested, only Lactobacillus casei strains HNK10 and L1-8, Lactobacillus plantarum Lc5 and Lactococcus lactis NN01 produced polygalacturonases (EC 3.2.1.) and/or pectin-esterases (EC 3.1.1.). Crude extracellular extracts of strain L1-8 were able to clarify pectin.

Identification, characterization, and partial purification of glucoamylase from Aspergillus niger (syn A. ficuum) NRRL 3135.

The crude extracellular extract of Aspergillus niger (syn A. ficuum) NRRL 3135 contains glucoamylase (exo-1,4-alpha-D-glucanohydrolase, EC 3.2.1.2). The enzyme, a glycoprotein, was purified 7-fold by ion-exchange chromatography, chromatofocusing, and conconavalin A affinity chromatography. The molecular weight of the enzyme was estimated to be 90 kDa by SDS-PAGE and gel permeation chromatography. The pI of the enzyme was 3.4. The temperature optimum of the enzyme was 60 degrees C and the pH optimum was 5.0. The Vmax values for soluble starch, maltose, maltotriose, maltotretraose, maltopentaose, and isomaltose were 55.2, 11.7, 32.3, 47.8, 59.2, 12.5 nKat glucose/sec, respectively and the Km values for the same substrates were 0.09%, 0.67 mM, 0.76 mM, 0.76 mM, 0.68 mM, and 122.01 mM, respectively.

Production of Proteases by Psychrotrophic Microorganisms

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-(L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52°C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.

Use of monoclonal antibodies to detect Mn(II)-peroxidase in birch wood degraded by Phanerochaete chrysosporium

A monoclonal antibody (Mab) produced to purified Mn(II)-peroxidase was visualized on and within cell corners of birch wood degraded by Phanerochaete chrysosporium using colloidal gold immuno-transmission electron microscopy techniques. Labelling of the fungal cell membrane and cell wall was also observed. The same Mab was used to visualize the penetration of extracellular fungal metabolite extracts, infiltrated into previously decayed wood. Binding of antibodies to the lignin-rich cell corner region of the middle lamella in wood decayed by P. chrysosporium was observed in sectioned wood blocks and in wood infiltrated with crude extracellular extracts from P. chrysospirium liquid cultures. When a control monoclonal antiserum, produced to extracellular metabolites of Postia (Poria) placenta and cross-reactive with fungal cellulase, was used in labelling, the cellulose rich region of the wood cell walls were labelled. Labelling in the middle lamella cell corners was only noted in what has been described as nonor poorly lignified cell corner regions.