Extracellular Culture Filtrate Research Articles

Yeast-Mycelial Dimorphism in Pichia pastoris SMD1168 Is Triggered by Nutritional and Environmental Factors

This study reports, for the first time, morphological transition from yeast-like to filamentous form, normally associated with pathogenicity/increased protein secretion, in Pichia pastoris SMD1168 strain. The response was recorded in response to nutritional and environmental cues. The factors affecting this switch were extracellular pH (under nitrogen starvation conditions), carbon and nitrogen source under nitrogen- and carbon-limiting conditions respectively. Under nitrogen-limiting conditions, addition of fructose and sucrose in the culture medium induced filamentous morphology in a segregated form whereas addition of galactose led to a mixture of yeast and the filamentous form of the cells. Under carbon-limiting conditions, isoleucine and proline forced a filamentous form whereas glycine, valine, alanine and phenylalanine promoted yeast-like morphology. Similar dimorphic shift was also displayed by a recombinant methanol slow utilizing (Muts) strain (SMD-GCSF Muts) producing human granulocyte colony-stimulating factor in response to change in the initial inoculum level. Analysis of the extracellular metabolome by GC-MS indicated that several amino acids (leucine, proline, tyrosine), carboxylic acids (phenylacetic-, propanoic acid), alcohols and butylamine were present at different levels in the culture broth of the two morphological forms. High accumulation of proline and butylamine was seen in the extracellular culture filtrate of the filamentous form of the yeast. Presence of quorum-sensing molecules (phenylethyl alcohol, dodecanol) suggested complex network of pathways involved in this morphological transition.

Antifungal Effects of Rhizospheric Bacillus Species Against Bayberry Twig Blight Pathogen Pestalotiopsis versicolor

Bayberry is an attractive, nutritious, and popular fruit in China. The plant fungal pathogen Pestalotiopsis versicolor XJ27 is the causative agent of bayberry twig blight disease, which severely affects bayberry production. Traditional control techniques, such as chemical fungicides, are being used to control this disease; however, these techniques cause environmental and health hazards. In this study, we screened sweet potato rhizospheric bacteria with biocontrol potentials against P. versicolor XJ27, the bayberry twig blight pathogen. Ten isolates showed inhibition; Bacillus siamensis S3 and Bacillus tequilensis S5 showed the highest fungal growth inhibition. The antagonistic bacterial culture suspensions of S3 and S5 inhibited the mycelial growth by 82.9% and 76.2%, respectively. Their extracellular culture filtrates had mycelial growth inhibition of 86.8% and 82.2%, respectively. In detached leaf assay, the extracellular culture filtrates of S3 and S5 inhibited the size of the leaf lesion by 82.3% and 76.2%, respectively. SEM and TEM imaging showed a severe hyphal-damaged structure caused by the antagonistic bacteria. The fungal inhibition mechanisms might employ the hydrolytic enzymes and lipopeptides produced by the bacteria. Both the S3 and S5 have chitinase and protease activity; they produce a series of lipopeptides such as surfactin, iturin, and mycosubtilin. Therefore, we can suggest these bacteria as biocontrol agents for bayberry twig blight disease as an alternative to fungicides based upon their attributes of antifungal activity.

Fungicidal Efficiency of Silver and Copper Nanoparticles Produced by Pseudomonas fluorescens ATCC 17397 Against Four Aspergillus Species: A Molecular Study

Nano-science has emerged as a powerful tool in the field of designing new antimicrobial substances. In the present study, a green and eco-friendly method for synthesizing silver and copper nanoparticles using Pseudomonas fluorescens was employed. The biosynthesis of metals nanoparticles was firstly identified by the color change of the extracellular culture filtrate and confirmed with the help of the study of UV–Vis spectroscopy. The synthesized nanoparticles have further been characterized by transmission electron microscopy, energy dispersive X-ray and fourier transformation infrared studies. The antifungal activity of AgNPs and CuNPs was evaluated in vitro and the results showed that AgNPs had the greater effect as compared to CuNPs and Aspergillus species were the most sensitive strains. Protein electrophoresis results of Aspergillus species demonstrated the change in the protein profiles of the four fungal species against their controls after treatment with AgNPs and CuNPs. In addition, ISSR PCR analysis using five primers (HB-08, 44B, HB-10, HB-15 and 98A) indicated the appearance and disappearance of DNA polymorphic bands and induction of a high genetic variability in response to the two treatments. The results confirmed that metal nanoparticles are a promising approach to control fungal pathogens and eliminate their toxicity; therefore their applications should gained significant importance.

Antifungal potential of actinomycete isolate Streptomyces exfoliates MT9 against wood-rotting fungi.

An actinomycete isolate, Streptomyces exfoliatus MT9 was assessed for in vitro antagonism against wood-rotting fungi. Strain MT9 showed strong antagonistic activity (ZOI ? 25 mm) towards various tested wood-rotting fungi. Extracellular production of antifungal metabolite(s) including primary and secondary was monitored up to 10 days of submerged fermentation. Antagonist S. exfoliatus MT9 produces fungal cell-wall lytic enzymes, namely chitinase (3.098 U ml-1), b-1,3 glucanase (2.4 U ml-1) and protease (144.0 U ml-1) and also showed antifungal activity towards tested P. chrysosporium MTCC 787 (12.0 mm) and P. placenta MTCC 144 (16.0 mm). Extracellular culture filtrate (ECF) of S. exfoliatus MT9 also exhibited strong antifungal activity (ZOI ≥ 25 mm) towards tested wood-rotting fungi and n-butanol was found to be the suitable solvent for complete extraction of antifungal metabolite(s) from ECF. Reduced antifungal activity of n-butanol extract against P. chrysosporium MTCC 787 (11.00 mm) and P. placenta MTCC 144 (10.00 mm) on ergosterol agar plate, no activity against bacteria and characteristic UV spectra at 224 nm revealed the polyene nature of antifungal metabolite(s) present in the n-butanol extract. A novel actinomycete isolate, S. exfoliatus MT9 is producing antifungal metabolite(s) that makes it suitable for biotechnological processes and has the potential to be used as a bioactive agent for controlling wood-rotting fungi.

Elicitation of gymnemic acid production in cell suspension cultures of Gymnema sylvestre R.Br. through endophytic fungi.

The enhancement of plant secondary metabolite production in cell suspension cultures through biotic or abiotic elicitation has become a potential biotechnological approach for commercialization or large-scale production of bioactive compounds. Gymnema sylvestre R.Br. is an important medicinal plant, rich in a group of oleanane triterpenoid saponins called gymnemic acid, well known for its anti-diabetic activity. Two endophytic fungal strains were isolated from the leaves of G. sylvestre and identified as Polyancora globosa and Xylaria sp. based on the PCR amplification and internal transcribed spacer (ITS 1-5.8S-ITS 2) sequencing of 18S rRNA gene. The process of elicitation of cell suspension cultures of G. sylvestre with dried powder of fungal mycelia (DPFM) and extracellular culture filtrate (ECF) of endophytic fungi consistently enhanced the accumulation of gymnemic acid and the DPFM was proved to be an effective elicitor when compared to the ECF. The DPFM elicited the gymnemic acid content in the range of 2.57–10.45-fold, while the ECF elicited the gymnemic acid content in the range of 2.39–7.8-fold. P. globosa, a novel and a rare endophytic fungal strain, has shown a great influence on the production of gymnemic acid. Cell suspension cultures elicited with DPFM of P. globosa produced higher amount of gymnemic acid content (124.23 mg/g dried cell weight) compared to the cultures elicited with DPFM of Xylaria sp. (102.24 mg/g DCW). But the cultures treated with consortium of DPFM of both fungi showed great influence on the production of gymnemic acid (139.98 mg/g DCW) than the cultures treated with DPFM alone. Similarly, cultures treated with consortium of ECF of both fungi produced more gymnemic acid content (94.86 mg/g DCW) compared with cultures treated with ECF of Xylaria sp. (77.93 mg/g DCW) and ECF of P. globosa (78.65 mg/g DCW) alone.

Biochemical characterization of a novel thermostable xyloglucanase from an alkalothermophilic Thermomonospora sp.

Xyloglucanase from an extracellular culture filtrate of alkalothermophilic Thermomonospora sp. was purified to homogeneity with a molecular weight of 144kDa as determined by SDS-PAGE and exhibited specificity towards xyloglucan with apparent K (m) of 1.67mg/ml. The enzyme was active at a broad range of pH (5-8) and temperatures (40-80°C). The optimum pH and temperature were 7 and 70°C, respectively. The enzyme retained 100% activity at 50°C for 60h with half-lives of 14h, 6h and 7min at 60, 70 and 80°C, respectively. The kinetics of thermal denaturation revealed that the inactivation at 80°C is due to unfolding of the enzyme as evidenced by the distinct red shift in the wavelength maximum of the fluorescence profile. Xyloglucanase activity was positively modulated in the presence of Zn(2+), K(+), cysteine, β-mercaptoethanol and polyols. Thermostability was enhanced in the presence of additives (polyols and glycine) at 80°C. A hydrolysis of 55% for galactoxyloglucan (GXG) from tamarind kernel powder (TKP) was obtained in 12h at 60°C and 6h at 70°C using thermostable xyloglucanases, favouring a reduction in process time and enzyme dosage. The enzyme was stable in the presence of commercial detergents (Ariel), indicating its potential as an additive to laundry detergents.

Rapid production of cellulase-free xylanases by solventogenic Clostridia from rumen

Xylanases without contaminating cellulases are produced by a wide variety of aerobic bacteria and fungi but anaerobic organisms producing such enzymes are rare. Clostridial species viz., Clostridium beijerinckii LU-1, C. acetobutylicum LU-1, C. butyricum and C. bifermentans LU-1 isolated from rumen produced xylanase (E.C. 3.2.1.8) of 1.5–4.2 U/ml on oat spelt xylan as sole carbon source. Celluloses absolutely did not support their growth. Among the carbon sources tested such as cellobiose, cellulose, filter paper cellulose (Whatman No. 1), methylnitrocellulose (MNC), AVICEL (microcrystalline cellulose), carboxymethylcellulose (CMC), starch and pectin did not support growth and enzyme activity. Among the xylans birch wood xylan and larch wood xylan did not support their growth. β-Glucosidase (E.C. 3.2.1.21), filter paper cellulase, carboxymethylcellulase (E.C. 3.2.1.4), methylnitrocellulase and avicelase (E.C. 3.2.1.74) were absent in the extracellular culture filtrate and no enzyme activity was found bound to the cells. In all these strains when grown on oat spelt xylan (1%, w/v), at pH 7 and 37 °C, the maximum enzyme production was observed within 21–24 h. In medium containing oat spelt xylan, there was rapid increase in protein productivity, accumulation of extracellular endo-β-xylanase concomitantly with a decline of 65–82% of residual xylan, subsequent decrease in cell growth, but the accumulated enzyme utilized the maximum amount of xylan in 24 h. C. bifermentans LU-1 produced cell-bound β-xylosidase (E.C. 3.2.1.37) 0.145 U/ml. Endoxylanases had an optimum pH of 5 and temperature 50 °C ( C. acetobutylicum LU-1 and C. butyricum LU-1) and ( C. beijerinckii LU-1) 60 °C. Xylanases were stable between pH 4 and 8 and temperature 30–50 °C. Xylanase from C. bifermentans LU-1 had an optimum pH of 4 and temperature of 70 °C. The pH optima of 5 and good stability between 4 and 8 for the endoxylanases of C. beijerinckii LU-1, C. acetobutylicum LU-1 and C. butyricum LU-1 is suitable for processes where pH changes are common.

Purification and properties of a low molecular weight 1,4-β- d-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp.

A low molecular weight 1,4-β- d-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS–PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC.

Decolorization of triphenylmethane and azo dyes by Citrobacter sp.

A Citrobacter sp., isolated from soil at an effluent treatment plant of a textile and dyeing industry, decolorized several recalcitrant dyes except Bromophenol Blue. More than 90% of Crystal Violet and Methyl Red at 100 μM were reduced within 1 h. Gentian Violet, Malachite Green and Brilliant Green lost over 80% of their colors in the same condition, but the percentage decolorization of Basic Fuchsin and Congo Red were less than the others, 66 and 26%, respectively. Decolorization of Congo Red was mainly due to adsorption to cells. Color removal was optimal at pH 7–9 and 35–40 °C. Decolorization of dyes was also observed with extracellular culture filtrate, indicating the color removal by enzymatic biodegradation.

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l−1 and 356 U l−1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l−1 min−1 using the extracellular culture filtrate.

Cloning of clustered Streptomyces viridosporus T7A lignocellulose catabolism genes encoding peroxidase and endoglucanase and their extracellular expression in Pichia pastoris.

A 4.1-kb fragment of chromosomal DNA from the lignocellulose-decomposing actinomycete Streptomyces viridosporus T7A was previously found to encode a lignin peroxidase gene. However, when cloned into Escherichia coli in pBSKS+, peroxidase activity was not expressed. When cloned in pIJ702 in Streptomyces lividans, the gene was expressed in a peroxidase positive background, owing to the production by S. lividans of its own extracellular peroxidase. To circumvent these problems, the DNA was cloned into the commercial expression vector pIC9 for extracellular expression in the yeast Pichia pastoris. Yeast transformants, however, expressed two activities, extracellular peroxidase and an extracellular endoglucanase. The enzymes were not expressed by the yeast cells alone or by yeast cells with pIC9 without the insert. Expression of the enzymes by only those transformants expressing the 4.1-kb DNA was confirmed by Western blot analyses, by nondenaturing activity gel staining, and by spectrophotometric enzyme assays of extracellular culture filtrates. Activity gel staining showed that the two activities resided in different proteins and the peroxidase expressed was similar to ALip-P3, one of the isoenzymes of lignin peroxidase of the S. viridosporus T7A wildtype. Other evidence indicated that in the transformants, the peroxidase and endoglucanase genes in the 4.1-kb insert were controlled by the methanol-inducible AOX1 yeast promoter in pIC9, since their expression was induced by methanol. In the best transformants, extracellular production of peroxidase by recombinant P. pastoris cultures was significantly higher than typically observed in S. viridosporus. The results also indicate that lignocellulose catabolism genes may be clustered on the S. viridosporus chromosome.

Phosphodiesterase as mycorrhizal P sources: I. Phosphodiesterase production and the utilization of DNA as a phosphorus source by the ericoid mycorrhizal fungus Hymenoscyphus ericae.

These results provide the first account of the breakdown and utilization of DNA by an ericoid mycorrhizal fungus, and of its phosphodiesterase activity in vitro. Hymenoscyphus ericae (Read) Korf & Kernan grew well on DNA as a sole source of phosphorus (P), achieving greater mycelium dry weight than on an equivalent concentration of P supplied as orthophosphate. Some characteristics of the production and activity of extracellular (culture filtrate) and cell-wall bound phosphodiesterase in H. ericae are reported. At least part of the phosphodiesterase activity is attributed to the exonuclease F nucleotide diesterase on the basis of its high affinity for a specific substrate of this enzyme, The pH optima for extra-cellular and cell-wall-bound diesterase are acidic (pH 4.0-5.5), with considerable activity maintained in the pH range typical of organic soils under ericaceous plants (pH 3.0-4.5). The production of phosphodiesterase was not substrate-induced, since highest specific activity for wall-bound enzyme was found in culture grown without organic or inorganic P. The results are discussed in relation to the ecology and biology of acid organic soils on which ericaceous plants are dominant.

Transglycosylation of extracellular.BETA.- glucosidase of Trichoderma pseudokoningii S38 and its function in regulation of cellulase biosynthesis.

Two β-glucosidase (βG) components, βG1 and βG2, purified from extracellular culture filtrate of Trichoderma pseudokoningii S38 by chromatography techniques, showed transglycosylation activity, the character of which was previously believed to belong to the membrane-bound βG. βG1 also hydrolyzed cellobiose (and other β-linked disaccharides) to produce glucose, and could hydrolyze p-nitrophenyl-β-D-glucoside (PNPG). However, the activity of hydrolysis of disaccharides and PNPG of βG2 was lower than that of βGl, but it had higher transglycosylation activity, showing different substrate specificities. The transglycosylation products originating from cellobiose by βGs, such as all of the other β-linked disaccharides and even cellotriose, were determined by HPLC. Among them, gentiobiose was believed to be the most effective inducer of cellulose biosynthesis in this strains. The induction ability of a certain disaccharide had a relationship with its amount in the traction mixture which was controlled by the dual activities of βG, hydrolysis and transglycosylation.

Increased ethanol production during growth of electric-field stimulated Kluyveromyces marxianus IMB3 during growth on lactose-containing media at 45�C

Following treatment of the thermotolerant ethanol producing yeast strain Kluyveromyces marxianus IMB3 with single electric field pulses of 2.4kV/cm, ethanol production was found to increase from 25 to 35% of the theoretical maximum yield within 20 hours, during growth on lactose-containing media (4% [w/v]). Following treatment with electric fields and addition of 1mM MnCl2 to lactose-containing media, ethanol production almost doubled within a 30 hour period. In addition, low levels of functional β-galactosidase activity could be detected in the extracellular culture filtrates, presumably as a result of electropermeabilization.

Decolorization of triphenylmethane dyes by the bird's nest fungus Cyathus bulleri

Decolorization of three triphenylmethane dyes by three bird's nest fungi—Cyathus bulleri, C. stercoreus, and C. striatus—was studied. Cyathus bulleri was found to be the most efficient in decolorization as demonstrated by the disappearance of the dyes from cultures, monitored by decreases in absorbance. Growth of the Cyathus spp. was not affected by the presence of dyes in the cultures. Decolorization of dyes was also observed with extracellular culture filtrates, indicating exocellular activity. Laccase activity, tested in replicate cultures, was found to be maximum during the decolorization period. Nitrogen in the medium had no effect on decolorization.

Increased efficiency of substrate utilization by exposure of the thermotolerant yeast strain, Kluyveromyces marxianus IMB3 to electric-field stimulation

In order to enhance cellobiose utilization and conversion of substrate to ethanol by the thermotolerant yeast strain, Kluyveromyces marxianus IMB3, the organism was exposed to short, intense electric pulses. When cells were treated with pulses measuring 0.25kV for 10mS, in the presence of cellobiose, ethanol production was found to increase by almost 40% above that found in fermentations containing non-treated cells. When the extracellular culture filtrate was assayed for β-glucosidase activity no significant difference in levels was detected between treated and control systems. Increasing the voltage of the pulses resulted in a decrease in ethanol production.

Production of ethanol by the thermotolerant yeastKluyveromyces marxianus IMB3 during growth on lactose-containing media

The thermotolerant yeast strain,Kluyveromyces marxianus IMB3 was shown to be capable of growth and ethanol production on lactose containing media at 45°C. On media containing 4% (w/v) lactose, ethanol production increased to 6.0g/l within 50h and this represented 29% of theoretical yield. During growth on lactose containing media the organism was shown to produce a cell-associated β-galactosidase and no significant enzyme could be detected in the extracellular culture filtrate. Addition of β-galactosidase, released fromKluyveromyces marxianus IMB3 cells, to active fermentations, resulted in increasing ethanol production to 53% of theoretical yield at 45°C.

Growth of a thermotolerant ethanol-producing strain of Kluyveromyces marxianus on cellobiose containing media

The thermotolerant yeast strain, Kluyveromyces marxianus 1MB 3, was shown to be capable of limited growth on cellobiose containing media at 45°C. Growth, sugar utilization and ethanol production were shown to increase in the presence of exogenously added thermostable fungal β-glucosidase. During active growth of the organism on cellobiose-containing media, β-glucosidase activity was detected in cell lysate preparations with only minor amounts of activity present in the extracellular culture filtrate. The results suggest that limitations in ethanol production by this organism during growth on cellobiose containing media may be overcome by addition of exogenously added β-glucosidase which results in increased substrate access to the biocatalytic unit.

Production, isolation and partial characterization of an amylase system produced by Talaromyces emersonii CBS 814.70

The thermophilic fungus Talaromyces emersonii CBS 814.70 was shown to produce an amylase system during growth on soluble starch. The effect of starch substrate on amylase production during growth of the organism has been examined. During fractionation of the proteins in the extracellular culture filtrate, it has been shown that those filtrates contain at least one form of α-amylase and one form of α-glucosidase. A purified α-amylase has been shown to have a wide pH optimum, ranging between 4.0 and 5.5 with significant quantities of activity remaining at pH 2.5. The enzyme was shown to be thermostable, having a half-life of over 30 min at 80°C. From the data obtained from our studies, it would appear that this enzyme may find use in a number of biotechnological applications.

Extracellular enzymatic activity of aquatic and aero-aquatic conidial fungi

Nineteen species of aquatic and areo-aquatic conidial fungi were tested for their ability to produce extracellular enzymes which degrade cellulose, starch, lipids, proteins and tannic acid. The cellulolytic activity was determined by using both solid and liquid media. The activity of other enzymes was examined using solid media. Two-thirds of the species were able to hydrolyze soluble cellulose (CMC) incorporated in solid and liquid media with varying degrees of activity. Extracellular culture filtrates ofAegerita candida, Helicodendron giganteum andH. tubulosum contained a Cl-Cx enzyme complex that could degrade both soluble cellulose (CMC) and crystalline cellulose (filter paper). Lipase activity was demonstrated by 11 species. Fourteen of the species showed activity for amylase and protease, but only 11 of the 16 were capable of degrading tannic acid.