Strain F4 Research Articles

Decolorization of dyes using white-rot fungi and radical-generating reactions

Decolorization of synthetic dyes was performed using cultures of white-rot fungi producing ligninolytic enzymes and radical-generating reactions that could be involved in the mechanism of fungal decolorization. Among the white-rot fungi tested, Pleurotus ostreatus exhibited the highest decolorization rates, and also the highest production of laccase and Mn-peroxidase. P. ostreatus strain f6 gave 69% decolorization of Eosin Yellowish, 96% of Evans Blue, 75% of Phenol Red (all at 1 mM) and 88% of Poly B-411 (20 ppm) during a 14-day treatment. Treatment with Cu/succinic acid/H2O2 resulted in 96% decolorization of Evans Blue and Poly B-411 within 24 h. However, only 48% and 2% decolorization was achieved with Phenol Red and Eosin Yellowish, respectively. Similar decolorization rates were also obtained when Cu was replaced with Co. The results show that treatment of dye-containing solutions with both fungal cultures and biomimetic catalytic reactions results in decolorization.

Identification of Lactic Acid Bacteria isolated from Opaque beer (<i>Chibuku</i>) for potential use as a starter culture

A study was carried out to identify lactic acid bacteria (LAB) isolated from chibuku that would be later assessed for potential as starter cultures. Thirty-eight isolates were Gram stained and the 20, which were Gram positive, were identified to genus level using morphological, physiological and biochemical tests. Five genera of lactic acid bacteria were identified and these were Lactobacillus (seven isolates coded B1, B2, C4, E3, E6, F1 and F4), Lactococcus (five isolates: E1, F5, G5, G6 and H1), Leuconostoc (three isolates: E2, D5, F6), Streptococcus (two isolates: G2 and G4) and Enterococcus (three isolates: B3, B4 and G3). From these genera, eleven isolates five from the genus Lactobacillus, three from Lactococcus and three from Leuconostoc were selected for identification to species level using API 50 CH kits. The Lactobacillus strains were identified as follows: two strains were Lb plantarum (C4 and F4), two strains Lb. delbrueckii (B2 and E3), one strain could not be assigned to a species and was termed Lactobacillus sp. E6. Two of the Lactococcus isolates were identified as Lc. lactis subsp. lactis (G6 and H1), while the third isolate was Lc. raffinolactis (strain F5). The three Leuconostoc strains were Ln. mesenteroides subsp. mesenteroides. Keywords: Lactic acid bacteria; Sorghum beer (chibuku); Starter culture; Identification J Food Tech in Africa (2002) 7, 93-97

Isolation and initial characterization of a bacterial consortium able to mineralize fluorobenzene.

Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the beta-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter(-1) and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.

Calcium-sensitive extracellular poly(alpha-L-guluronate)lyase from a marine bacterium Pseudomonas sp. strain F6: Purification and some properties

: Poly(α-L-guluronate)lyase, as one of alginate lyases, was purified from the culture medium of a marine bacterium, Pseudomonas sp. strain F6, to an electrophoretically homogeneous state. The enzyme was shown to have a molecular mass of 36 kDa by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and was most active at around pH 7.5 and was stable between pH 6.5 and pH 8.5. In the thermal stability experiments, the enzyme's activity diminished through an intermediate state with increasing incubation temperatures and was finally lost when heated at 100°C for 15 min. The addition of hen egg-white lysozyme to the enzyme decreased thermal stability dramatically. The apparent retention of enzyme activity (approximately 50%) was observed after the addition of 6 M guanidine hydrochloride and 8 M urea. Enzyme activity was lost completely with 10 mMSDS, while the ordered structure, which is considered likely to be β-structure, was markedly created. The similar conformational feature has also been created in marine bacterial and mollusc enzymes and the β-structure is commonly observed in polyuronate lyases. The divalent cation (Ca2+) promoted the activity of the calcium chelator-treated enzyme significantly, suggesting that Ca2+ is involved in the formation of the active intermediate between the acidic uronate(s) and amino acid side-chain(s) of the enzyme.

Ligninolytic characteristics of Pleurotus ostreatus strain F6 and its monokaryotic protoplast derivative P19.

A stable isolate of Pleurotus ostreatus P19 differing in some morphological and physiological characteristics from its parental wild-type strain F6 was obtained via protoplast isolation during the preparation of strains with altered ligninolytic abilities. The isolate is monokaryotic, does not form clamp-connections, and produces much higher activities of enzymes involved in lignin modification (laccase, manganese peroxidase). Cellulase activity was comparable to that of wild-type strain F6, but the xylanase activity was slightly higher in isolate P19. However, this monokaryotic derivative degrades lignin at a slightly lower rate than its parental strain F6. Electron microscopy observations of wood degradation as a function of mycelium growth were performed on three zones of birch wafers delimited according to the distance from the point of inoculation. The different stages of fungal mycelium growth showed differences in the ultrastructural patterns of the decay not only between the strains P19 and F6, but also depending on the distance from the point of inoculation. This suggests a spatio-temporally controlled secretion of enzymes along the hyphae. The enhanced ability of P19 to degrade the condensed forms of lignin in middle lamellae is correlated to its higher laccase activity.

Ligninolytic characteristics of <i>Pleurotus ostreatus</i> strain F6 and its monokaryotic protoplast derivative P19

Ligninolytic characteristics of <i>Pleurotus ostreatus</i> strain F6 and its monokaryotic protoplast derivative P19

Screening of Pleurotus ostreatus isolates for their ligninolytic properties during cultivation on natural substrates.

Thirteen basidiospore-derived isolates of Pleurotus ostreatus f6 strain differing in the level of ligninolytic enzyme production and other characteristics (mycelium extension rate, colony morphology) from the parental strain were cultivated on natural substrates. Under these conditions ligninolytic enzyme activity, loss of organic mass, polycyclic aromatic hydrocarbons (PAHs) degradation and colonization of sterile and nonsterile soil were studied. The activity of ligninolytic enzymes was substantially higher in straw than in liquid culture, although the differences between the isolates were less pronounced on this substrate. Some of the isolates showed a very good ability to decompose the lignocellulosic substrate (straw) and a relatively high loss of organic mass was found after 50 days of cultivation in these strains. The original strain f6 and isolates B13 and B26 successfully degraded all seven tested PAH compounds present in experimental soil samples, but the higher or lower ligninolytic enzyme production of isolates tested had no substantial effect on the extent of the degradation. In our screening, six basidiospore-derived isolates growing well in nonsterile soil were found, which could be suitable for the prospective biotechnological exploitation.

Variability of ligninolytic enzyme activities in basidiospore isolates of the fungusPleurotus ostreatus in comparison with that of protoplast-derived isolates

Variability of production of all tested ligninolytic enzymes (laccase, peroxidase and manganese-dependent peroxidase) was substantially higher in isolates derived from basidiospores of strain F6 of the white-rot basidiomycetePleurotus ostreatus than the relatively low variability of the dikaryotic mycelial colonies of this strain, and also higher than the variability of protoplast-derived isolates of the same strain. The difference was caused mainly but not completely by the monokaryotic nature of the isolates. To reach the best fructification of the fungus, two fructification media were chosen from twenty two tested and a modified technique of fructification was used.

Evidence obtained with monoclonal antibodies that O antigen is the major antigen responsible for the cross-reactivities between serotypes 4 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae.

Monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae serotype 4 (reference strain M62 and field isolate F6) were produced and characterized. Three hybridoma clones were raised against strain M62, and 13 were raised against strain F6. The predominant antibody class was immunoglobulin M (IgM), although IgG2a and IgG2b were also obtained. Three of the MAbs produced to field isolate F6 (5C5, 1E10, and 5H7) did not recognize the reference strain of serotype 4, another (6F7) was reactive with both reference strains of serotypes 4 and 7, and the remaining 12 MAbs reacted only with the reference strain of the homologous serotype. All epitopes recognized by MAbs, except for one (6F7), were sensitive to periodic acid oxidation, and all of them were resistant to boiling in the presence of sodium dodecyl sulfate and reducing conditions, as evidenced by immunodot. Enhanced chemioluminiscence-immunoblot assays revealed that 10 MAbs (3E12, 5B8, 7C3, 6F7, 7F5, 7E6, 5G4, 4F1, 7E10, and 4B8) recognized a ladder-like banding pattern, which is in accordance with the O side chain antigen of lipopolysaccharide (LPS), while the remaining 6 MAbs (5C5, 5H7, 1E10, 6D11, 6B4, and 5E4) blotted with high-molecular-weight regions composed of a single banding pattern. The suitability of MAbs for serotyping of 78 field isolates was also examined. A high correlation was found between the results previously established by indirect hemagglutination with polyclonal sera and those obtained by enzyme-linked immunosorbent assay with MAbs. According to the different immunoreactivity of MAbs, three groups were established: group I (MAbs 3E12, 5B8, 7C3, 6F7, and 7F5), group II (MAbs 7E6, 5G4, 4F1, 7E10, and 4B8), and group III (MAbs 5C5, 5H7, and 1E10). MAbs 6D11, 6B4, and 5E4 could not be included in any of the described above. At least six different immunodominant epitopes on the O antigen of the A. pleuropneumoniae serotype 4 LPS were identified. Finally, the implications of the effect of the O antigen of LPS in cross-reactions between serotypes 4 and 7 are clearly evidenced.

Production of cellulolytic and xylanolytic enzymes by an Arthrographis species

A fungal isolate, Arthrographis sp. strain F4, when grown in shake-flask culture, produced cellulolytic and xylanolytic enzymes optimally at 30°C with an initial pH of 5.0 to 6.0. Coarsely-ground filter paper was the most suitable carbon substrate for production of the enzymes. Inorganic nitrogen sources gave higher activities of the enzymes than organic nitrogen sources: NH4NO3 and yeast extract was the most effective combination. Significant stimulation (P<0.05) of enzyme production was achieved with 0.1% (v/v) Tween 80.

Preliminary studies on a xylanase from an Arthrographis species

An Arthrographis sp. strain F4 xylanase was purified by acetone fractionation, ion-exchange on DEAE-Sephadex A-50 and Sephadex G-200 gel-filtration techniques. Its relative molecular mass ( M r) was estimated to be 28 100. The xylanase was optimally active at 55°C, pH 5.5, and stable at 40°C and pH 5.0–6.0. Significant inhibition ( P < 0.05) of the enzyme was observed with Mn 2+, Hg 2+, Cu 2+ or Ag +, but not with Ba 2+, Ca 2+ or Co 2+ ( P > 0.05). The K m value for oat splets xylan was 7.7 mg ml −1.

Preliminary studies on a xylanase from anArthrographisspecies

An Arthrographis sp. strain F4 xylanase was purified by acetone fractionation, ion-exchange on DEAE-Sephadex A-50 and Sephadex G-200 gel-filtration techniques. Its relative molecular mass (Mr) was estimated to be 28 100. The xylanase was optimally active at 55°C, pH 5.5, and stable at 40°C and pH 5.0–6.0. Significant inhibition (P 0.05). The Km value for oat splets xylan was 7.7 mg ml−1.

Purification and properties of an oligo-1,6- d-glucosidase from an alkalophilic Bacillus species

An isomaltose-hydrolyzing α- d-glucosidase from the alkalophilic Bacillus designated strain F5 was purified to an electrophoretically homogeneous state. The molecular weight of the purified glucosidase was 60 000 by SDS—poly(acrylamide) gel electrophoresis, and 63 000 by Sephacryl S-200 gel-filtration chromatography. The enzyme was most active for isomaltose at pH 6.0–6.5 and 45°, and stable up to 50° at pH 7.0 and in the range of pH 6.0–9.0 at 50° by 10-min incubation. The apparent V max and K m values for isomaltose were 34.5 μmol.min −1.mg −1 of protein, and 3.33mM. Panose and isomaltotriose are the best substrates for this enzyme. The restricted substrate specificity indicated the assignment of the enzyme to be an oligo-1,6-glucosidase (dextrin 6-α-glucanohydrolase; EC 3.2.1.10), but it was suggested that it could be a new type of oligo-1,6-glucosidase on the basis of its action on a series of (1→4)-α-malto-oligosaccharides.

Nucleotide sequence of alkalophilic Bacillus oligo-1,6-glucosidase gene and the properties of the gene product by Escherichia coli HB 101.

The nucleotide sequence of alkalophilic Bacillus oligo-1, 6-glucosidase gene was determined . The coding sequence on a 3.1 kb DNA fragment in pMC21 showed an open reading frame of 1527 by encoding 509 amino acids. The amino acid sequence from position 2 to 11 corresponds exactly to the NH2-terminal sequence of the purified enzyme protein from strain F5. The molecular weight (60, 142) of the protein derived from 1524 by open reading frame coincided with the molecular weight (60, 000) of the parent strain. On the other hand, the molecular weight of purified oligo-1, 6-glucosidase produced by E. coli HB101 carrying plasmid pMC21 showed 65, 000 by SDS-PAGE. The enzymatic properties, i. e., pH and temperature optima, pH and thermal stabilities, and substrate specificities, were almost the same as those obtained from an alkalophilic Bacillus sp. (strain F5).

Nodulation, Nitrogen Fixation, and Hydrogen Oxidation by Pigeon Pea Bradyrhizobium spp. in Symbiotic Association with Pigeon Pea, Cowpea, and Soybean

The pigeon pea strains of Bradyrhizobium CC-1, CC-8, UASGR(S), and F4 were evaluated for nodulation, effectiveness for N(2) fixation, and H(2) oxidation with homologous and nonhomologous host plants. Strain CC-1 nodulated Macroptilium atropurpureum, Vigna unguiculata, Glycine max, and G. soja but did not nodulate Pisum sativum, Phaseolus vulgaris, Trigonella foenum-graecum, and Trifolium repens. Strain F4 nodulated G. max cv. Peking and PI 434937 (Malayan), but the symbioses formed were poor. Similarly, G. max cv. Peking, cv. Bragg, PI 434937, PR 13-28-2-8-7, and HM-1 were nodulated by strain CC-1, and symbioses were also poor. G. max cv. Williams and cv. Clark were not nodulated. H(2) uptake activity was expressed with pigeon pea and cowpea, but not with soybean. G. max cv. Bragg grown in Bangalore, India, in local soil not previously exposed to Bradyrhizobium japonicum formed nodules with indigenous Bradyrhizobium spp. Six randomly chosen isolates, each originating from a different nodule, formed effective symbioses with pigeon pea host ICPL-407, nodulated PR 13-28-2-8-7 soybean forming moderately effective symbioses, and did not nodulate Williams soybean. These results indicate the six isolates to be pigeon pea strains although they originated from soybean nodules. Host-determined nodulation of soybean by pigeon pea Bradyrhizobium spp. may depend upon the ancestral backgrounds of the cultivars. The poor symbioses formed by the pigeon pea strains with soybean indicate that this crop should be inoculated with B. japonicum for its cultivation in soils containing only pigeon pea Bradyrhizobium spp.

A polynucleotide coenzyme of oxidative phosphorylation III mechanism of action

The aim of the present study was to investigate pyrene degradation by a functional strain F14, which was constructed through protoplast fusion between Sphingomonas sp. GY2B and Pseudomonas sp. GP3A. The ability of F14 to degrade pyrene increased from 18% to 46% with the decrease of pyrene concentration from 100 mg L−1 to 15 mg L−1 within 10 days. When pyrene was in binary mixture with phenanthrene or naphthalene, about 42% and 78% of pyrene were biodegraded by F14, respectively, indicating the presence of naphthalene or phenanthrene enhanced pyrene degradation, and the enhanced effect of 2-ring naphthalene was more obvious than that of 3-ring phenanthrene. Detection of metabolites 4, 5-dihydropyrene during pyrene degradation further confirmed the degradation capability of fusion strain F14. The present study showed that fusion strain F14 have a promising application in bioremediation of PAHs contaminated environment.