77 research outputs found
Dechlorination of 1,2– dichloroethane by Pseudomonas aeruginosa OK1 isolated from a waste dumpsite in Nigeria
As part of our attempt at isolating and stocking some indigenous microbial species, we isolated a bacterium from a waste dumpsite with appreciable dechlorination activity. 16S rDNA profiling revealed the isolate to be a strain of Pseudomonas aeruginosa and the sequence has been deposited in the NCBI nucleotide sequence database (accession number AJ550306). The bacterium utilized 0.1% (v/v) 1, 2 – dichloroethane (1, 2 – DCE) as sole source of carbon and attained peak cell density of 6.0 × 107 cfu/ml in 48 h. It also has a proportionate increase in chloride release during this period resulting in the release of 80% free Cl-. The bacterium also had dehalogenase activities against other chlorinated organics such as monochloroacetic acid, trichloroacetic acid, dichloromethane, trichloromethane and tetrachloromethane at pH 7.5 and 9.0. Optimum temperature for dehalogenase activity against 1, 2 – DCE was 35oC.
Key Words: Dechlorination, 16S rDNA, bioremediation, Pseudomonas aeruginosa OK1.
African Journal of Biotechnology Vol.3(10) 2004: 508-51
Rapid depletion of dissolved organic sulphur (DOS) in freshwaters
Sulphur (S) is a key macronutrient for all organisms, with similar cellular requirements to that of phosphorus (P). Studies of S cycling have often focused on the inorganic fraction, however, there is strong evidence to suggest that freshwater microorganisms may also access dissolved organic S (DOS) compounds (e.g. S-containing amino acids). The aim of this study was to compare the relative concentration and depletion rates of organic 35S-labelled amino acids (cysteine, methionine) with inorganic S (Na235SO4) in oligotrophic versus mesotrophic river waters draining from low nutrient input and moderate nutrient input land uses respectively. Our results showed that inorganic SO42− was present in the water column at much higher concentrations than free amino acids. In contrast to SO42−, however, cysteine and methionine were both rapidly depleted from the mesotrophic and oligotrophic waters with a halving time < 1 h. Only a small proportion of the DOS removed from solution was mineralized and excreted as SO42− (< 16% of the total taken up) suggesting that the DOS could be satisfying a demand for carbon (C) and S. In conclusion, even though inorganic S was abundant in freshwater, it appears that the aquatic communities retained the capacity to take up and assimilate DOS
Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase
A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1
Biochemical and structural insights into enzymatic depolymerization of polylactic acid and other polyesters by microbial carboxylesterases
Polylactic
acid (PLA) is a biodegradable polyester derived from
renewable resources, which is a leading candidate for the replacement
of traditional petroleum-based polymers. Since the global production
of PLA is quickly growing, there is an urgent need for the development
of efficient recycling technologies, which will produce lactic acid
instead of CO<sub>2</sub> as the final product. After screening 90
purified microbial α/β-hydrolases, we identified hydrolytic
activity against emulsified PLA in two uncharacterized proteins, ABO2449
from <i>Alcanivorax borkumensis</i> and RPA1511 from <i>Rhodopseudomonas palustris</i>. Both enzymes were also active
against emulsified polycaprolactone and other polyesters as well as
against soluble α-naphthyl and <i>p</i>-nitrophenyl
monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete
or extensive hydrolysis of solid PLA with the production of lactic
acid monomers, dimers, and larger oligomers as products. The crystal
structure of RPA1511 was determined at 2.2 Ã… resolution and revealed
a classical α/β-hydrolase fold with a wide-open active
site containing a molecule of polyethylene glycol bound near the catalytic
triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins
demonstrated that the catalytic triad residues are important for the
hydrolysis of both monoester and polyester substrates. We also identified
several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220)
and ABO2449 (Phe38 and Leu152), which were not essential for activity
against soluble monoesters but were found to be critical for the hydrolysis
of PLA. Our results indicate that microbial carboxyl esterases can
efficiently hydrolyze various polyesters making them attractive biocatalysts
for plastics depolymerization and recycling
Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail
Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (p NPbetaG) and p NP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using p NPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of p NPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases
Halorhabdus tiamatea: Proteogenomics and glycosidase activity measurements identify the first cultivated euryarchaeon from a deep-sea anoxic brine lake as potential polysaccharide degrader.
Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis AX-2T from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea SARL4BT from the Shaban deep-sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H. tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H. tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant-derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low-oxygen conditions. Likewise, proteomics indicated quinone-mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light-dependent enzymes (e.g. bacteriorhodopsin), suggesting that H. tiamatea evolution was mostly not governed by a cold, dark, anoxic deep-sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase-rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments
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