24 research outputs found
Revisit to the biodesulfurization capability of hyperthermophilic archeon Sulfolobus Solfataricus P2 revealed DBT consumption by the organism in an oil/water two-phase liquid system at high temperatures
The ability of hyperthermophilic archaeon, Sulfolobus solfataricus P2, to grow on organic and inorganic sulfur sources was investigated. A sulfur free mineral
medium has been employed with different sources of carbon. Results showed that
inorganic sulfur sources display growth curve patterns significantly different from the curves obtained with organic sulfur sources. Solfataricus has an ability to utilize DBT and its derivatives, but it lacks BT utilization. Solfataricus utilizes DBT at a rate of 1.23 μmol 2-HBP h-1 g DCW-1 even at 78 o40 C, at which DBT is known to be unstable. After enabling DBT stabilization using a two-phase culture system, stable microbial growth was achieved showing a desulfurization rate of 0.34 μM DBT g DCW-1 h-143 . Solfataricus offers beneficial properties compared to the other desulfurizing mesophilic/moderate thermophilic bacteria due to its capacity to utilize DBT and its derivatives under hyperthermophilic conditions
Biodesulfurization of DBT by a hyperthermophilic archaeon sulfolobus solfataricus P2
Combustion of fossil fuels leads to the atmospheric emission of sulfur oxides that contribute to acid rain. Significant quantities of sulfur (up to 70%) in petroleum is found as recalcitrant heterocyclic organosulfurs [dibenzothiophene (DBT) and substituted DBTs]. Current sulfur removal processes involve hydrodesulfurization, which requires high temperatures and pressure for efficient catalysis. Biodesulfurization is an alternative method for oil and
coal industry offering low costs and relatively easy operating conditions. The present study describes the usage of a hyperthermoacidophilic archaeon S. sulfotaricus P2 in the utilization of sulfur compounds from variety of organic and inorganic compounds at high temperatures, between 75 and 85◦C and low pH,
3.0. To establish optimal sulfur free conditions, carbon sources containing
arabinose, ethanol, glucose, mannose and mannitol have been employed to find the most suitable sources. Growths on the sulfur sources such as DBT, dibenzothiophene sulfone, BT, 4,6- dimethyldibenzothiophene, sodium sulfite, potassium disulphite, and potassium persulphate were investigated. 0.3mm of all sulfur supplements except DBT led to increases in the growth rate from 1.35 to 2.0-fold. Further investigations revealed the maximum DBT tolerance as 0.1mm. It was found that 88.5% of DBT was consumed and maximum desulfurization rate was obtained as 1.23mol 2-HBP hour−1 gDCW−1 within 16.5 hours. Our results also revealed that DBT sulfone consumption is the rate limiting step in the overall DBT usage process for S. solfataricus. To the best of our knowledge,this is the first report showing the DBT desulfurization kinetics analysis of S. solfataricus
Biodesulphurized subbituminous coal by different fungi and bacteria studied by reductive pyrolysis. Part 1: Initial coal
One of the perspective methods for clean solid fuels production is biodesulphurization. In order to increase the effect of this approach
it is necessary to apply the advantages of more informative analytical techniques. Atmospheric pressure temperature programming
reduction (AP-TPR) coupled with different detection systems gave us ground to attain more satisfactory explanation of the effects of
biodesulphurization on the treated solid products.
Subbituminous high sulphur coal from ‘‘Pirin” basin (Bulgaria) was selected as a high sulphur containing sample. Different types of
microorganisms were chosen and maximal desulphurization of 26% was registered. Biodesulphurization treatments were performed with
three types of fungi: ‘‘Trametes Versicolor” – ATCC No. 200801, ‘‘Phanerochaeta Chrysosporium” – ME446, Pleurotus Sajor-Caju and
one Mixed Culture of bacteria – ATCC No. 39327. A high degree of inorganic sulphur removal (79%) with Mixed Culture of bacteria
and consecutive reduction by 13% for organic sulphur (Sorg) decrease with ‘‘Phanerochaeta Chrysosporium” and ‘‘Trametes Versicolor”
were achieved.
To follow the Sorg changes a set of different detection systems i.e. AP-TPR coupled ‘‘on-line” with mass spectrometry (AP-TPR/MS),
on-line with potentiometry (AP-TPR/pot) and by the ‘‘off-line” AP-TPR/GC/MS analysis was used. The need of applying different
atmospheres in pyrolysis experiments was proved and their effects were discussed. In order to reach more precise total sulphur balance,
oxygen bomb combustion followed by ion chromatography was used
Analysis of expressed sequence tags (ESTs) from Agrostis species obtained using sequence related amplified polymorphism
Bentgrass (Agrostis spp.), a genus of the Poaceae family, consists of more than 200 species and is mainly used in athletic fields and golf courses. Creeping bentgrass (A. stolonifera L.) is the most commonly used species in maintaining golf courses, followed by colonial bentgrass (A. capillaris L.) and velvet bentgrass (A. canina L.). The presence and nature of sequence related amplified polymorphism (SRAP) at the cDNA level were investigated. We isolated 80 unique cDNA fragment bands from these species using 56 SRAP primer combinations. Sequence analysis of cDNA clones and analysis of putative translation products revealed that some encoded amino acid sequences were similar to proteins involved in DNA synthesis, transcription, and signal transduction. The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (GenBank accession no. EB812822) was also identified from velvet bentgrass, and the corresponding protein sequence is further analyzed due to its critical role in many cellular processes. The partial peptide sequence obtained was 112 amino acids long, presenting a high degree of homology to parts of the N-terminal and C-terminal regions of cytosolic phosphorylating GAPDH (GapC). The existence of common expressed sequence tags (ESTs) revealed by a minimum evolutionary dendrogram among the Agrostis ESTs indicated the usefulness of SRAP for comparative genome analysis of transcribed genes in the grass species
Purification and characterization of recombinant durum metallothionein domain constructs
Metallothioneins (MTs) are small proteins with high cysteine content and high binding capacity for metals like Zn, Cu and Cd. MTs exist in a wide range of organisms and are classified in one super- family according to the distribution of cysteine motifs in their sequences. Type 1 plant MTs, similar to their mammalian counterparts, have the cysteine motifs clustered in the N-and C-termini constituting the β- and -domains respectively. The two domains are connected by an unusually long (about 42 amino acids) hinge region whose structural and functional properties are unclear. Recent studies indicate that, despite the classification into a single family, all MTs do not have a single unifying function, and while some MTs help metal homeostasis others play a role in detoxification of heavy metals (e.g. Cd and As).
We identified an mt gene in Cd resistant durum wheat coding for a type 1 MT (dMT) and the recombinant protein (dMT) was over-expressed in E. coli as GST fusion (GSTdMT). For detailed structural and functional investigations GST-fusion constructs of βhinge-, hinge- and the hinge-domains of dMT were over-expressed in E. coli. Proteins were purified and results of characterization by size exclusion chromatography, SDS- and native-PAGE, UV-VIS absorption spectroscopy, atomic absorption spectroscopy, dynamic light scattering, and small-angle solution X-ray scattering will be presented. Studies on the isolated domains indicate distinct metal-binding properties and structural features for these regions. GST fusion of the hinge appears to be stable reflecting an intrinsic structural organization for this domain. These findings will be discussed in terms of relevance for the native structure of dMT
Revisiting the biodesulfurization capability of hyperthermophilic archaeon Sulfolobus solfataricus P2 revealed DBT consumption by the organism in an oil/water two-phase liquid system at high temperatures
The ability of the hyperthermophilic archaeon Sulfolobus solfataricas P2 to grow on organic and inorganic sulfur sources was investigated. A sulfur-free mineral medium was employed with different sources of carbon. The results showed that inorganic sulfur sources display growth curve patterns significantly different from the curves obtained with organic sulfur sources. Solfataricas has the ability to utilize DBT and its derivatives, but it lacks BT utilization. Solfataricas utilizes DBT at a rate of 1.23 mu mol 2-HBP h(-1) g DCW-1 even at 78 degrees C, at which DBT is known to be unstable. After enabling DBT stabilization using a two-phase culture system, stable microbial growth was achieved showing a desulfurization rate of 0.34 mu M DBT g DCW-1 h(-1). Solfataricas offers beneficial properties compared to the other desulfurizing mesophilic/moderate thermophilic bacteria due to its capacity to utilize DBT and its derivatives under hyperthermophilic conditions
Identifying, cloning and structural analysis of differentially expressed genes upon Puccinia infection of Festuca rubra var. rubra
Differentially expressed genes in response to rust infection (Puccinia sp.) in creeping red fescue (Festuca rubra var. rubra) were identified and quantified using the mRNA differential display technique. The differentially induced genes were identified as homologs of mitogen-activated protein kinase (MAPK) 3 of Arabidopsis thaliana, stem rust resistance protein Rpg1 of barley and Hsp70 of Spinacia oleracea. The change in the steady state expression levels of these genes in response to rust infection was tested by Northern blot analysis and further quantified by real-time PCR. A steady accumulation of transcripts in the course of rust infection was observed. Full-length transcript of a fescue MPK-3 was obtained by RACE PCR. Its corresponding cDNA encodes a protein with a predicted MW of 42.5 kDa which was mapped onto the structural model of homologs MAPK to illustrate the corresponding MAPK signature motifs. This study, for the first time, presents evidence on the rust infection dependent metabolic pathways in creeping red fescue
Recombinant Expression and Characterization of A.thaliana Heterotrimeric G protein alpha subunit; GPA1
Current research on plant heterotrimeric G proteins is mainly focused on mutant / overexpression studies performed in model organisms, i.e. A. thaliana and O. sativa. These studies provide information about the pathways involving these proteins; however, the mechanism of G protein activation via signaling molecules remains unknown. Similarly, information on biochemical and biophysical characteristics of the plant heterotrimeric G-proteins is lacking, whereas for the mammalian counterparts, such studies have contributed unique insights into understanding their functional roles. Following this, A.thaliana heterotrimeric G protein subunit (GPA1) is cloned into P.pastoris expression system and the recombinant protein is purified. Initial analyses reveal that GPA1 is membrane bound and N-terminally blocked indicating the presence of lipid modifications. The purified protein is GDP bound and can bind to GTP. Circular dichroism polarimetry analyses show that the dominant structure is helical and activation by receptor mimetics leads to a decrease in helical content; similar to mammalian counterparts. It appears that, contrary to mammalian G-alphas, the intrinsic fluorescence of GPA1 decreases in the presence of GTP and increases in the presence of GDP. Full characterization of GAPA1, including its nucleotide binding kinetics and dynamics, will help to establish a better understanding of the functional mechanisms of heterotrimerc G-protein activation in plants and will shed light on the level of similarity of plant and mammalian hetetrotrimeric G protein signaling pathways
Functional and structural investigations of a metallothionein from durum wheat, dMT
Plant metallothioneins (MTs) are low molecular weight (7-
8 kDa), sulphydryl-rich, aromatic residue lacking metalbinding
proteins which have two terminal metal-binding
clusters separated by a conserved distinctively long hinge
region of about 50 amino acids. The length and conserved
sequence of the hinge region distinguishes plant MTs from
their mammalian counterparts, suggesting additional roles
other than metal binding and detoxification. Homology
modeling indicates mammalian-like folds for the metalbinding
domains of dMT, and ab initio calculations yield a
DNA-binding like structural motif for the hinge region1.
Here, we study the structural features of full-length dMT
Abstracts
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and its constructs in unstructured, metal-free (apo) and
metal-bound (holo) states using various biophysical and
biochemical characterization techniques and we investigate
putative DNA-binding interactions of the hinge region
using functional assays (e.g. whole-genome PCR
technique) for the apo- and holo-protein. The Cd-binding
dMT is expressed in Escherichia coli as a Glutathione-STransferase
fusion protein and cleaved from this tag for
further analyses. The exact Cd-bound state of dMT and
oxidation of thiols are kept under control through
preparation of apo-protein from purified Cd-dMT prior to
reconstitution by titration of equivalent amounts of Cd
solution under anaerobic conditions. 4 mole equivalence of
Cd could saturate dMT as followed by spectral changes of
circular dichroism and UV-Vis absorption spectroscopy.
This finding is further confirmed by parallel inductively
coupled plasma optical emission spectroscopy
measurements. Small angle X-ray solution scattering
measurements and gel-filtration chromatography revealed
an oligomeric form for the Cd-bound dMT whereas apo
dMT is monomeric as also shown by native state
electrospray ionization mass spectrometry experiments.
Taken together, our results suggest that structural
rearrangements driven by metal-binding result in
oligomerization/complex formation that may lead to
functional variations.
Acknowledgements
This work is supported by a joint TUBITAK and JULICH
grant, TBAG-U-157 105T535.
1. Bilecen et al., 2005, J. Biol. Chem. 280,13701-1