Littérature scientifique sur le sujet « Desulfurization, Biodesulfurization, dsz enzymes, 4S pathway »

ABSTRACT Dibenzothiophene (DBT) and its derivatives can be microbially desulfurized by enzymes DszC, DszA, and DszB, which are encoded by the operon dszABC and contribute to the conversion in tandem. We investigated the expression characteristics of the dsz operon. Our results revealed that the levels of transcription and translation of dszA, dszB, and dszC decreased according to the positions of the genes in the dsz operon. Furthermore, the translation of dszB was repressed by an overlapping structure in the dsz operon. In order to get better and steady expression of the Dsz enzymes and optimize the metabolic flux of DBT, we rearranged the dsz operon according to the catalytic capabilities of the Dsz enzymes and expressed the rearranged dsz operon, dszBCA, in Rhodococcus erythropolis. After rearrangement, the ratio of dszA, dszB, and dszC mRNAs in the cells was changed, from 11:3.3:1 to 1:16:5. Western blot analysis revealed that the levels of expression of dszB and dszC had been enhanced but that the expression of dszA had decreased. The desulfurization activity of resting cells prepared from R. erythropolis DRB, which carried the rearranged dsz operon, was about 12-fold higher than that of resting cells of R. erythropolis DRA, which carried the original operon in a similarly constructed vector.

Dibenzothiophene (DBT) is an organic sulfur compound which remains in oil after hydrodesulfurization (HDS) process and can be removed by biodesulfurization (BDS). A new strain of Paenibacillus validus (strain PD2) was isolated from oil contaminated soils that is able to desulfurize DBT. HPLC analysis and Gibbs assay showed that this strain was capable to convert DBT to 2-Hydroxybiphenyl (2-HBP) as final product. The presence of dszC gene confirmed that DBT desulfurization occurred through the 4S pathway. Maximum growth and the highest induction in dsz operon obtained in the presence of dimethyl sulfoxide (DMSO) as sole sulfur source. DBT concentration, temperature and pH were optimized statistically for growing and resting cells by using Response Surface Methodology (RSM). All parameters in growing cells had a significant effect on 2-HBP production during BDS of DBT by P. validus PD2, although in resting cells temperature in range of 20-40 degrees C was not a significant factor. Maximum BDS for growing cells was obtained at 0.41 mM DBT concentration, pH 6.92 and temperature 31.23 degrees C. For resting cells, optimum pH, temperature and DBT concentration were 6.62, 27.73 degrees C and 7.86 mM respectively. The results of this study showed that high concentrations of DBT could be desulfurized by P. validus strain PD2 in model-oil. Thus, the isolated strain could be introduced as a proper candidate for biodesulfurization of organic sulfur in the oil industry.

ABSTRACTMicrobial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubatingRhodococcus erythropolisIGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 μM with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes' activities (IC50s) for DszA, DszB, and DszC were measured to be 60 ± 5 μM, 110 ± 10 μM, and 50 ± 5 μM, respectively. The fact that the IC50s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation.

A number of actinobacteria of the genus Gordonia are able to use dibenzothiophene (DBT) and its derivatives as the only source of sulfur, which makes them promising agents for the process of oil biodesulfurization. Actinobacteria assimilate sulfur from condensed thiophenes without breaking the carbon–carbon bonds, using the 4S pathway encoded by the dszABC operon-like structure. The genome of the new dibenzothiophene-degrading hydrocarbon-oxidizing bacterial strain Gordonia amicalis 6-1 was completely sequenced and the genes potentially involved in the pathways of DBT desulfurization, oxidation of alkanes and aromatic compounds, as well as in the osmoprotectant metabolism in strain 6-1 and other members of the genus Gordonia, were analyzed. The genome of G. amicalis strain 6-1 consists of a 5,105,798-bp circular chromosome (67.3% GC content) and an 86,621-bp circular plasmid, pCP86 (65.4% GC content). This paper presents a comparative bioinformatic analysis of complete genomes of strain 6-1 and dibenzothiophene-degrading Gordonia strains 1D and 135 that do not have the dsz operon. The assumption is made about the participation in this process of the region containing the sfnB gene. Genomic analysis supported the results of phenomenological studies of Gordonia strains and the possibility of their application in the bioremediation of oil-contaminated environments and in the purification of oil equipment from oil and asphalt-resin-paraffin deposits.

L’enorme quantità di pneumatici fuori uso accumulati costituisce un grave problema ambientale. Per porvi rimedio si cercano soluzioni che mirano al riutilizzo della gomma naturale (NR), principale materia prima utilizzata nella produzione di pneumatici. Per poter essere riutilizzata, la NR viene generalmente macinata finemente e sottoposta ad un trattamento di devulcanizzazione, cioè alla rottura dei ponti trasversali zolfo-zolfo. Diversi metodi chimici o meccanici sono utilizzabili per devulcanizzare il polverino. Tuttavia, ciascuno di essi presenta degli svantaggi. Sarebbe quindi auspicabile la messa a punto di processi in cui reazioni specifiche siano realizzate in condizioni moderate di temperatura e pressione. In questo scenario l’utilizzo di biocatalizzatori potrebbe costituire un’alternativa valida e di minor impatto ambientale. Questo studio si occupa di verificare la possibilità di trattamenti enzimatici di “biodevulcanizzazione”. Non essendo noti enzimi in grado di devulcanizzare la gomma, questo lavoro di tesi è partito dall’analisi di microrganismi isolati da campioni ambientali e con potenziali proprietà desolforanti che sono state saggiate su un substrato modello, il dibenzotiofene (DBT). Il primo screening sui microrganismi ha portato alla selezione di un nuovo ceppo di Rhodococcus sp. AF21875. L’attività desolforante di questo microrganismo è stata studiata con due approcci paralleli, da un lato verificando la presenza di geni codificanti per enzimi che desolforano il DBT e dall’altro individuando nuovi enzimi con abilità desolforanti. In batteri attivi in processi di desolforazione, i geni dsz codificano per 4 enzimi: DszA, DszB, DszC e DszD. Nel DNA genomico di Rhodococcus sp. AF21875 è stata verificata la presenza dei quattro geni dsz, che sono stati clonati per consentire la produzione ricombinante delle proteine corrispondenti in un ceppo del batterio Escherichia coli. Tre delle proteine, DszA, DszC e DszD, sono abbondantemente espresse in forma solubile e sono state purificate con successo. In previsione di un impiego biotecnologico delle proteine DszA, DszC e DszD è stata intrapresa l’analisi di alcune caratteristiche strutturali e di stabilità. In particolare, è stata analizzata la composizione della struttura secondaria e la stabilità al calore mediante dicroismo circolare; la stabilità in presenza di diversi solventi organici, attraverso spettrofluorimetria. L’attività degli enzimi è stata monitorata mediante cromatografia liquida (HPLC) che consente di rilevare la formazione di 2-idrossibifenile (HBP) come prodotto finale di reazione. L’attività di desolforazione dei quattro enzimi è stata infine saggiata su gomma naturale vulcanizzata. È emerso che il trattamento enzimatico provoca modificazioni chimiche della gomma. Sebbene le analisi condotte evidenzino cambiamenti di modesta entità e non associabili univocamente ad un processo di desolforazione, gli enzimi individuati costituiscono un buon punto di partenza per approcci di ingegneria proteica volti a migliorare l’attività e la stabilità degli enzimi Dsz da Rhodococcus sp. AF21875. Inoltre, per individuare nuove attività enzimatiche è stata eseguita un’analisi di proteomica differenziale delle cellule di Rhodococcus sp. AF21875. Da cellule cresciute in terreno privo o addizionato di DBT sono state estratte le proteine totali che sono state analizzate mediante elettroforesi bidimensionale. Quando Rhodococcus sp. AF21875 cresce in presenza di DBT, produce un pool di proteine che non si ritrovano tra le proteine espresse in assenza di DBT. Tre delle proteine sono state analizzate tramite digestione triptica in gel e analisi di spettrometria di massa. Questa analisi ha permesso di identificare due enzimi che non sono coinvolti nel metabolismo dello zolfo ma appartengono ad una stessa via catabolica distinta da quella cui appartengono gli enzimi Dsz.
The environmental hazard posed by the accumulation of huge amounts of used tires might be partly relieved by the implementation of methods for recycling natural rubber (NR) from waste tires. This approach requires rubber grinding and a process of devulcanization that breaks the sulfur-sulfur crosslinks among polymer chains. Several chemic al or mechanical methods are already used to devulcanize ground-rubber. However, each of them has drawbacks related either to the lack of specificity or to the use of hazardous chemicals. It would therefore be desirable to develop processes in which selective and specific reactions are carried out in mild conditions of temperature and pressure, without the use of hazardous compounds. In this view, the use of biocatalysts could be a valuable and ecological alternative. This study explores the possibility of applying enzymes to devulcanize rubber in a process of “biodevulcanization”. Since enzymes active in rubber devulcanization were not available at the beginning of this thesis, this research started with the analysis of microorganisms isolated from environmental samples contaminated with waste tires. The desulfuring properties of several bacteria were tested on the model substrate dibenzothiophene (DBT). A first in-vivo screening of microorganisms allowed the selection of a new strain of Rhodococcus sp. referred as AF21875. This microorganism was studied with two aims: assessing the presence of a metabolic pathway for DBT desulfurization already described in other bacteria and identifying new metabolic abilities and enzymes. In bacteria active in desulfurization, four enzymes co-operate in the reaction of desulfurization: DszA, DszB, DszC and DszD. The presence of the four corresponding dsz genes in the genomic DNA of Rhodococcus sp. AF21875 has been assessed. The four genes have been cloned in a strain of the bacterium Escherichia coli to allow for the production of recombinant Dsz enzymes. The three recombinant proteins DszA, DszC and DszD are soluble and were successfully purified. More difficult was the production of DszB that is poorly expressed in any experimental condition. In view of a biotechnological application, structural and stability studies were carried out on DszA, DszC and DszD enzymes. In particular, we investigated secondary structure and heat stability by circular dichroism, while protein stability in the presence of different organic solvents was studied by spectrofluorimetry. Enzymes activity on DBT was assessed by high performance liquid chromatography (HPLC) by detecting the formation of 2 -hydroxybiphenyl (HBP), the reaction product of DBT desulfurization. The desulfurization activity of the four enzymes was then tested on vulcanized natural rubber using Rubber Process Analyzer and Fourier Transform Infrared Spectroscopy to detect chemical modifications induced by the enzymatic treatment. These analyses revealed minor changes. Other studies should be performed to attribute such modifications to desulfurization. Overall, Dsz enzymes from Rhodococcus sp. AF21875 were found to be an interesting starting point for the application of protein engineering approaches aimed to improve not only their activity but also their stability. A differential proteomic analysis of Rhodococcus sp. AF21875 was performed to identify enzymatic activities related to sulfur metabolism and different from Dsz proteins. Total proteins, extracted from cells grown either in the presence or in the absence of DBT, were separated by two-dimensional electrophoresis, showing that DBT induces a few changes in the proteome of Rhodococcus sp. AF21875. Three proteins, belonging to a metabolic pathway different from the Dsz one were identified by in-gel tryptic digestion and mass spectrometry.