MXPA98002175A - Utilization of dszd in the disappearance of dbt by rhodococcus sp. ig - Google Patents

Abstract

The invention relates to the discovery that the reaction rate of the desulfurization of fossil fuels is increased by the addition of an oxidoreductase to the biocatalyst. The invention is directed to a method for improving the rate of desorption of a fossil fuel containing organic sulfur compounds, comprising the following steps: a) contacting the fossil fuel with an aqueous phase containing a biocatalyst capable of unfolding carbon and sulfur bonds and an amount of oxidoreductase, forming therefrom a fossil fuel and an aqueous phase mixture, b) maintaining the mixture of step (a) under conditions sufficient for the cleavage of the carbon and sulfur bonds of the molecules of organic sulfur by means of the biocatalyst, resulting in a fossil fuel having a reduced content of organic sulfur, and c) separating the fossil fuel having a reduced content of organic sulfur from the resulting aqueous phase. The invention also relates to a recombinant microorganism containing one or more recombinant DNA molecules that encode a biocatalyst capable of de-sulfurizing a fossil fuel containing organic sulfur molecules, and encoding an oxireductase. The invention likewise refers to a composition comprising (a) a biocatalyst capable of de-sulfurizing a fossil fuel containing organic sulfur molecules and (b) an oxidoreducta

Description

UTILIZATION OF DszD IN THE DISAPPEARANCE OF DBT BY Rhodococcus sp. IGTS8 BACKGROUND OF THE INVENTION The microbial desalination of fossil fuels has been an active research area for more than fifty years. The purpose of these investigations has been to develop biotechnology-based methods for the pre-combustion of sulfur from fossil fuels, such as coal, crude and petroleum distillates. The driving forces for the development of dewatering methods are increasing levels of sulfur in fossil fuels and increasingly stringent regulations on sulfur emissions. Monticello et al., "Practical Considerations in Biodesulfurization of Petroleum", 3rd IGT International Symposium on Gas, Oil, Coal and Environmental Biotechnology (3-5 Dec, 1990), New Orleans, LA. Many biocatalysts and processes for de-sulfurizing fossil fuels have been developed, including those described in US Pat. Nos. 5,356,801, 5,358,870, 5,358,813, 5,198,341, 5,132,219, 5,344,778, 5,104,801 and 5,002,888, incorporated herein by reference. The economic analyzes indicate that a limitation in the commercialization of the technology is to improve the reaction rates and the specific activities of the biocatalysts, such as the bacteria and enzymes involved in the desulphurization reactions. The reaction rates and the specific activities (sulfur removed / hour / gram of biocatalyst) that have been described in the literature are much lower than those necessary for optimal commercial technology. Therefore, it is desired to obtain improvements in the longevity and in the specific activity of the biocatalyst. COMPENDIUM OF THE INVENTION The invention relates to the discovery that a class of proteins, one of which was recently purified from Rhodococcus sp. IGTS8, activates two monooxygenases (DszC and DszA) involved in the desazuframiento of fossil fuels. Neither DszC nor A are enzymatically active when they are purified to homogeneity; however, after the addition of this additional protein (here called DszD), the enzymatic activity is restored. The function of this protein is thought to be coupling the oxidation of NADH to the oxygenation of the substrate molecule. A search of the sequence databases revealed that DszD is equivalent to another recently identified Rhodococcus protein, ThcE, which is induced by growth in the presence of atrazine, thiocarbamate herbicides and primary alcohols. Based on the sequence similarities, ThcE appears to be a member of group III alcohol dehydrogenases, or oxidoreductases, called alcohol: N, N'-dimethyl-3-nitrosoaniline oxidoreductases. DszD has a monomeric molecular weight of approximately 50,000 (by SDS-PAGE), but behaves as a multimeric protein (decamer) in HPLC size exclusion chromatography. the activation of DszC and A by DszD follows a saturation kinetics. Thus, the invention relates to the discovery that the rate of microbial desulfurization of fossil fuels is increased or activated or depends on the addition of an oxidoreductase to the biocatalyst or to the reaction medium. The invention is directed to a method for increasing the rate of desulfurization of a fossil fuel containing organic sulfur compounds, consisting of the following steps: a) contacting the fossil fuel with an aqueous phase containing a biocatalyst or biocatalyst capable of excising the carbon-sulfur bonds (such as Dsz A, Dsz B and / or Dsz C) and a potentiating amount of the speed of an oxidoreductase, thus forming a mixture of fossil fuel and aqueous phase; b) maintaining the mixture of step (a) under conditions sufficient for the excision of the carbon-sulfur bonds of the organic sulfur molecules by the biocatalyst, thus giving rise to a fossil fuel having a reduced content of organic sulfur, and c) separating the fossil fuel having a reduced organic sulfur content from the resulting aqueous phase. The invention also relates to the increase in the rate of the reaction catalyzed by DszA and / or DszC with an oxidoreductase speed-enhancing amount. This can be achieved, for example, by adding the oxidoreductase to a biocatalyst or causing the expression or overexpression of the oxidoreductase in a biocatalyst. In yet another embodiment, the invention relates to a recombinant microorganism containing one or more recombinant DNA molecules encoding a biocatalyst capable of catalyzing one or more steps in a process to de-sulfur a fossil fuel containing organic sulfur molecules and encoding an oxidoreductase. The invention includes a composition comprising (a) a biocatalyst capable of catalyzing one or more steps in a process for the desulfurization of a fossil fuel containing organic sulfur molecules and (b) an oxidoreductase. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphic illustration of the DszC and A activity after ion exchange chromatography. DszC (15 μg) was added to each fraction and studied for the conversion of DBT to DBTO and DBT02. DszA (5 μg) was added to each fraction and studied for the conversion of DBT sultone to BHBF. The endogenous DszC activity was also studied. Figure 2 is a graphic illustration of the DszC activity after Superdex 75 size exclusion chromatography. DszC (15 μg) was added to each fraction and studied for the conversion of DBT to DBT02.

DszA activity after Superdex 75 size exclusion chromatography. DszA (5 μg) was added to each fraction and studied for the conversion of DBT sultone to BHBF. Figure 3 is an electrophoretic gel illustrating the SDS-PAGE (14% acrylamide) of the purification of DszD. Band 1 presents the molecular weight patterns (Biorad, 200, 116, 97.4, 66, 45, 31, 21.5 and 14.5 kDa); band 2, crude cell lysate; band 3, after Q-sepharose; band 4, after Toyopearl-DEAE; band 5, after MonoQ, and band 6, after Superdex 75. Figure 4 illustrates the activation of DszC by the addition of increasing amounts of DszD. A fixed amount of DszC (0.33 nmol) was titrated with increasing amounts of DszD. Figure 5 illustrates the activation of DszA by increasing the amounts of DszD. A fixed amount of DszA (0.16 nmol) was titrated with increasing amounts of DszD. Figure 6 establishes the DNA sequence and the putative amino acid sequence of the ThcE gene (DszD). DETAILED DESCRIPTION OF THE INVENTION In petroleum extraction and refinery techniques, it is generally understood that the term "organic sulfur" refers to organic molecules that have a hydrocarbon skeleton to which one or more sulfur atoms (so-called heteroatoms) are covalently attached. . These sulfur atoms can be attached directly to the hydrocarbon skeleton, for example by one or more carbon-sulfur bonds, or they can be present in a substituent attached to the hydrocarbon backbone of the molecule, for example a sulfate group. Reference is made to the general class of organic molecules that have one or more sulfur heteroatoms as "organosulfur compounds". The hydrocarbon portion of these compounds can be aliphatic, aromatic or partially aliphatic and partially aromatic. Reference is made to condensed cyclic or condensed organosulfur compounds in which one or more sulfur heteroatoms are directly or indirectly attached to the adjacent carbon atoms in the hydrocarbon skeleton by carbon-sulfur bonds as "sulfur bearing heterocycles" . Reference is made to the sulfur that is present in many types of sulfur-bearing heterocycles such as "thiophenic sulfur", in view of the -membered aromatic ring in which the sulfur heteroatom is present. The simplest of said heterocarbons carrying sulfur is thiophene, which has the composition C4H4S. Sulfur-bearing heterocycles are known to be stable to conventional desulphurisation treatments, such as hydrodesulfurization (HDA). Sulfur-bearing heterocycles may have relatively simple or relatively complex chemical structures. In complex heterocycles, multiple condensed aromatic rings are present, one or more of which may be heterocyclic. The difficulty of desazuframiento increases with the structural complexity of the molecule. That is to say, that the refractory behavior is more accentuated in the heterocycles carrying sulfur complexes, such as dibenzothiophene (DBT, C12H8S). DBT is a sulfur-bearing heterocycle having a condensed multiple aromatic ring structure wherein a -membered thiophenic ring is flanked by two six-membered benzyl rings. Much of the residual post-HDS organic sulfur in the fossil fuel refinery intermediaries and in the fuel products is thiophenic sulfur. Most of this residual thiophene sulfur is present as DBT and derivatives thereof having one or more alkyl or aryl groups attached to one or more carbon atoms present in one or both flanking benzyl rings. The DBT itself is accepted in the relevant techniques as a composite model illustrating the behavior of the class of compounds that includes DBT and its derivatives in reactions involving thiophenic sulfur. Monticello and Finnerty, Annual Reviews in Microbiology 33: 371-389 (1985), in 372-373. DBT and its derivatives may represent a significant percentage of the total sulfur content of particular crude oils, carbons and asphalts. For example, it has been described that these sulfur-bearing heterocycles represent up to 70% by weight of the total sulfur content of West Texas crude and up to 40% by weight of the total sulfur content of some crude oils in the Middle East. Thus, the DBT is considered to be particularly relevant as a model compound for the forms of thiofenic sulfur found in fossil fuels, such as crude oils, carbons or asphalts of specific geographical origin and various refining intermediaries and fuel products manufactured from them. idem Another characteristic of DBT and its derivatives is that, after a release of fossil fuel into the environment, these sulfur-bearing heterocycles persist for long periods of time without significant biodegradation. Gundlach et al., Science 221: 122-129 (1983). It is desirable, therefore, to separate these organosulfur compounds from fossil fuels or other carbon materials containing them. A fossil fuel or carbon material which is suitable for the treatment of desulphurization according to the present invention is one containing organic sulfur. The said fossil fuel is referred to as "fossil fuel substrate". Substrate fossil fuels that are rich in thiophene sulfur are particularly suitable for desulphurization according to the method described herein. Examples of such fossil fuel substrates include heavy crude from Cerro Negro or Orinoco; Athabascan tar and other types of asphalt; petroleum refining fractions, such as light crudes, heavy atmospheric gas oil and No. 1 diesel oil, and coal-derived liquids manufactured from sources such as coal Pocahontas # 3, Lewis-Stock, Australian Glenooe or Wyodak. Biocatalytic desalination, or DAB, is the cleavage, release or removal of sulfur from organosulfur compounds, including refractory organosulfur compounds, such as sulfur bearing heterocycles, as a result of oxidative (preferably selectively) cleavage of carbon bonds -sulfur in said compounds by a biocatalyst. The DAB treatment gives rise to the de-sulfurized hydrocarbon skeleton of the first refractory organosulfur compound, together with inorganic sulfur substances which can be easily separated from one another by known techniques, such as fractional distillation or extraction with water. For example, DBT is "converted" to hydroxybiphenyl when it is subjected to DAB treatment. The DAB is carried out by biocatalyst (s).

Biocatalysts include one or more non-human organisms (e.g., recombinant and non-recombinant, viable and non-viable microorganisms) that functionally express one or more enzymes that direct, alone or together, the removal of sulfur from the organosulfur compounds. , including sulfur bearing heterocycles, by oxidation of sulfur and / or excision of carbon-sulfur bonds in said compounds; one or more enzymes obtained from said organisms; or a mixture of said organisms and enzymes. The organisms that exhibit one or more biocatalytic activities necessary for the desulfurization of a fossil fuel or other carbon material are here designated Dsz +. The organisms that lack such biocatalytic activity are here called Dsz-. A "biocatalyst" is defined herein as a biological material, or a material of biological origin, which has the ability to catalyze one or more reactions in the presence of appropriate cofactors and / or coenzymes, for example. The invention relates to the improved removal of sulfur from carbonaceous materials, such as fossil fuels, containing organic sulfur molecules, consisting of adding an amount that increases the speed of an oxidoreductase to the biocatalyst capable of de-sulfurizing the carbonaceous material. The biocatalysts employed herein are, in general, known in the art. Several researchers have described the genetic modification of natural bacteria in mutant strains capable of catabolizing DBT. Kilbane, J.J., Resour. Cons Recycl. 3: 69-79 (1990); Isbister, J.D. and R.C. Doyle, US Patent No. 4,562,156 (1985), and Hartdegan F.J. et al., Chem. Eng. Progress 63-67 (1984). Many of these mutants de-sulfur DBT in a non-specific manner. Therefore, a portion of the fuel value is lost through this microbial action. Isbister and Doyle described the derivation of a mutant Pseudomonas strain that appeared to be capable of selectively releasing sulfur from DBT. Kilbane has described the mutagenesis of a mixed bacterial culture, thus producing a bacterium capable of selectively releasing sulfur from DBT by an oxidative pathway. This crop was composed of bacteria that can be obtained from natural sources, such as sewage sludge, waste water from the oil refinery, garden soil, coal, soil contaminated with tar, etc. and that they are maintained in culture under conditions of continuous sulfur deprivation in the presence of DBT. The culture was then exposed to the chemical mutagen l-methyl-3-nitro-1-nitrosoguanidine. The major catabolic product of DBT metabolism by this mutant culture was hydroxybiphenyl; the sulfur was released as water-soluble inorganic sulfate and the hydrocarbon portion of the molecule remained essentially intact as monohydroxy-biphenyl. Kilbane, J.J., Resour. Cons Recycl. 3: 69-79 (1990), whose teachings are incorporated herein by reference. Kilbane has also isolated a mutant strain of Rhodococcus from this mixed bacterial culture. This mutant, IGTS8 or ATCC No. 53968, is a particularly preferred biocatalyst for use in the present invention. The isolation and characteristics of this mutant are described in detail in J.J. Kilbane, US Patent No. 5,104,801, whose teachings are incorporated herein by reference. This microorganism has been deposited in the American Type Culture Collection (ATCC), 12301 Park Lawn Drive, Rockville, Maryland, USA. 20852, under the terms of the Budapest Treaty and has been designated Deposit No. ATCC 53968. A suitable preparation of ATCC biocatalyst No. 53968 is a culture of live microorganisms, generally prepared as described in US Pat. UU No. 5,104,801, and mutants or derivatives thereof (see, for example, US Patent No. 5,358,869). Cell-free enzyme preparations obtained from ATCC No. 53968 or their mutants can also be used generally as described in US Pat. Nos. 5,132,219, 5,344,778 and 5,358,870. These enzyme preparations can still be purified and used. Other examples of microorganisms that appear to behave in the same or similar manner include the microbial consortium (a mixture of several microorganisms) described by Kilbane (1990), 3 Resour. Conserv. Recycl. 69-79; the microorganisms described by Kilbane in US Pat. No. 5,002,888 (granted March 26, 1991), 5,104,801 (granted April 14, 1992), 5,344,778, 5,132,219, 5,198,341, 5,344,778, 5,356,813, 5,356. 801, 5,358,869, 5,358,870 [also described by Kilbane (1990), Biodesulfurization: Future Prospecte in Coal Cleaning, in Proc. 7th Annual Pittsburgh International Coal Conference, 373-382] and 5,198,341 (granted March 30, 1993) and by Omori et al. (1992), Desulfurization of dibenzothiophene by Corynebacteri um sp. strain SY1, 58 Appl. Env. Microbiol. (No. 3) 911-915, and Izumi et al., Applied and Environmental Microbiology ^ 0: 223-226 (1994), all of which are incorporated herein by reference. Each of the above microorganisms can function as a biocatalyst in the present invention, since each one produces one or more enzymes (protein biocatalysts) that carry out the specific chemical reaction (s) by means of the which the sulfur of refractory organosulfur compounds is excised. Mutational or genetically engineered derivatives of any of the foregoing microorganisms can also be used, as exemplified in US Pat. cited above, as a biocatalyst here, provided that the appropriate biocatalytic function is maintained. Additional microorganisms suitable for use as a biocatalyst or source of biocatalyst in the desulphurization process now described can be derived from natural microorganisms by known techniques. As indicated above, these methods include the cultivation of preparations of microorganisms obtained from natural sources, such as sewage sludge, waste water from the oil refinery, garden soil, coal or soil contaminated with tar, under culture conditions. selective in which microorganisms grow in the presence of refractory organosulfur compounds, such as sulfur bearing heterocycles, as the sole source of sulfur; the exposure of microbial preparation to chemical or physical mutagens; or a combination of these methods. Said techniques are referred to by Isbister and Doyle in U.S. Pat. No. 4,562,156 (granted December 31, 1985), by Kilbane in 3 Resour. Conserv. Recycl. 69-79 (1990), US Pat. Nos. 5,002,888, 5,104,801 and 5,198,341, and by Omori et al. In 58 Appl. Env. Microbiol. (No. 3) 911-915 (1992), all of them incorporated by reference. As explained above, enzymes are protein or peptide biocatalysts that can be produced by living cells. The enzymes promote, direct or facilitate the appearance of a reaction or series of specific chemical reactions (referred to as a route), in general, without consuming themselves as a result of them. The enzymes may include one or more polypeptide chains unmodified or modified post-translationally or synthetically or fragments or portions thereof, which catalyze the desired reaction or series of reactions when they are in the presence of appropriate coenzymes, co-factors or additional coreactants. The reaction or series of reactions relevant to an embodiment of the present invention culminates in the cleavage of the sulfur from the hydrocarbon skeleton of a refractory organosulfur compound, such as a sulfur-bearing heterocycle. The hydrocarbon skeleton of the first refractory organosulfur compound remains substantially intact. The microorganisms or enzymes used as biocatalysts in the present invention preferably and advantageously do not consume the hydrocarbon skeleton of the first refractory organosulfur compound as a carbon source for growth. As a result, the fuel value of the substrate fossil fuels exposed to the DAB treatment does not deteriorate. Although live microorganisms (for example, a culture) can be used here as a biocatalyst, this is not necessary. Enzymatic biocatalytic preparations useful in the present invention include used microbial, extracts, fractions, subfractions or purified products obtained by conventional means and are capable of carrying out the desired biocatalytic function. In general, said enzyme preparations are substantially free of intact microbial cells. Kilbane and Monticello describe enzyme preparations that are suitable for use herein in U.S. Pat. No. 5,132,219 (issued July 21, 1992) and 5,358,870 (filed June 11, 1992), for example. Rambosek et al. describe recombinant microorganisms and enzymatic preparations obtained by engineering Rhodococcus sp. ATCC No. 53968 and suitable for use herein in U.S. Pat. 5,356,813. In a particularly preferred embodiment, the biocatalyst is overexpressed in the recombinant host cell (such as a cell that contains more than one copy of the gene or genes). For example, the desulfurization of dibenzothiophene by Rhodococcus sp. IGTS8 has been shown to involve at least three enzymes (designated DszA, B and C), of which it is now appreciated that DszA and C are monooxygenases. As such, in a particularly preferred embodiment, the biocatalyst includes one or more of the enzymes, Dsz A, Dsz B and / or Dsz C. The biocatalytic enzyme preparations suitable for use herein may optionally be attached to a solid support, for example a membrane, a filter, a polymeric resin, particles or glass beads or ceramic beads or particles. The use of immobilized enzyme preparations facilitates the separation of the biocatalyst from the reaction medium, such as the treated fossil fuel that has been devoid of refractory organosulfur compounds. The specific activity of a given biocatalyst is a measure of its biocatalytic activity per unit mass. Thus, the specific activity of a particular biocatalyst depends on the nature or identity of the microorganism used or used as a source of biocatalytic enzymes, as well as on the procedures used to prepare and / or store the biocatalytic preparation. The concentration of a particular biocatalyst can be adjusted as desired for use in particular circumstances. For example, when a culture of live microorganisms (eg, ATCC No. 53968) is used as a biocatalytic preparation, a suitable culture medium lacking in another source of sulfur other than sulfur-bearing heterocycles with suitable microorganisms can be inoculated. and ferment it until reaching a desired cultivation density. The resulting culture can be diluted with additional medium or other suitable buffer, or the microbial cells present in the culture can be recovered, for example, by centrifugation and resuspended at a higher concentration than that of the original culture. The concentrations of microorganism and enzymatic biocatalyst can be adjusted in a similar way. In this way, appropriate volumes of biocatalytic preparations having specific activities and / or predetermined concentrations can be obtained. As indicated above, a protein (called DszD) has now been purified from Rhodococcus sp. IGTS8 that activates and potentiates the activity of two integral monooxygenases in the bio-degradation pathway (DszC and DszA). The function of this protein is thought to be to couple the oxidation of NADH to the oxygenation of the substrate molecules by DszA and DszC. A search of the sequence databases revealed that DszD is equivalent to another recently isolated Rhodococcus protein, ThcE, which, as described, is induced by growth in the presence of atrazine, thiocarbamate herbicides and primary alcohols. ThcE is a member of group III alcohol dehydrogenases, or oxidoreductases, termed alcohol: N, N'-dimethyl-3-nitrosoaniline oxidoreductases, and has been described by Nagy et al., Arch. Microbiol. (1995) 163: 439-446, which is incorporated herein by reference in its entirety. DszD has a monomeric molecular weight of approximately 50,000 (by SDS-PAGE), but behaves as a multimeric protein (decamer) in size-exclusion HPLC chromatography. The activation of DszC and A by DszD follows a saturation kinetics. In view of the above-described discovery, DBT desulfurization can be increased by the addition of an oxidoreductase. Suitable oxidoreductases include the monooxygenase reductase, or the oxidoreductase alcohols, such as the N, N'-dimethyl-4-nitrosoaniline (NDMA) -dependent oxidoreductases (MNO) alcohol. It has been reported that group III alcohol dehydrogenases, or oxidoreductases, oxidize a primary alcohol and reduce an electron acceptor, such as the non-physiological compound NDMA. They generally contain a closely bound, but non-covalent, molecule of NADp that mediates the electron transfer between an alcohol and the electron acceptor (e.g., NDMA). As defined herein, the term "oxidoreductase" includes endogenous or wild-type enzymes, recombinantly produced enzymes, fusion proteins, active fragments, mutants, or combinations thereof that possess the ability to increase and / or activate the activity of DszA and / or DszC. . Mutants include allelic variants, amino acid or site-directed mutations or derivatives (such as those prepared using recombinant DNA technology). Alternatively, the mutants can be prepared using other chemical or physical mutagenesis techniques with the host microorganism. The enzyme is preferably isolated from Rhodococcus or from rhodococic origin, such as IGTS8 or Rhodococcus sp. N186 / 21 Other preferred embodiments include recombinant oxidoreductases having a highly homologous amino acid sequence (such as at least about 90%) to the amino acid sequence of these enzymes. Alternatively, homologous oxidoreductases, such as those that can be isolated from Amycola topsis methanolics and Mycobacterium gas tri. As indicated above, oxidoreductases that can be employed herein include those that are generally known in the art and can be used directly as found in nature (eg, a microbial fraction containing the protein or enzyme) , obtained commercially or recombinantly prepared. For example, the DNA and amino acid sequences of DszD are cited by Nagy et al., Arch. Microbiology (1995) 163: 439-446 (and illustrated in Figure 6) and can be used to transform a suitable host microorganism as is well known in the art and is discussed in US Pat. No. 5,356,801, for example. The DNA sequence can be isolated from a suitable Rhodococcus using well-known techniques, such as PCR. In another embodiment, the oxidoreductase can be overexpressed by the dewatering microorganism (such as IGTS8). This can be achieved, for example, by mutagenesis. Suitable mutagens include radiation, for example ultraviolet radiation, and chemical mutagens, such as N-methyl-N 1 -nitrosoguanidine, hydroxylamine, ethyl methanesulfonate and nitrous acid. Mutagenesis and subsequent selective study of mutants that harbor greater enzymatic activity can be performed according to methods generally known in the art. When oxidoreductase is recombinant, the protein can be prepared and, preferably, overexpressed in situ, such as by the addition of a recombinant microorganism containing one or more copies of a DNA sequence encoding oxidoreductase. In a particularly preferred embodiment, the recombinant microorganism encoding oxidoreductase also possesses one or more enzymes capable of catalyzing one or more reactions in the biodegradation of a fossil fuel, particularly DszA and / or DszC. For example, the DNA encoding the oxidoreductase, under the control of a suitable promoter, can be transformed into IGTS8 or another microorganism capable of de-sulfurizing a fossil fuel. In another example, the DNA encoding the oxidoreductase is either simultaneous (eg, presented in a single plasmid or vector) or independently transformed into a host cell common with the DNA encoding the biocatalyst (s) or enzymes of desazuframiento. The DNA encoding the oxidoreductase may be, for example, under the control of the same or a different promoter than the DNA encoding the biocatalyst capable of de-sulfurizing the fossil fuel. In one embodiment, the oxidoreductase DNA is incorporated or ligated to the IGTS8 gene pool or desorption operon. The oxidoreductase is added to the reaction mixture in a rate enhancing amount. "Velocity enhancing amount", as defined herein, is an amount that will significantly increase the reaction rate of the biocatalyst, as originally obtained, including activation of the biocatalyst. For example, when the biocatalyst is IGTS8, a cell-free fraction or purified enzyme preparation thereof, a "rate enhancing amount" of oxidoreductase is an amount of oxidoreductase that, in addition to that inherently present in the biocatalyst as obtained , it will significantly increase the rate of desazuframiento. The rate of desorption can be increased, for example, by at least 25%, 50% or 100% compared to the speed using the biocatalyst per se. In one embodiment, the oxidoreductase is added to the reaction medium in an amount that reaches or approaches the saturation kinetics. The microorganism that harbors the DNA sequence encoding DszD can grow under conditions that maximize gene expression. The Rhodococcus species containing the gene can grow in the presence of an alcohol (such as ethanol, ethanolamine, glycerol or propanol), aldehydes (such as propionaldehyde), thiocarbamate or atrazine, for example. These compounds can induce or increase the expression of the gene in the microorganism. As summarized above, the invention described herein relates in one aspect to a DNA molecule or fragment thereof containing a gene or genes encoding an oxidoreductase and / or a biocatalyst capable of de-sulfurizing a fossil fuel containing organosulfur compounds. The DNA molecule or fragment thereof can be purified and isolated DNA obtained from, for example, a natural source, or it can be recombinant DNA (heterologous or foreign) that is, for example, present in a non-human host organism. The DNA can be isolated by well-known techniques, such as PCR, by designing oligonucleotide primers from the nucleotide sequence indicated in Figure 6. The recombinant DNA molecules of the present invention include DNA resulting from insertion into their chain, by chemical or biological means, of one or more genes coding for a biocatalyst capable of selectively excising the carbon-sulfur bonds and an oxidoreductase, said gene not being originally present in that chain. The recombinant DNA includes any DNA synthesized by methods that use restriction nucleases, nucleic acid hybridization, DNA cloning, DNA synthesis or any combination of the foregoing. Methods of construction can be found in Maniatis et al. and in other methods known to those skilled in the art. Methods for the construction of the DNA plasmids or vectors of the present invention include those described in Maniatis et al. and other methods known to those skilled in the art. The terms "DNA plasmid" and "vector" are intended to include any replication-competent plasmid or vector that is capable of having foreign or exogenous DNA inserted by chemical or biological means, and then when transformed into an appropriate host organism non-human, to express the product of the foreign or exogenous DNA insert (for example, to express the biocatalyst and oxidoreductase of the present invention). In addition, the plasmid or vector must be receptive to the insertion of a DNA molecule or fragment thereof containing the gene or genes of the present invention, whose gene or genes encode a biocatalyst, as defined above. Methods for the construction of plasmid vectors of DNA include those described in Maniatis et al. and other methods known to those skilled in the art. The plasmids of the present invention include any DNA fragment that contains a gene or genes encoding an oxidoreductase and / or a biocatalyst. The term "plasmid" is intended to include any DNA fragment. The DNA fragment must be transmissible, for example, to a host microorganism by transformation or conjugation. Methods for the construction or extraction of DNA plasmids include those described in Maniatis et al. and other methods known to those skilled in the art. The transformed non-human host organisms of the present invention can be created by various methods by those skilled in the art. For example, electroporation can be used, as explained by Maniatis et al. By the term "non-human host organism" is meant any non-human organism capable of capturing and expressing foreign, exogenous or recombinant DNA. Preferably, the host organism is a bacterium, more preferably a pseudomonas. In the biocatalytic desorption stage, the carbon-containing material or fossil fuel containing sulfur-bearing heterocycles is combined with the biocatalyst and the oxidoreductase. The relative amounts of the biocatalyst and oxidoreductase and the carbonaceous material, such as a fossil fuel, can be adjusted to suit particular conditions, or to produce a particular level of residual sulfur in the treated de-sulfur material. The amount of biocatalyst preparation to be combined with a given amount of substrate will reflect the nature, concentration and specific activity of the particular biocatalyst (s) and oxidoreductase used, as well as the nature and relative abundance of the biocatalyst (s). organic and inorganic sulfur compounds present in the substrate and the degree of desulphurization sought or considered acceptable. The method of desulfurization of a fossil fuel of the present invention involves two aspects. First, a host organism or biocatalytic preparation obtained therefrom and oxidoreductase are contacted with a fossil fuel to be de-sulfurized. This can be done in any suitable container, possibly equipped with a stirring or mixing device. The mixture is thoroughly mixed and allowed to incubate for a sufficient time to allow the excision of a significant number of carbon-sulfur bonds in the organosulfur compounds., thus producing a de-sulfurized fossil fuel. In one embodiment, an aqueous emulsion or microemulsion is produced with an aqueous culture of the organism or enzymatic fraction and the fossil fuel, allowing the organism to propagate in the emulsion, while the expressed biocatalyst excises the carbon-sulfur bonds.

Variables such as temperature, mixing speed and rate of desorption will vary according to the biocatalyst organism and / or oxidoreductase used. The parameters can be determined through only a routine experimentation. Several techniques for monitoring the speed and degree of desulfurization are well known and easily acquired by experts in this field. Basal and time course samples of the incubation mixture can be collected and can be prepared for a determination of the residual organic sulfur in the fossil fuel. The disappearance of the sulfur from the organosulfur compounds, such as DBT, in the sample that is being subjected to biocatalytic treatment can be monitored using, for example, X-ray fluorescence (FRX) or atomic emission spectrometry (flame spectrometry). Preferably, the molecular components of the sample are first separated, for example, by gas chromatography. The process and the biocatalytic compositions (including the recombinant microorganisms) of the claimed invention give rise to a significant and unexpected improvement on the desulphurization processes described above. It has been seen that, in vi tro, reactions catalyzed by purified DszA and DszC proteins are activated by the addition of oxidoreductase. This is particularly unexpected in view of recent discussions in the literature suggesting that FAD binds directly to DszC (Denome et al., J. Bacteriol., 176: 6707-6716, 1994) and that they suggest that NADH is the single cofactor required by the system (Ohshiro et al., FEMS Microbiol. Lett. 118: 341-344, 1994). Others suggest that DszABC are the only enzymes responsible for the occurrence of dessulfurization (Piddington et al., Appl. Env. Microbiol., 67: 468-475, 1995). Without being limited to any particular mechanism or theory, it is believed that the route of the desulphurization reaction is as indicated below: Here, it is believed that oxidoreductase is a short chain of electron transport that delivers the reducing equivalents of NADH (or another electron donor) to the enzymes, DszC and / or DszA (possibly, a physiological electron acceptor of oxidoreductase) . It is believed that the DszC enzyme is responsible for the biocatalysis of the oxidation reaction of DBT to DBT02. It is believed that the DszA enzyme is responsible for the oxygenation of DBT02 to phenol-enylsulfite (FFS). It is particularly preferred to add the cofactor, FMN, to the reaction medium, as well as an electron donor, NADH or NADPH. The addition of a regeneration system of NADH or NADPH to convert NAD + to NADH is also preferred, according to methods known in the art. The invention will now be further illustrated by the following examples. EXAMPLES Growth of Rhodococcus sp. IGTS8 A sample of frozen stock of Rhodococcus sp. IGTS8, strain CPE-648, containing plasmid pEN0K3 (genotype of DszA-B-C +) as described by Piddington et al. . { Appl. Environ. Microbiol. 61: 468-475 (1995)) in 500 ml of rich medium in a 2000 ml shake flask for 48 hours at 30 ° C. This culture was used to inoculate (4% inoculum) a NBS fermenter of 15 liters in the same medium. This culture was grown for 48 hours at 30 ° C, while controlling the pH (between 6.8 and 7.3), with stirring and dissolved oxygen (> 50% saturated). Finally, a 5% inoculum was transferred to a production-scale fermenter (Chemap 300 liters) containing half of basal salts, 0.5 g / L of Ivanhoe antifoam, 8 g / L of ethanol and dimethyl sulfoxide 1 , 5 mM. The culture was grown for 45 hours, reaching an optical density of 11, with a doubling time of 4.3 hours during the first 24 hours of the operation. The cell suspension was concentrated through a centrifugation, resulting in the production of approximately 2 hours., 5 kg of wet cell paste. The paste was stored at -70 ° C until its use for purification. Purification of DszD 150 g (wet cell paste) of Rhodococcus grown above in 25 mM NaPi, pH 7.5 (buffer A) containing 100 mM NaCl, 0.5 mM DTT, 1 mM PMSP and DNase were resuspended. The cell suspension was passed twice through a French pressure cell (at 20,000 psi) and then centrifuged at 30,000 x g for 45 minutes (5 ° C) to remove unruptured cells and cell debris. All subsequent chromatography steps were performed at 4 ° C using an FPLC system from Pharmacia. The supernatant was loaded onto a Q-sepharose column (2.6 cm x 20 cm) equilibrated with buffer A containing 100 mM NaCl. Following loading, the column was washed extensively with the same buffer until the OD 280 of the eluent was close to zero. The column with a linear gradient of 100 mM NaCl at 500 mM NaCl in buffer A was developed for 180 minutes, at a flow rate of 5 ml / minute, and fractions of 10 ml were collected. Fractions exhibiting DszD activity were pooled and dialysed overnight against buffer A. The dialysate was loaded on a Toyopearl DEAE-650M column (2.6 cm x 10 cm) equilibrated with buffer A. The column was developed with a linear gradient of 0 to 200 mM NaCl for 90 minutes, at a flow rate of 4 ml / minute, and fractions of 4 ml were collected. Fractions containing DszD activity were pooled and dialyzed overnight against buffer A. The dialysate was loaded onto a Pharmacia MonoQ column equilibrated with buffer A. The column was developed with a linear gradient of 160 to 300 mM NaCl for 30 minutes. minutes, at a flow rate of 0.5 ml / minute, and fractions of 0.5 ml were collected. Fractions showing DszD activity were pooled and concentrated to 0.2 ml using Amicon microconcentrators (molecular weight cut-off of 10 kDa). The concentrated sample was then applied to a size exclusion column Superdex 75 from Pharmacia equilibrated with buffer A containing 100 mM NaCl. The column was eluted with the same buffer at a flow rate of 0.2 ml / minute and fractions of 0.2 ml were collected. Fractions containing DszD activity were pooled and concentrated using the microconcentrators and the protein was stored on ice until use. Analysis by SDS-PAGE (14% polyacrylamide) of the final preparation showed a single band with a monomeric molecular weight of approximately 50,000 Da. Enzymatic tests The DszD activity was measured by monitoring the production of DBTO and DBT02 from DBT, catalyzed by the combination of DszC and DszD. The DszC was obtained from an expression system in E. coli, previously described. The assay (in 25 mM NaPi, pH 7.5, 100 mM NaCl and 0.5 mM DTT) contained DszC (between 6 and 15 μg), 3 mM NADH, 10 μM FMN, DBT 100 μM and the sample containing DszD. The test mixture was allowed to incubate at 30 ° C with agitation at 300 rpm for some period of time (typically 15 to 60 minutes). The reaction was stopped by the addition of acetonitrile (up to 50%) and the products were analyzed by reverse phase HPLC. Activation of DszA by DszD was studied in the same way (DszA was also obtained from an expression system in E. coli), except for the fact that the substrate was DBT sultone and the product was 2,2 '-dihydroxy-biphenyl (BHBF). Results: Purification of DszD Figure 1 shows the activity profile DszD of the fractions from the first anion exchange column (Q-Sepharose). As can be seen from these data, the activity starts around fraction 20 and extends to approximately fraction 60. The activation of DszA and C occurs in these reactions; moreover, the endogenous DszC activity is also present in these fractions (notably, fractions 40 to 50). Fractions 40 to 60 were pooled and re-separated in Toyopearl-DEAE. A pattern of activity similar to the Q-sepharose column was observed after chromatography on Toyopearl-DEAE, except for the fact that the activity eluted at a lower salt concentration and that the endogenous DszC activity appeared at later fractions (a small amount of activity in fraction 40). This was further substantiated by Western analysis, which showed that DszC elutes with a peak between fraction 45 and 50 (data not shown). Fractions 15 to 35 were collected and applied to the MonoQ column. Active fractions from this column were pooled, concentrated and re-separated by chromatography on a Superdex 75 FPLC column. The activity profile of this column is shown in Figure 2. This figure shows that both DszA and C are activated by protein (s) in the same fractions. Analysis of SDS-PAGE (Figure 3) showed that the final preparation consisted of a single polypeptide with a molecular weight of approximately 50,000. The HPLC analysis using a TSK3000SW size exclusion column from TosoHaas in a Hewlett Packard 1050 HPLC system showed a single protein peak, eluting at a mass of approximately 500,000 Da, indicating that the native protein is with the highest probability a decamer. Activation by DszD of DszC and DszA Figure 4 shows that the activation of DszC by DszD follows a saturation kinetics. By increasing the ratio between DszD and C, a higher rate of DBT02 formation is observed. A representation of the initial velocity versus DszD: DszC shows that saturation is reached. Figure 5 shows the result of the activation of DszA by the same preparation. The same effect is observed, that is, when adding more DszD, an increase in the reaction rate of DszA occurs. Amino acid sequence of DszD DszD was subjected to N-terminal sequence and the following sequence was obtained (one-letter amino acid abbreviations): H2N-AIELNQIWDFPIKEFHPFPRALMGVGAHDIIGVEAKNLGFKRTLLM-COOH (SEQ ID NO: 3) A search in the sequence databases resulted in a 100% match with a Rhodococcus protein termed ThcE (Nagy et al., Arch. Microbiol. 253: 439-446 (1995)). The DNA sequence and the putative amino acid sequences of the open reading frames are indicated in Figure 6. This protein has a high homology with alcohol: N, N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases found in other Gram-positive organisms that are involved in the oxidation of alcohols and in the concomitant reduction of an electron acceptor. The physiological electron acceptor in those organisms is unknown. EQUIVALENTS Those skilled in the art will know, or will be able to determine, using only routine experimentation, many equivalents of the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

Claims (33)

1. CLAIMS 1. A method for increasing the rate of bio-degradation of a fossil fuel containing organic sulfur molecules, consisting of the following steps: a) contacting the fossil fuel with an aqueous phase containing a biocatalyst capable of selectively excising the carbon-sulfur bonds and an additional amount enhancing the speed of a group III alcohol dehydrogenase, thus forming a mixture of fossil fuel and an aqueous phase; b) maintaining the mixture of stage a) under conditions sufficient for the excision of the carbon-sulfur bonds of the organic sulfur molecules by the biocatalyst, thus giving rise to a fossil fuel having a reduced content of organic sulfur, and c) Separate fossil fuel having a reduced organic sulfur content from the resulting aqueous phase.
2. 2. The method of Claim 1, wherein the alcohol dehydrogenase of group III is N, N'-dimethyl-4-nitrosoaniline-dependent oxidoreductase alcohol.
3. 3. The method of Claim 1, wherein the group III alcohol dehydrogenase is of Rhodococcus origin. Four . The method of Claim 3, which further comprises adding NADH or NADPH and flavin. 5. The method of Claim 4, wherein the fossil fuel is a liquid hydrocarbon. 6. The method of Claim 5, wherein the biocatalyst capable of excising carbon-sulfur bonds is a microorganism. The method of Claim 6, wherein the microorganism contains a recombinant DNA molecule that encodes one or more enzymes capable of selectively excising carbon-sulfur bonds. 8. The method of Claim 7, wherein the recombinant DNA molecule is derived from Rhodococcus sp. ATCC 53968. 9. The method of Claim 5, wherein the biocatalyst capable of excising carbon-sulfur bonds is a cell-free fraction. 10. The method of Claim 9, wherein the biocatalyst is a cell-free fraction of Rhodococcus sp. ATCC 53968. The method of Claim 5, wherein the biocatalyst consists of one or more enzymes or fractions of enzymes derived from a microorganism that has the ability to selectively excise carbon-sulfur bonds. 12. The method of Claim 11, wherein the microorganism is Rhodococcus sp. ATCC 53968. 13. The method of Claim 5, wherein the biocatalyst capable of excising carbon-sulfur bonds and the alcohol dehydrogenase group III are recombinantly produced by a single microorganism. 14. A DNA molecule consisting of DNA encoding a group III alcohol dehydrogenase and DNA encoding a biocatalyst capable of selectively excising the carbon-sulfur bond of an organic sulfur molecule. 15. The DNA molecule of Claim 14, wherein the oxidoreductase is N, N'-dimethyl-4-nitrosoaniline-dependent oxidoreductase alcohol. 16. The DNA molecule of Claim 14, wherein the group III alcohol dehydrogenase is of Rhodococcus origin. 17. The DNA molecule of Claim 16, wherein the DNA molecule encoding the biocatalyst is derived from Rhodococcus sp. ATCC 53968. 18. A microorganism containing a recombinant DNA molecule encoding: a) a group III alcohol dehydrogenase and b) one or more enzymes that catalyze one or more steps in the selective excision of a carbon-sulfur bond of a organic sulfur molecule. 19. The microorganism of Claim 18, wherein the group III alcohol dehydrogenase is N, N'-dimethyl-4-nitrosoanilone-dependent oxidoreductase alcohol. 20. The microorganism of Claim 18, wherein the DNA encoding group III alcohol dehydrogenase is of Rhodococcus origin. 21. The microorganism of Claim 20, wherein the DNA encoding one or more bio-wasting enzymes is derived from Rhodococcus sp. ATCC 53968. 22. A composition consisting of: a) a biocatalyst capable of selectively excising the carbon-sulfur bonds of organic sulfur molecules and b) an additional amount of a group III alcohol dehydrogenase. 23. The composition of Claim 22, wherein the oxidoreductase is N, N'-dimethyl-4-nitrosoaniline-dependent oxidoreductase alcohol. 24. The composition of Claim 22, wherein the DNA encoding group III alcohol dehydrogenase is of Rhodococcus origin. 25. The composition of Claim 24, wherein the biocatalyst is Rhodococcus sp. ATCC 53968 or enzymes thereof. 26. The composition of Claim 25, which further contains flavin and NAD or NADH. 27. A method for increasing the rate of selective oxidation of the sulfur atom of an organic sulfur compound, wherein said organic sulfur compound is a component of a carbonaceous material, consisting of the following steps: a) contacting the carbonaceous material with an aqueous phase containing a biocatalyst capable of selectively oxidizing the sulfur atom of an organic sulfur molecule and a rate enhancing amount of a group III alcohol dehydrogenase; b) maintaining the mixture of step a) under conditions sufficient for the oxidation of the sulfur atom of the organic sulfur compound by the biocatalyst. the organic sulfur compounds by the biocatalyst. The method of Claim 27, wherein the alcohol dehydrogenase of group III is N, N'-dimethyl-4-nitrosoaniline-dependent oxidoreductase alcohol. 29. The method of Claim 27, wherein the alcohol dehydrogenase group III is of Rhodococcus origin. 30. The method of Claim 29, wherein the biocatalyst is a monooxygenase. 31. The method of Claim 30, wherein the biocatalyst is a DszA or DszC. 32. The method of Claim 31, further comprising adding NADH or NADPH and flavin. 33. The method of Claim 32, wherein the sulfur-containing compound is a substituted or unsubstituted dibenzothiophene.