Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Petroleum oil or shale oil treating
Reexamination Certificate
1999-05-03
2001-08-14
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Petroleum oil or shale oil treating
C435S189000, C435S252300, C435S252330, C435S281000, C536S023200
Reexamination Certificate
active
06274372
ABSTRACT:
BACKGROUND OF THE INVENTION
The microbial desulfurization of fossil fuels has been an area of active investigation for over fifty years. The object of these investigations has been to develop biotechnology based methods for the pre-combustion removal of sulfur from fossil fuels, such as coal, crude oil and petroleum distillates. The driving forces for the development of desulfurization methods are the increasing levels of sulfur in fossil fuel and the increasingly stringent regulation of sulfur emissions. Monticello et al., “Practical Considerations in Biodesulfurization of Petroleum,” IGT's 3d Intl. Symp. on Gas, Oil, Coal and Env. Biotech., (Dec. 3-5, 1990) New Orleans, La.
Many biocatalysts and processes have been developed to desulfurize fossil fuels, including those described in U.S. 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. Economic analyses indicate that one limitation in the commercialization of the technology is improving the reaction rates and specific activities of the biocatalysts, such as the bacteria and enzymes that are involved in the desulfurization reactions. The reaction rates and specific activities (sulfur removed/hour/gram of biocatalyst) that have been reported in the literature are much lower than those necessary for optimal commercial technology. Therefore, improvements in the longevity and specific activity of the biocatalyst are desirable.
SUMMARY OF THE INVENTION
The invention relates to the isolation and characterization of a novel NADH:FMN oxidoreductase (FMN\FMNH
2
=oxidize\reduced flavin mononucleotide; NAD
+
\NADH=oxidize\reduced nicotinamide adenine dinucleotide) from the bacterium Rhodococcus strain sp. IGTS8 (ATCC 53968), and the observation that this enzyme activates two monooxygenases, DszA and DszC, involved in the desulfurization of fossil fuels. Neither DszA nor DszC is active when purified, but enzymatic activity is restored upon addition of this novel enzyme. It is believed that this enzyme serves to couple the oxidation of NADH to substrate oxygenation by catalyzing the reaction:
FMN+NADH→FMNH
2
+NAD
+
.
Subsequently, FMNH
2
interacts with O
2
to form an activated oxygen species which is then transferred to dibenzothiophene (DBT) by DszC and to dibenzothiophene-5,5-dioxide (DBTO
2
) by DszA.
The invention is thus drawn to the isolated NADH:FMN oxidoreductase (also referred to herein as ‘flavin reductase’) and isolated homologues and fragments thereof. The novel flavin reductase of the present invention has been purified to homogeneity and has an apparent monomeric molecular weight of 25 kDa. The enzyme contains no tightly bound flavin cofactor, but appears to utilize flavin mononucleotide (FMN) as a dissociable substrate. The enzyme activates the monooxygenase enzymes DszA and DszC in a process displaying saturation kinetics.
In another embodiment, the enzyme includes an isolated nucleotide sequence, such as a DNA or RNA sequence or molecule, encoding the novel flavin reductase, or a homologue or fragment thereof. The invention also includes a recombinant microorganism containing one or more recombinant nucleic acid molecules which encode the novel flavin reductase. The microorganism can further comprise one or more recombinant nucleic acid molecules which encode a biocatalyst capable of catalyzing one or more steps in a process for desulfurizing a fossil fuel containing organic sulfur molecules.
In a further embodiment, the invention provides a method of using the novel flavin reductase in the biocatalytic desulfurization of a fossil fuel containing organic sulfur compounds. The method comprises the steps of (1) contacting the fossil fuel with an aqueous phase containing a biocatalyst capable of cleaving carbon—sulfur bonds (such as DszA, DszB and/or DszC) and a rate-enhancing amount of the flavin reductase of the present invention, thereby forming a fossil fuel and aqueous phase mixture; (2) maintaining the mixture under conditions sufficient for cleavage of the carbon—sulfur bonds of the organic sulfur molecules by the biocatalyst, thereby resulting in a fossil fuel having a reduced organic sulfur content; and (3) separating the fossil fuel having a reduced organic sulfur content from the resulting aqueous phase.
The invention also relates to a method of enhancing the rate of biodesulfurization of a carbonaceous material containing organosulfur compounds. The method comprises the step of adding the flavin reductase to a biodesul-furization system in a rate-enhancing amount. This can be accomplished by adding the flavin reductase to the biocatalyst or by causing the expression or overexpression of the flavin reductase in a microorganism which serves as a biodesulfurization catalyst.
The invention also relates to a composition for use in biodesulfurization of a carbonaceous material containing organosulfur molecules, such as a fossil fuel. The composition comprises (a) a biocatalyst capable of desulfurizing a fossil fuel containing organic sulfur molecules and (b) the flavin reductase.
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Childs John D.
Gray Kevin A.
Squires Charles H.
Enchira Biotechnology Corporation
Hamilton Brook Smith & Reynolds P.C.
Nashed Nashaat T.
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