Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Petroleum oil or shale oil treating
Patent
1996-02-27
1998-12-08
Prouty, Rebecca E.
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Petroleum oil or shale oil treating
435281, 435189, 4352523, 4353201, 536 232, 935 22, C12N 902, C12N 120, C10G 3200, C07H 2104
Patent
active
058468137
DESCRIPTION:
BRIEF SUMMARY
This application is the national stage of PCT/US95/15864, filed Dec. 5, 1995, which claims priority to U.S. provisional application 60/004,105, filed Sep. 21, 1995.
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 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 desulfurization of fossil fuels. Neither DszC nor A are enzymatically active when purified to homogeneity; however, upon the addition of this additional protein (designated DszD herein), enzymatic activity is restored. The function of this protein is believed to couple the oxidation of NADH with 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 upon sequence similarity, ThcE appears to be a member of the group III alcohol dehydrogenases, or oxidoreductases, designated alcohol: N,N'-dimethyl-3-nitrosoaniline oxidoreductases. DszD has a monomer molecular weight of approximately 50,000 (by SDS-PAGE) but behaves as a multimeric protein (decamer) on HPLC size exclusion chromatography. The activation of DszC and A by DszD follows saturation kinetics.
Thus, the invention relates to the discovery that the rate of microbial desulfurization of fossil fuels is enhanced or activated by or dependent upon the addition of an oxidoreductase to the biocatalyst or reaction medium. The invention is drawn to a method for enhancing the rate of desulfurizing a fossil fuel containing organic sulfur compounds, comprising the steps of: biocatalyst or biocatalysts capable of cleaving carbon-sulfur bonds (such as Dsz A, Dsz B and/or Dsz C) and a rate-enhancing amount of an oxidoreductase, thereby forming a fossil fuel and aqueous phase mixture; 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 the resulting aqueous phase.
The invention also relates to enhancing the rate of the reaction catalyzed by DszA and/or DszC with a rate enhancing amount of oxidoreductase. This can be accomplished, for example, by adding the oxidoreductase to a biocatalyst or by causing 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 which enc
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Gray Kevin A.
Monticello Daniel J.
Squires Charles H.
Energy BioSystems Corporation
Prouty Rebecca E.
Stole Einar
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