Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-12-08
2002-08-27
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S232000, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06440704
ABSTRACT:
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
This invention relates to the isolation of a nucleic acid sequence that encodes an enzyme capable of removing carboxyl groups from aromatic rings. In particular the enzyme decarboxylates 2,3-dihydroxybenzoic acid to form catechol.
The decarboxylation reaction involves the non-oxidative removal of a carboxyl group from an aromatic ring. Aromatic rings containing a carboxyl group are chemically challenging to work with because the carboxyl group is a deactivating group. Deactivating groups make electrophilic substitutions on aromatic rings difficult. Therefore, the removal of a deactivating carboxyl group from an aromatic ring has a great deal of potential in the chemical industry.
Catechol is an aromatic compound utilized in the development of pharmaceuticals such as L-DOPA(L-3,4-dihydroxyphenylalanine) and adrenaline, agrobiochemicals such as carbofuran, and antioxidants such as 4-tert-butyl catechol and veratrol. Additionally, catechol is utilized in the production of flavorants such as vanilla and polymerization inhibitors. The current global noncaptive market for catechol is 20.5×10
6
Kg/yr.
Current commercial production of aromatics has several drawbacks. One disadvantage relates to the starting material utilized in aromatic production. Most aromatics are synthesized from benzene, toluene and xylene which are derived from petroleum or natural gas fossil fuel feedstocks. For example, catechol is currently produced by distillation of coal tar or the hydroxylation of phenol. Both of these methods require fossil fuels as starting material. Fossil fuels are nonrenewable and therefore more expensive than renewable resources. In addition, many countries do not have a large national supply of fossil fuels for the derivation of aromatic compounds.
In addition to cost, the use of fossil fuels has a negative impact on the environment. Petroleum based processes for the production of aromatics often require hazardous starting materials. One example of a hazardous starting material is benzene, which is a carcinogen. These processes also produce hazardous waste by-products that can inadvertently leak into the environment. Hazardous waste by-products also need to be disposed of or stored, adding to the costs of operation.
Another problem with current aromatic compound production is that the synthetic procedures involve harsh reaction conditions. Current methods for the removal of carboxyl groups from aromatic rings involve high temperatures and acid conditions in the presence of metal catalysts. These extreme reaction conditions are expensive energy consuming procedures that pose industrial and environmental safety concerns and dramatically increase the cost of the aromatics.
Therefore, a method for the production of aromatics, and specifically catechol, is needed to overcome the problems associated with current methods utilized in the production of aromatics.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide an alternative method for the removal of carboxyl groups form aromatic rings. In particular, it is an object of the present invention to provide a method for the decarboxylation of 2,3-dihydroxybenzoic acid to form catechol. Specifically, the invention provides an isolated nucleic acid sequence encoding a stable enzyme that will catalyze the non-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catechol.
Another object of the present invention is to provide an isolated nucleic acid sequence encoding a protein that catalyzes the non-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catechol and that hybridizes, under stringent conditions, to SEQ ID NO:1. SEQ ID NO:1 comprises a nucleic acid sequence that encodes
Aspergillus niger
2,3-dihydroxybenzoic acid decarboxylase.
Yet another object of the present invention is to provide an isolated nucleic acid sequence encoding a protein that catalyzes the non-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catechol and that hybridizes under stringent conditions to a nucleic acid sequence corresponding to an amino acid sequence of SEQ ID NO:2.
A further object of the present invention is to provide fragments of the nucleic acid sequence encoding 2,3-dihydroxybenzoic acid decarboxylase that hybridize to SEQ ID NO: 1 and that code for products that maintain biological activity necessary to decarboxylate 2,3-dihydroxybenzoic acid. These fragments can be either recombinant or synthetic or a combination thereof.
A further object of the present invention is to provide a recombinant vector comprising a nucleic acid sequence encoding a protein that catalyzes the non-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catechol. The definition of a vector for the purposes of this invention is any nucleic acid sequence into which a foreign nucleic acid sequence may be inserted wherein the nucleic acid sequence containing the foreign nucleic acid sequence may be used to introduce the foreign nucleic acid sequence into a host cell. This vector may also comprise regulatory elements operably linked to the nucleic acid sequence.
A further object of the present invention is to provide various cells transformed with the vector comprising a nucleic acid sequence encoding a protein that catalyzes the non-oxidative decarboxylation of 2,3-dihydroxybenzoic acid to catechol.
A further object of the present invention is to provide methodology for the production of the various products of the present invention. Examples include isolated nucleic acid sequences encoding 2,3-dihydroxybenzoic acid decarboxylase, isolated 2,3-dihydroxybenzoic acid decarboxylase, isolated SEQ ID NO:1, isolated SEQ ID NO:2, and the protein product of SEQ. ID. NO.:1.
A further object of the present invention is to illustrate the use of an enzyme for the synthesis of catechol and the decarboxylation of aromatic acids. This approach reduces environmental concerns associated with traditional methods for the production of aromatics.
2,3-dihydroxybenzoic acid decarboxylase of the present invention is suited to accomplish these and other related objects of the present invention by catalyzing a reaction whereby a carboxyl group is removed from an aromatic ring. Specifically, a carboxyl group is removed from 2,3-dihydroxybenzoic acid to produce catechol and carbon dioxide. This decarboxylation is unusual in that it involves a non-oxidative decarboxylation from an aromatic nucleus and does not require a cofactor. This enzyme is found in the fungus
Aspergillus niger
and functions in the pathway for the metabolism of indole. This enzyme is listed in the Enzyme Commission by the no. EC.4.1.1.46
Aspergillus niger
is a known fungus and is readily available to those with ordinary skill in the art. Additionally,
Aspergillus niger
has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. The deposit was filed with ATCC on Aug. 10, 1998. The culture is identified as
Aspergillus niger
CSVInd and by the ATCC Accession No. 74460. Additionally, the pET22b(+)DHBD vector, as described in this specification, has been deposited with the ATCC, 10801 University Blvd., Manassas, Va. 20110-2209. The deposit was filed with ATCC on Aug. 10, 1998. The vector is identified as pET22b(+)DHBD and by the ATCC Accession No. 203104. These deposited materials are available pursuant to all requirements of the United States Patent and Trademark Office.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows and in part will be apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention.
REFERENCES:
patent: 5272073 (1993-12-01), Frost et al.
patent: 5616496 (1997-04-01), Frost et al.
patent: 5629181 (1997-05-01), Frost et al.
patent: 6043076 (2000-03-01), Ramakrishnan
Lee, C. et al., Science, vol. 239, pp. 1288-1291, Mar. 1988.*
Mierendorf, R. et
Board of Regents of University of Nebraska
Nashed Nashaat T.
Shook Hardy & Bacon LLP
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