Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2001-01-12
2002-01-22
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S189000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06340579
ABSTRACT:
TECHNICAL FIELD
This invention relates to &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase as an enzyme which converts &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde into 2-pyrone-4,6-dicarboxylic acid, a gene encoding this enzyme, a recombinant vector containing this enzyme, and a transformant carrying this gene; and also to a process for producing the enzyme and 2-pyrone-4,6-dicarboxylic acid by using the enzyme.
BACKGROUND ART
&agr;-Hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase is an enzyme which converts &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde into 2-pyrone-4,6-dicarboxylic acid, and plays an important role in the production of 2-pyrone-4,6-dicarboxylic acid useful as raw materials for polymers. Therefore, it is extremely important for the production of 2-pyrone-4,6-dicarboxylic acid to determine the gene of &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase and further to mass-produce &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase encoded by the gene.
On the other hand, remarkable developments have been achieved in recent years on recombinant DNA technology as mass production technology for proteins such as enzymes, resulting in successful mass production of numerous enzymes, physiologically-active proteins and the like by using recombinant DNA technology. Concerning &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase, however, the gene which encodes the enzyme has not been isolated yet.
An object of the present invention is to provide a technique for mass-producing 2-pyrone-4,6-dicarboxylic acid by isolating the gene of &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase and then enhancing the activity of &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semi-aldehyde dehydrogenase encoded by the gene.
DISCLOSURE OF THE INVENTION
The present inventors have proceeded with extensive research on microbiological production of 2-pyrone-4,6-dicarboxylic acid. As a result, the present inventors have succeeded in isolating, from a microorganism which contains &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase that converts &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semi-aldehyde into 2-pyrone-4,6-dicarboxylic acid, the gene of &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase by using recombinant DNA technology and further in determining its base sequence. In addition, the present inventors have also found a process for forming a recombinant vector of the gene and a transformant carrying the gene and then producing the enzyme and 2-pyrone-4,6-dicarboxylic acid, leading to the completion of the present invention.
The present invention, therefore, provides &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or an amino acid sequence with one or more amino acids deleted, substituted or added in the first-mentioned amino acid sequence, a gene encoding the enzyme, a recombinant vector containing the gene, and a transformant carrying the gene.
The present invention also provides a process for producing 2-pyrone-4,6-dicarboxylic acid, which comprises cultivating the transformant in the presence of &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde.
Further, the present invention also provides a process for producing the enzyme, which comprises cultivating the transformant and collecting &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase from the resulting culture.
Use of the transformant according to the present invention permits production of &agr;-hydroxy-&ggr;-carboxymuconic acid-&egr;-semialdehyde dehydrogenase in a large amount, thereby making it possible to perform industrial production of 2-pyrone-4,6-dicarboxylic acid.
REFERENCES:
Noda, Y, et al. (1990) J. Bacteriol. 172(5), 2704-2709.*
Yrjala, K., et al. (1997) FEMS Microbiol. Lett. 154, 403-408.*
Riegert, U., et al. (1998) J. Bacteriol. 180(11), 2849-2853.*
Masai, E., et al. (1999) J. Bacteriol 181(1), 55-62.
Fukuda Masao
Hotta Yasushi
Katayama Yoshihiro
Masai Eiji
Nishikawa Seiji
Cosmo Research Institute
Patterson Jr. Charles L.
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