Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Utility Patent
1997-12-01
2001-01-02
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C435S320100, C435S325000, C435S252300, C435S128000, C435S109000, C536S023100, C536S023200, C536S024320
Utility Patent
active
06168940
ABSTRACT:
BACKGROUND OF THE INVENTION
This application claims priority from Japanese Patent Application No. 333018/1996, filed Nov. 29, 1996 and Japanese Patent Application 60077/1997, filed Feb. 28, 1997.
The present invention relates to a protein having ethylenediamine-N,N′-disuccinic acid: ethylenediamine lyase activity and a gene coding for the protein.
Diaminoalkylene-N,N′-disuccinic acids are not only important as intermediates for synthesis of medicines and agricultural chemicals but also unique in terms of having an ability to capture heavy metals. Also, since optical isomers of these compounds are expected to have a possibility of being susceptible to biodegradation when they have been released to the natural world, these compounds may be used as a chelating agent, a builder for detergent, etc.
Diaminoalkylene-N,N′-disuccinic acids can be synthesized easily from various amines and maleic or fumaric acid. However, in the case of optically active diaminoalkylene-N,N′-disuccinic acids, optically active aspartic acid or the like is needed as a starting material for the organosynthesis of such compounds. For example, optically active diaminoethylene-N,N′-disuccinic acid can be prepared from L-aspartic acid and dibromoethane [John A. Neal et al., Inorganic Chem. 7, 2405 (1968)]. However, L-aspartic acid and dibromoethane are relatively expensive raw materials and, thus, the product prepared from these materials cannot be supplied in various fields at a low cost.
On the other hand, diaminoalkylene-N,N′-disuccinic acids have also been isolated and identified from culture solutions of some actinomycetes [T. Nishikiori et al., J. Antibiotics 37, 426 (1984)]. However, the productivity of these actinomycetes is extremely low and, thus, a method of producing a diaminoalkylene-N,N′-disuccinic acid using such a bacterium is not practical in industry.
Under these circumstances, the inventor has previously proposed a novel method for efficiently producing optically active S,S-diaminoalkylene-N,N′-disuccinic acids from fumaric or maleic acid and various diamines by utilizing a catalytic action of microorganisms (Japanese Unexamined Patent Publication No. 9-140390; EP-0731171A; EP-0805211A).
It is an object of the present invention to further improve the catalytic action of the microorganisms.
SUMMARY OF THE INVENTION
The inventor has succeeded in improving the yield of a diaminoalkylene-N,N′-disuccinic acid in microorganism cells by cloning a gene for an ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase and allowing multiple copies of this gene to exist in cells of a microorganism by genetic recombination to thereby enhance the catalytic ability of the microorganism greatly. Thus, the present invention has been achieved.
The invention provides a DNA comprising a nucleotide sequence coding for a polypeptide having ethylenediamine-N,N′-disuccinic acid: ethylenediamine lyase activity, the polypeptide having the amino acid sequence of SEQ ID NO: 1 which may have a deletion, substitution or addition of at least one amino acid. The DNA comprising a nucleotide sequence coding for a polypeptide having ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase activity may be a DNA comprising a nucleotide sequence coding for a polypeptide having the amino acid sequence of SEQ ID NO: 1 or its analogue derived from the degeneracy of genetic codes. The nucleotide sequence coding for the polypeptide may have the nucleotide sequence of SEQ ID NO: 2.
In another aspect of the invention, the invention provides a DNA which hybridizes with the nucleotide sequence of SEQ ID NO: 2 or a fragment thereof and which comprises a nucleotide sequence coding for a polypeptide having ethylenediamine-N,N′-disuccinic acid: ethylenediamine lyase activity. Examples of the fragment of the nucleotide sequence of SEQ ID NO: 2 include a fragment of 326 bases ranging from nucleotide No. 176 to nucleotide No. 501 in the nucleotide sequence of SEQ ID NO: 2. The hybridization may be performed at 25-40° C. and at a formamide concentration of 10-50%. The conditions of washing may be appropriately determined depending on the detection method to be used.
Furthermore, the invention also provides a DNA fragment of 200-350 bp which can be amplified with synthetic DNAs consisting of the nucleotide sequences of SEQ ID NOS: 4 and 5, respectively. This DNA fragment can be used for obtaining a gene analogue to the ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase gene or for judging whether a gene obtained is identical with the ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase gene or not.
The invention also provides a recombinant plasmid containing the DNA comprising a nucleotide sequence coding for a polypeptide having ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase activity.
The invention further provides a host transformed with the recombinant plasmid. The host may be a microorganism such as
E. coli
. The transformed host can be used to produce diaminoalkylene-N,N′-disuccinic acid and, thus, the invention also provides a method for producing diaminoalkylene-N,N′-disuccinic acid using the transformed host.
Moreover, the invention provides a polypeptide having ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase activity which has the amino acid sequence of SEQ ID NO: 1 that may have a deletion, substitution or addition of at least one amino acid. The deletion, substitution or addition can be performed by a well-known technique, site-specific mutagenesis (see, for example, Nucleic Acid Research, Vol. 10, No. 20, pp. 6487-6500, 1982).
The cloned ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase gene can be introduced in multiple copies into microorganism cells by genetic recombination. Thus, it is possible to enhance the catalytic ability of the microorganism greatly to thereby improve the yield of a diaminoalkylene-N,N′-disuccinic acid.
REFERENCES:
patent: 5874262 (1999-02-01), Z{umlaut over (a)}hner et al.
patent: WO 9636725 (1996-11-01), None
Nishikiori, T., et al., Production by Actinomycetes of (S,S)-N, N′-Ethylenediaminedisuccinicacid, an Inhibitor of Phospholipase C, Journal of Antiboitics, vol. 37, No. 1, pp. 426-427 (1984).
Hoyer, L., et al., The ARG4 gene ofCandida albicans, Gene, vol. 142, pp. 213-218 (1994).
Schmidt, G. et al., GenBank Database, Accession No. X56135, Jul. 26, 1991.*
Achutamurthy Ponnathapu
Davidson Davidson & Kappel LLC
Nitto Chemical Industry Co. Ltd.
Tung Peter P.
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