Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
1999-08-31
2001-04-10
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S233000, C435S252330, C536S023700, C536S023200
Reexamination Certificate
active
06214590
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a 2-aminothiazoline-4-carboxylate racemase gene, a recombinant DNA, a transformant or transductant, a 2-aminothiazoline-4-carboxylate racemase enzyme, a process for preparing the enzyme, and a process for preparing L-cysteine or L-cystine using the enzyme.
2. Prior Art
Bacteria belonging to the genus Pseudomonas are known as a representative of microorganisms converting DL-2-aminothiazoline-4-carboxylate, hereinafter abbreviated as DL-ATC, into L-cysteine (Japanese Patent Application Laid-open Publication No. 51-70881), and the cells thereof have hitherto used in the synthesis reactions of L-cysteine (Japanese Patent Publication No. 53-25037). Racemization of DL-ATC is required for efficient conversion of DL-ATC into L-cysteine and enzymatic racemization of DL-ATC is considered to take place in the above mentioned bacteria. However, these conventional bacteria only produce insufficient amounts of racemase and the efficiency of producing L-cysteine from starting DL-ATC is poor. Further, there has been no report on isolation of the above mentioned enzyme or the cloning of its gene.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a 2-aminothiazoline-4-carboxylate racemase gene, a process for preparing the 2-aminothiazoline-4-carboxylate racemase enzyme by expressing the gene, and a process for preparing L-cysteine or L-cystine using said enzyme.
In order to achieve such an object, the present inventors have made great efforts to enhance amounts of desired enzyme produced by cloning a racemase enzyme gene from bacteria possessing the enzyme and enhancing its genetic amplification, transcription and translation activities. Thus, the present invention has been completed.
Accordingly, the present invention provides a DNA fragment comprising a 2-aminothiazoline-4-carboxylate racemase gene and having the following restriction enzyme cleavage map and 4.8 kilo base pairs (kb):
Also, the present invention provides a 2-aminothiazoline-4-carboxylate racemase gene coding for the following protein (a) or (b):
(a) a protein comprising the amino acid sequence as shown in SEQ ID NO:2; or
(b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted from, replaced in or added to the amino acid sequence (a), and having a 2-aminothiazoline-4-carboxylate racemase activity.
An example of the above mentioned gene includes the gene having a base sequence of from 191st to 937th bases of SEQ ID NO:1.
Further, the present invention provides a recombinant DNA comprising the 2-aminothiazoline-4-carboxylate racemase gene or a DNA fragment of the gene in a vector DNA.
Still further, the present invention provides a transformant or transductant comprising the above mentioned recombinant DNA.
According to the present invention, a process for preparing a 2-aminothiazoline-4-carboxylate racemase is also provided which comprises cultivating the above mentioned transformant or transductant in a culture medium and collecting the 2-aminothiazoline-4-carboxylate racemase from the culture medium.
Further provided is a 2-aminothiazoline-4-carboxylate racemase comprising the following protein (a) or (b):
(a) a protein comprising the amino acid sequence as shown in SEQ ID NO:2; or
(b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted from, replaced in or added to the amino acid sequence (a), and having a 2-aminothiazoline-4-carboxylate racemase activity.
According to the present invention, there is further provided a process for preparing L-cysteine or L-cystine comprising contacting a DL-2-aminothiazoline-4-carboxylate with the 2-aminothiazoline-4-carboxylate racemase of claim
2
or
7
to produce L-cysteine or L-cystine.
REFERENCES:
Ryu et al. The stability of L-ATC hydrolase participating in the L-cysteine production. Biotechnol. Lett. 1995, vol. 17, pp. 275-280.*
Nippon Nogeikagaku Kaishi, Mar. 5, 1998, Japan.
Fronda Christian L.
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Nashed Nashaat T.
Shiba Toshikazu
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