Mannose isomerase and DNA encoding the enzyme

Details Mannose isomerase and DNA encoding the enzyme Mannose isomerase and DNA encoding the enzyme

2001-10-11

2004-01-06

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S105000, C435S233000, C435S252300, C536S023200

Reexamination Certificate

active

06673581

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a protein having mannose isomerase activity, DNA encoding the protein, a recombinant DNA comprising the DNA, a transformant carrying the recombinant DNA, a process for producing mannose isomerase by using the transformant, and processes for producing fructose, mannose, xylulose and lyxose by using the transformant.
BACKGROUND ART
Mannose isomerase is known to be present in microorganisms belonging to the genera Pseudomonas [J. Biol. Chem., 218, 535 (1956); Japanese Published Unexamined Patent Application No. 292578/94; U.S. Pat. No. 5,124,262 (1992)], Mycobacterium [J. Bacteriol., 101, 777 (1970)], Xanthomonas [Agric. Biol. Chem., 28, 605 (1964)], Streptomyces [Agric. Biol. Chem., 31, 435 (1967)], Acinetobacter (Japanese Published Unexamined Patent Application No. 9986/96) and Escherichia [J. Gen. Microbiol., 97, 257 (1976)]. However, DNA encoding this enzyme has not yet been isolated from any of these microorganisms.
Mannose isomerase derived from
Escherichia coli
has been purified and its properties have been studied [J. Gen. Microbiol., 124, 219 (1981)], but its amino acid sequence has not been analyzed yet. The nucleotide sequence of the chromosomal DNA of
Escherichia coli
has been determined [Science, 277, 1453 (1997)], and on the basis of the database of Yale University,
E. coli
Genetic Stock Center, the mannose isomerase gene is assumed to be present at around 87.6 minutes on the chromosome. However, the gene has not been specified or isolated.
With regard to the production of carbohydrates by utilizing mannose isomerase, there are known processes for producing mannose by utilizing mannose isomerase derived from a bacterium of the genus Pseudomonas (Japanese Published Unexamined Patent Application No. 292587/94) and mannose isomerase derived from a bacterium of the genus Acinetobacter (Japanese Published Unexamined Patent Application No. 9986/96). These processes, however, require a long reaction time because of low enzyme activity per cell, and thus are not suitable for industrial production of mannose.
So far, no process has been known for efficiently producing carbohydrates by utilizing mannose isomerase.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a protein having mannose isomerase activity, DNA encoding the protein, a process for producing a protein having mannose isomerase activity by using the DNA, and efficient processes for producing fructose, mannose, xylulose and lyxose by using the protein.
The present inventors made an intensive investigation to attain the object. As a result, they have succeeded in isolating DNA encoding mannose isomerase from a microorganism belonging to the genus Escherichia, and have found it possible to efficiently produce carbohydrates such as mannose by utilizing the isolated DNA. The present invention has been completed on the basis of this result.
The present invention relates to the following (1)-(14).
(1) A protein consisting of an amino acid sequence wherein one or more amino acid residues are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having mannose isomerase activity. The protein comprising an amino acid sequence wherein one or more amino acid residues are deleted, substituted or added and having mannose isomerase activity can be produced by site-directed mutagenesis described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as Molecular Cloning, Second Edition); Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter referred to as Current Protocols in Molecular Biology); Nucleic Acids Research, 10, 6487 (1982); Nucleic Acids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Proc. Natl. Acad. Sci. USA, 82, 488 (1985); Gene, 34, 315 (1985), etc.
Examples of the proteins produced by the above method are proteins obtained by introducing a site-directed mutation into DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 1.
The number of amino acid residues which are deleted, substituted or added is not specifically limited, but is preferably within the range of one to several decades, more preferably one to several.
(2) A DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(3) A DNA encoding a protein comprising an amino acid sequence wherein one or more amino acid residues are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having mannose isomerase activity.
(4) A DNA having the nucleotide sequence shown in SEQ ID NO: 2.
(5) A DNA which hybridizes to the DNA according to any of the above (2)-(4) under stringent conditions and which encodes a protein having mannose isomerase activity.
The expression “DNA which hybridizes under stringent conditions” as used herein refers to DNA which is obtained by colony hybridization, plaque hybridization, Southern hybridization and so on using the DNA according to any of the above (2)-(4) as a probe. Such DNA can be identified, for example, by performing hybridization at 65° C. in the presence of 0.7-1.0 M NaCl using a filter with colony- or plaque-derived DNA immobilized thereon and then washing the filter at 65° C. using 0.1 to 2-fold concentrated SSC solution (1-fold concentrated SSC solution: 150 mM sodium chloride and 15 mM sodium citrate).
Hybridization can be carried out according to the methods described in laboratory manuals such as Molecular Cloning, Second Edition; Current Protocols in Molecular Biology; and DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995). The hybridizable DNA is, for example, a DNA having at least 80% homology, preferably 95% or more homology to the nucleotide sequence shown in SEQ ID NO: 2.
(6) A recombinant DNA which is obtained by inserting the DNA according to any of the above (2)-(5) into a vector.
(7) A transformant which is obtained by introducing the recombinant DNA according to the above (6) into a host cell.
(8) The transformant according to the above (7) which is
Escherichia coli.
(9) A process for producing a protein having mannose isomerase activity, which comprises culturing the transformant according to the above (7) or (8) in a medium, allowing the protein having mannose isomerase activity to form and accumulate in the culture, and recovering the protein from the culture.
(10) A process for producing fructose, which comprises allowing a culture of a transformant obtained by introducing DNA encoding a protein having mannose isomerase activity or a treated matter thereof as an enzyme source and mannose to be present in an aqueous medium, allowing fructose to form and accumulate in the aqueous medium, and recovering fructose from the aqueous medium.
(11) A process for producing mannose, which comprises allowing a culture of a transformant obtained by introducing DNA encoding a protein having mannose isomerase activity or a treated matter thereof as an enzyme source and fructose to be present in an aqueous medium, allowing mannose to form and accumulate in the aqueous medium, and recovering mannose from the aqueous medium.
(12) A process for producing xylulose, which comprises allowing a culture of a transformant obtained by introducing DNA encoding a protein having mannose isomerase activity or a treated matter thereof as an enzyme source and lyxose to be present in an aqueous medium, allowing xylulose to form and accumulate in the aqueous medium, and recovering xylulose from the aqueous medium.
(13) A process for producing lyxose, which comprises allowing a culture of a transformant obtained by introducing DNA encoding a protein having mannose isomerase activity or a treated matter thereof as an enzyme source and xylulose to be present in an aqueous medium, allowing lyxose to form and accumulate in the aqueous medium, and recovering lyxose from the aqueous medium.
(14) The process according to any o

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