Novel thermostable gluconate dehydratase and use thereof

Abstract

The present invention relates to a novel thermostable gluconate dehydratase from the thermoacidophilic archaeon Sulfolobus solfataricus , a coding sequence, and an expression system. The gluconate dehydratase has a molecular weight of about 320,000 to 380,000 daltons as the native protein, and about 40,000 to 50,000 daltons as the monomer protein, and catalyzes the dehydration reaction of aldonic acids to 2-keto-3-deoxy derivatives at temperatures of less than 120° C. The gluconate dehydratase can be produced from native or recombinant host cells and thereby used in the pharmaceutical, agricultural, and other industries.

1 . An isolated or purified polynucleotide encoding a gluconate dehydratase, wherein the gluconate dehydratase comprises a polynucleotide having at least a 50% identity to a nucleic acid sequence encoding a polypeptide comprising amino acid sequences of SEQ ID NO:2, or a polynucleotide complementary to the polynucleotide having at least a 50% identity to a polynucleotide encoding an polypeptide comprising amino acid sequences of SEQ ID NO:2. 
     
     
         2 . The polynucleotide according to  claim 1 , wherein the polynucleotide is DNA. 
     
     
         3 . The polynucleotide according to  claim 1 , wherein the polynucleotide is RNA. 
     
     
         4 . The polynucleotide according to  claim 1 , wherein the polynucleotide comprises nucleotide sequences of SEQ ID NO:1 
     
     
         5 . A polypeptide comprising an amino acid sequence which is at least 50% identical to amino acid sequences of SEQ ID NO:2, wherein the polypeptide catalyzes dehydration of aldonic acid to 2-Keto-3-deoxy aldonic acid. 
     
     
         6 . An expression construct comprising the polynucleotide of  claim 2 , wherein the polynucleotide is operably linked to and under the regulatory control of a transcription and translation regulatory sequence. 
     
     
         7 . An organism transformed with an expression construct according to  claim 6 . 
     
     
         8 . The organism according to  claim 7 , wherein the organism is selected from the group consisting of a prokaryote, a eukaryotic cell, and a cell derived thereof. 
     
     
         9 . The organism according to  claim 7 , wherein the organism is  Escherichia coli  BL21(DE3)/pGNH (KCTC10619BP). 
     
     
         10 . A method for preparing a protein, comprising:
 (a) preparing a vector comprising a polynucleotide of  claim 1 , operably linked to and under the regulatory control of a transcription and translation regulatory sequence;   (b) introducing the vector into a host cell and selecting a transformant expressing the protein;   (c) culturing the transformant under a condition which permits the protein to be expressed; and   (d) purifying the protein from the cultures,   
       wherein the protein catalyzes a dehydration of aldonic acid to 2-keto-3-deoxy aldonic acid. 
     
     
         11 . A method of preparing an organism expressing a protein, comprising:
 (a) preparing a vector comprising a polynucleotide of  claim 1 , operably linked to and under the regulatory control of a transcription and translation regulatory sequence;   (b) introducing the vector into a host cell; and   (c) selecting a transformant expressing the protein,   
       wherein the protein catalyzes dehydration of aldonic acid to 2-keto-3-deoxy aldonic acid. 
     
     
         12 . A method of purifying a gluconate dehydratase, comprising:
 conducting chromatography of a culture solution or a cell from a gluconate dehydratase producing microorganism through a column packed with resin attached to one or more kinds of functional groups selected from the group consisting of carboxy, carboxymethyl, sulpho, sulphomethyl, sulphoprophyl, aminoethyl, diethylaminoethyl, trimethylaminomethyl, triethylaminoethyl, dimethyl-2-hydroxyethylaminomethyl, diethyl-2-hydroxypropylaminoethyl, phospho, alkyl and hydroxylapatite, and   
       wherein the matrix of the resin is selected from the group consisting of agarose, cellulose, dextran, polyacrylate, and polystyrene. 
     
     
         13 . The method according to  claim 12 , wherein the microorganism is thermoacidophilic archaea species. 
     
     
         14 . The method according to  claim 12 , wherein the microorganism belongs to  Sulfolobus  genus. 
     
     
         15 . The method according to  claim 12 , wherein the microorganism is selected from the group consisting of  Sulfolobus solfataricus, Sulfolobus acidocaldarius, Sulfolobus shibatae, Sulfolobus tokodaii, Sulfolobus metallicus, Sulfolobus hakonensis, Sulfolobus brierleyi, Sulfolobus islandicus, Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus yangmingensis, Sulfolobus  sp.,  Thermoplasma acidophilum, Thermoplasma volcanium, Ferroplasma acidophilum , and  Sulfolobus  strains AMP12/99, CH7/99, FF5/00, MV2/99, MVSoil3/SC2, NGB23/00, NGB6/00, NL8/00, NOB8H2, RC3, RC6/00, or RCS1/01. 
     
     
         16 . A method for producing a 2-keto-3-deoxy aldonic acid from aldonic acid, comprising:
 contacting the gluconate dehydratase to aldonic acid in water or an aqueous solvent at a temperature from 0° C. to 120° C. and a pH of 1.5 to 12, wherein the blend ratio of a gluconate dehydratase to aldonic acid is 1 ug: 0.01 to 1 mol.   
     
     
         17 . The method according to  claim 16 , wherein the gluconate dehydratase is selected from the group consisting of an isolated native gluconate dehydratase, a chemically synthesized gluconate dehydratase, a recombinant gluconate dehydratase, and derivatives thereto. 
     
     
         18 . The method according to  claim 16 , wherein the aldonic acid is selected from the group consisting of D-gluconate, D-Galactonate, D-Galactoheptonate, D-Arabonate, D-glucuronate, L-gulonate, D-tartarate, D-glucarate, L-isovalerate, L-threonate, D-ribonate, L-tartarate, D-gulonate, and D-galactarate.