| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | March 6, 2001 |
| PATENT TITLE |
L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase obtained from gluconobacter oxydans T-100 |
| PATENT ABSTRACT |
A novel L-sorbose dehydrogenase (SDH) and a novel L-sorbosone dehydrogenase both derived from Gluconobacter oxydans T-100, a DNA which encodes the SDH and/or SNDH, an expression vector which contains the DNA, a host cell transformed by the expression vector and a process for producing the SDH and/or SNDH, which comprises culturing the host cell in a medium and recovering the SDH and/or SNDH from the resulting culture. The SDH and SNDH of the present invention are useful enzymes having preferable properties for the production of 2-keto-L-gulonic acid, as well as L-ascorbic acid. According to the production method of the present invention, the SDH and SNDH having such preferable properties can be produced in large amounts by genetic engineering. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 17, 1998 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A recombinant L-sorbosone dehydrogenase having an amino acid sequence of SEQ ID NO: 2. 2. An L-sorbosone dehydrogenase produced by a process comprising: culturing a host cell transformed by an expression vector containing a DNA encoding the L-sorbosone dehydrogenase having the amino acid sequence of SEQ ID NO: 2 in a culture medium; and isolating said L-sorbosone dehydrogenase from the culture medium. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
TECHNICAL FIELD The present invention relates to a novel L-sorbose dehydrogenase (hereinafter referred to as SDH) and a novel L-sorbosone dehydrogenase (hereinafter referred to as SNDH) both derived from Gluconobacter Oxydans T-100. More particularly, the present invention relates to a novel SDH, a novel SNDH, a DNA encoding same, an expression vector containing said DNA, a host cell transformed (transfected) with said expression vector, and the production of the SDH and SNDH by culturing the host cell. The SDH and SNDH of the present invention are enzymes useful for producing 2-keto-L-gulonic acid. BACKGROUND ART 2-Keto-L-gulonic acid (hereinafter referred to as 2KLGA) is a key intermediate in the synthesis of L-ascorbic acid. For industrial production, 2KLGA is chemically synthesized from L-sorbose by oxidation according to the Reichstein's method. On the other hand, it is well known that many microorganisms have an ability to convert L-sorbose to 2KLGA through a two-step enzymatic oxidation by SDH and SNDH. Namely, SDH catalyzes the. oxidation of L-sorbose to L-sorbosone, and SNDH catalyzes the oxidation of L-sorbosone to 2KLGA. However, because of the low productivity of 2KLGA obtained by using these microorganisms, they have not been applied to the industrial production yet. It is desirable to provide efficient and simplified methods for the production of 2KLGA. DISCLOSURE OF THE INVENTION In an attempt to accomplish the above-mentioned objects, the inventors of this invention conducted intensive studies to find an SDH and an SNDH having preferable properties, and succeeded in producing a novel SDH and a novel SNDH having desirable properties for producing 2KLGA and developed the studies, which resulted in the completion of the present invention. Accordingly, the present invention relates to a novel SDH derived from Gluconobacter oxydans T-100 (FERM BP-4188), which is characterized by: (1) an ability to catalyze the conversion of L-sorbose into L-sorbosone, (2) a molecular weight of 58,000 dalton (SDS-PAGE), and (3) an N-terminal amino acid sequence of Thr--Ser--Gly--Phe--Asp--Tyr--Ile--Val--Val--Gly--Gly--Gly--Ser--Ala(SEQ ID NO: 5). Further, the present invention relates to an SDH having an amino acid sequence shown in the Sequence Listing, Sequence No. 1 to be mentioned later. The present invention also relates to a novel SNDH derived from Gluconobacter oxydans T-100, which is characterized by: (1) an ability to catalyze the conversion of L-sorbosone into 2KLGA, (2) a molecular weight of 50,000 dalton (SDS-PAGE), and (3) an N-terminal amino acid sequence of Asn--Val--Val--Ser--Lys--Thr--Val--Xaa--Leu (SEQ ID NO: 6, Xaa being an unidentified amino acid). The present invention further relates to an SNDH having an amino acid sequence shown in the Sequence Listing, Sequence No. 2. The present invention also relates to a DNA which encodes the above-mentioned SDH and/or SNDH, an expression vector which contains said DNA, a host cell transformed (transfected) by said expression vector and a process for producing the SDH and/or SNDH, which comprises culturing said host cell (transformant) in a medium and recovering the SDH and/or SNDH from the resulting culture. The Gluconobacter oxydans T-100 to be used in the present invention is a 2KLGA-high-producing mutant derived from Gluconobacter oxydans G716 (wild strain) by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis in a conventional manner. The Gluconobacter oxydans G716 was isolated from a persimmon, and has the following morphological and physiological properties. The method described in Bergey's Manual of Systematic Bacteriology Vol. 1 (1984) and the method described in Manual for Identification to Medical Bacteria (S. T. Cowan, 2nd. Edition, 1985) were principally employed for the taxonomic study. 1. Morphological properties The Gluconobacter oxydans G716 is a Gram-negative, motile bacterium. The cell shapes are rod, occurring both singly and in pairs, and rarely in chains. Morphological characteristics of Gluconobacter oxydans G716 Gram stain negative color of colony pale spore negative cell shape rod motility positive flagella 3-8 polar flagella 2. Physiological characteristics Physiological characteristics of the Gluconobacter oxydans G716 are summarized in the following table. Physiological characteristics of Gluconobacter oxydans G716 Conditions Characteristics growth in air + at 4.degree. C. - at 22.degree. C. + at 30.degree. C. + at 40.degree. C. - catalase + oxidase - gelatin liquefaction - nitrate reduction - aesuclin hydrolysis - acid formation L-arabinose + D-cellobiose + Dulcitol + D-galactose + D-glucose + glycerol + D-mannitol + D-mannose + D-xylose + D-lactose - maltose - D-raffinose - rhamnose - D-sorbitol - sucrose - D-trehalose - mol% G + C of the DNA 60.0 Ubiquinone Q10 The organism is aerobic, showing no growth under anaerobic conditions. Optimum temperature is 22 to 30.degree. C., showing no growth at 40.degree. C. and 4.degree. C. The best medium for growth is SY medium that is composed of 2.5% sorbitol and 0.5% yeast extract (pH 6.4). Strong ketogenesis occurs from glucose and glycerol. The Gluconobacter oxydans T-100 to be used in the present invention has the morphological and physiological properties identical to those of Gluconobacter oxydans G716. The new SDH and new SNDH of the present invention can be prepared by recombinant DNA technology, polypeptide synthesis and the like. In case where recombinant DNA technology is employed, the new SDH and/or new SNDH can be prepared by culturing a host cell transformed (transfected) with an expression vector containing a DNA encoding the amino acid sequence of the new SDH and/or new SNDH in a nutrient medium and recovering the same from the obtained culture. Particulars of this process are explained in more detail in the following. The host cell includes, for example, microorganisms such as bacteria (e.g. Escherichia coli, Gluconobacter oxydans and Bacillus subtilis), yeast (e.g. Saccharomyces cerevisiae), animal cell lines and cultured plant cells. Preferred examples of the microorganisms include bacteria, especially strains belonging to the genus Escherichia (e.g. E. coli JM109 ATCC 53323, E. coli NM538 ATCC 35638, E. coli HB101 ATCC 33694, E. coli HB101-16 FERM BP-1872 and E. coli 294 ATCC 31446) and the genus Bacillus (e.g. Bacillus subtilis ISW1214), yeast, especially strains belonging to the genus Saccharomyces (e.g. Saccharomyces cerevisiae AH22), and animal cell lines [e.g. mouse L929 cell and Chinese hamster ovary (CHO) cell]. When a bacterium, especially E. coli or Bacillus subtilis is used as a host cell, expression vector is usually composed of at least promoter, initiation codon, DNA encoding amino acid sequence(s) of the new SDH and/or new SNDH, termination codon, terminator region, and replicatable unit. When a yeast or an animal cell is used as a host cell, the expression vector is preferably composed of at least promoter, initiation codon, DNA encoding amino acid sequences of signal peptide and the new SDH and/or new SNDH and termination codon, and it is possible that enhancer sequence, 5'- and 3'-noncoding region of the new SDH, 5'- and 3'-noncoding region of the new SNDH, splicing junctions, polyadenylation site and replicatable unit are also inserted. The promoter for expressing the new SDH and/or new SNDH in bacteria comprises, for example, promoter and Shine-Dalgarno (SD) sequence (e.g. AAGG). Preferable promoters include, for example, conventionally employed promoters (e.g. PL-promoter and trp-promoter for E. coli) and promoter of the SNDH chromosomal gene. The promoters for expressing the new SDH and/or new SNDH in yeast include, for example, the promoter of the TRP1 gene, the ADHI or ADHII gene, and acid phosphatase (PH05) gene for S. cerevisiae. The promoters for expressing the new SDH and/or new SNDH in mammalian cells include, for example, SV40 early or late-promoter, HTLV-LTR-promoter, mouse metallothionein I (MMT)-promoter and vaccinia-promoter. Preferable initiation codon includes, for example, methionine codon (ATG). The signal peptide includes, for example, signal peptides of other enzymes conventionally employed (e.g. signal peptide of the native t-PA and signal peptide of the native plasminogen). The DNA encoding the signal peptide or the new SDH and/or new SNDH can be prepared in a conventional manner, such as a partial or whole DNA synthesis using DNA synthesizer and a method comprising preparing from Gluconobacter oxydans genomic DNA in a conventional manner such as PCR procedure or DNA probe procedure described in Molecular Cloning (mainly in Chapters 11 and 14, Cold Spring Harbor Laboratory Press, 1989, USA). The termination codon includes, for example, conventionally employed termination codons (e.g. TAG and TGA). The terminator region includes, for example, natural or synthetic terminator (e.g. terminator of the new SDH chromosomal gene and synthetic fd phage terminator). The replicatable unit is a DNA compound capable of replicating the whole DNA sequence belonging thereto in host cell, and may include natural plasmid, artificially modified plasmid (e.g. DNA fragment prepared from natural plasmid) and synthetic plasmid. In the present invention, the replicatable unit can be appropriately selected according to the microorganism to be used as a host cell. Preferable examples of the plasmid include plasmid pBR322 and artificially modified plasmid thereof (DNA fragment obtained from a suitable restriction enzyme treatment of pBR322) for E. coli, yeast 2 .mu. plasmid and yeast chromosomal DNA for yeast, plasmid pRSVneo ATCC 37198, plasmid pSV2dhfr ATCC 37145, plasmid pdBPV-MMTneo ATCC 37224 and plasmid pSV2neo ATCC 37149 for mammalian cells. The enhancer sequence includes, for example, the enhancer sequence (72 b.p.) of SV40. The polyadenylation site includes, for example, the polyadenylation site of SV40. The splicing junction includes, for example, the splicing junction of SV40. The promoter, initiation codon, DNA encoding amino acid sequence of the new SDH and/or new SNDH, termination codon(s) and terminator region can consecutively and circularly be linked with an adequate replicatable unit (plasmid), using, if desired, adequate DNA fragment(s) in a conventional manner (e.g. digestion with restriction enzyme, ligation using T4 DNA ligase) to give the expression vector of the present invention. When mammalian cells are used as host cells, it is possible that enhancer sequence, promoter, 5'-noncoding region of the cDNA of the new SDH and/or new SNDH, initiation codon, DNA encoding the signal peptide, DNA encoding amino acid sequence(s) of the new SDH and/or new SNDH, termination codon(s), 3'-noncoding region of the cDNA of the new SDH and/or new SNDH, splicing junctions and polyadenylation site are consecutively and circularly linked with an adequate replicatable unit in the above manner to give an expression vector. The transformant of the present invention can be prepared by introducing the expression vector obtained above into a host cell. Introduction of the expression vector into the host cell (transformation, hereinafter used as also meaning transfection) can be carried out in a conventional manner (e.g. Kushner method for E. coli, calcium phosphate method for mammalian cells and microinjection). For the production of the new SDH and/or new SNDH by the process of this invention, the thus-obtained transformant containing the expression vector is cultured in an aqueous nutrient medium. The nutrient medium to be used may contain carbon source(s) (e.g. glucose, glycerine, mannitol, fructose and lactose) and inorganic or organic nitrogen source(s) (e.g. ammonium sulfate, ammonium chloride, hydrolysate of casein, yeast extract, polypeptone, Bactotrypton and beef extract). If desired, other nutritious sources such as inorganic salts (e.g. sodium or potassium biphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate and calcium chloride), vitamins (e.g. vitamin B.sub.1), and antibiotics (e.g. ampicillin, kanamycin) may be added to the medium. For the culture of mammalian cells, Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) supplemented with fetal calf serum and antibiotic is often used. The culture of the tranformant is usually carried out at pH 5.5-8.5 (preferably pH 7-7.5) and 18-40.degree. C. (preferably 20-30.degree. C.) for 5-50 hours. When the thus-produced new SDH and/or new SNDH exist(s) in the culture solution, culture filtrate (supernatant) is obtained by filtration or centrifugation of the culture. The new SDH and/or new SNDH can be purified from the culture filtrate by a method generally employed for the purification and isolation of natural or synthetic proteins (e.g. dialysis, gel filtration, affinity column chromatography using anti-SDH monoclonal antibody or anti-SNDH monoclonal antibody, column chromatography on a suitable adsorbent and high performance liquid chromatography). When the produced new SDH and/or new SNDH exist(s) in periplasm and cytoplasm of the cultured transformant, cells are collected by filtration and centrifugation, and the cell wall and/or cell membrane thereof are/is destroyed by, for example, treatment with supersonic waves and/or lysozyme to give debris. The debris can be dissolved in a suitable aqueous solution (e.g. 8 M aqueous urea and 6 M aqueous guanidium salt). From the solution, the new SDH and/or new SNDH can be purified in a conventional manner as exemplified above. If it is necessary to refold the new SDH and/or new SNDH produced in E. coli by the method of the present invention, the refolding can be carried out in a conventional manner. If the SDH activity and/or new SNDH activity exist(s) in the transformant, the following can be exemplified as the materials obtained by processing the culture (hereinafter the material is referred to as processed material). (1) Raw cells: separated from the culture in a conventional manner such as filtration and centrifugation: (2) dried cells: obtained by drying said raw cells of (1) above in a conventional manner such as lyophilization and vacuum drying; (3) cell-free extract: obtained by destroying said raw cells of (1) above or dried cells of (2) above in a conventional manner (e.g. autolysis of the cells using an organic solvent, grinding the cells with alumina, sea sand etc., and treating the cells with supersonic waves); (4) enzyme solution: obtained by purification or partial purification of said cell-free extracts of (3) above in a conventional manner (e.g. column chromatography); and (5) immobilized cells or enzyme: prepared by immobilizing said raw cells of (1) above or dried cells of (2) above or the enzyme of (4) above in a conventional manner (e.g. a method using acrylamide, glass bead, ion exchange resin etc.). If the SDH activity and/or new SNDH activity exist(s) in a culture filtrate of the transformant, the culture filtrate (supernatant), purified enzyme solution and immobilized enzymes can be exemplified as the processed materials of the culture. The assay of the new SDH activity in crude mixture such as sonicated cell lysate or its processed material obtained in each purification step can be usually conducted according to T. SUGISAWA et al. (Agric. Biol. Chem., 55, 363-370, 1991) using L-sorbose as a substrate and 2,6-dichlorophenolindophenol (hereinafter referred to as DCIP) as an electron acceptor in phosphate buffer (pH 7.0). The enzyme activity is measured as the reduction rate of DCIP, which is determined by the decrease of absorbance at 600 nm. One enzyme unit is defined as the amount of the enzyme that catalyzes the reduction of 1 .mu.mol DCIP per minute. Preferable pH of the reaction mixture, concentration of L-sorbose and DCIP, reaction time and reaction temperature may vary with the medium or its processed material to be used. Generally, the reaction is carried out at pH 7 to 10, preferably pH 8 to 9, at 5 to 50.degree. C., preferably 20 to 45.degree. C. for 0.5 to 24 hours. The new SDH enzyme activity may be also assayed by determining the amount of the reaction product, L-sorbosone labelled with benzamidine hydrochloride, using post column high performance liquid chromatography (HPLC) with fluorescent detection (Ex. 315 nm, Em. 405 nm). The activity of novel SNDH present in crude mixtures, such as ultrasonication cell lysate and treated substances obtained in respective purification steps, can be also assayed by a general method by SUGISAWA et al. (Agric. Biol. Chem., 55, 363-370, 1991). In this case, SNDH activity is assayed by measuring the amount of NADH produced using nicotinamido adenine dinucleotide (hereinafter referred to as AND) as an electron acceptor and L-sorbosone as a substrate in phosphate buffer (pH 7.0), which is determined by the absorbance at 340 nm. One enzyme unit is defined as the amount of the enzyme that produces 1 .mu.mol NADH per minute. Preferable pH of the reaction mixture, concentrations of L-sorbosone and AND to be used, reaction time and reaction temperature may vary with a medium and processed materials to be used. Generally, the reaction is carried out at pH 7 to 10, preferably pH 8 to 9, at 5 to 50.degree. C., preferably 20 to 45.degree. C. for 0.5 to 24 hours. The new SDH and new SNDH of the present invention are enzymes having preferable properties useful for producing 2KLGA, and therefor L-ascorbic acid. According to the present invention, the new SDH and new SNDH having such preferable properties can be produced in large amounts by genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a restriction enzyme map of pUC18SD180. FIG. 2 is a restriction enzyme map of plasmid pUC19SD5 containing DNA encoding the novel SDH of the present invention and DNA encoding the novel SNDH of the present invention. FIG. 3 shows the construction of plasmid pUC19SD5. The present invention is explained in more detail in the following. In the following Examples, plasmids, enzymes such as restriction enzyme, T4 DNA ligases, and other materials were obtained from commercial sources and used according to the indication by suppliers. Operations employed for the cloning of DNA, transformation of host cells, cultivation of transformants, recovery of the new SDH and/or new SNDH from the obtained culture, and the like are well known in the art or can be adapted from literatures. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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