TWI298743B - Microorganism producing 5'-xanthylic acid and production method of 5'-xanthylic acid using the same - Google Patents

Description

1298743 v IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to a microorganism capable of producing 5, xanthanic acid and a method for producing 5'-xanthene using the same. In particular, the present invention relates to a mutant strain of Corynebacterium ammoniagenes CJXFT0301 (1^(:€]\/1-105 3 0), which produces a shoulder-stick axe mill (:^ and 81110401 (KCCM-10609) ), which has resistance to sulfamethoxazole, leads to an increase in the synthesis of dihydropteryric acid and ultimately to an increase in the content of tetrahydrofolate, and therefore, the microorganism of the present invention can be directly fermented during the same fermentation period. A higher concentration and higher yield of 5'-xanthic acid than the CJXFT0301 (KCCM-10530) strain is produced. And the present invention also relates to a method for producing 5'-xanthic acid using the mutant protein. 5'-xanthic acid, a metabolic intermediate in the synthesis of nucleotides, is of physiological importance in animals and plants, and is widely used in the fields of food and pharmaceutical industries, and for various medical purposes. 5'-xanthic acid system - attractive as a flavor-enhancing flavoring agent: one of the group of nucleotides, because it can highly enhance the flavor of food when it is combined with sodium glutamate. - Mountain W mouth; ^, a room between the house, and in the production of 5, - electric emperor a person of the 40th private horse wing & 呤 呤 nucleotides of the raw material is important. Before, the microbial fermentation method has been Guanglian is used to produce 5 nucleotides with high flavoring activity and high commercial value. In microbial fermentation, 7 1298743 Λ produces 5'_黄°票呤酸' and then 5, , tannic acid carries ', is _ bird feces 嘌呤 nucleotides. Therefore, based on the demand for 5, _ island 呤 呤 呤 苦, 5, · yellow. Ticket citrate needs to be produced. * - Some: some methods It is known to be used for the production of 5,-xanthine'. These methods include a method of synthesizing or degrading yeast ribonucleic acid by a chemical to produce 5, island faecal acid, and the 5,-guano The guanidine acid is deaminated and comprises a fermentation method in which the scutellaria is supplemented in the fermentation medium as a precursor, using a mutant strain of the microorganism, or supplementing the antibiotic in the medium (Japanese patent) Case Sh〇42_1477 and Heart 44-20390) or surfactant (Japanese Patent Office) No. 42_3825 and Sho No. 42-3838. As mentioned above, many methods for producing 5,_xanthic acid have been known, and the economical production of 咼 wave and high yield of 5,_xanthic acid is the most important. The target is directly industrially fermented by the direct fermentation of the microbial mutant strain of the f ^ method (4). The xanthate is industrially advantageous. In this respect, the inventors intend to develop a significant increase in the 5,-guano sputum nucleus. Microbial mutations in the production of glycosidic acid • The strain, which is capable of producing the maximum amount of 5,-guanine nucleotides, by improving the properties of the well-known cultivar cjxFT0301 (KCCM_1〇53〇). Therefore, the inventors have found a novel mutant strain which is established by imparting resistance to sulfonamide to the conventional strain of CJXFT0301 (KCCM-l〇530), and the mutation is established. The strain is capable of producing high concentration and high yield of 5 xanthic acid by direct fermentation during the same fermentation, which also leads to the production of the present invention. 8 1298743 SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a microorganism having resistance to sulfonamide and capable of producing high concentration and high yield of 5'-xanthic acid by direct fermentation. Another object of the present invention is to provide a method for producing a high concentration and a high yield of 5, xanthanic acid by culturing the microorganism. [Embodiment] The present invention relates to a CJXSul0401 (KCCM-10609) which has resistance to sulfonamide and can produce a high concentration and a high yield of 5'-xanthene. Further, the present invention provides a method for producing 5'-xanthic acid by culturing a fan-shaped window CJXSul0401 (KCCM-10609) and directly accumulating a high concentration and a high yield of 5'-xanthic acid in a culture liquid. . The microorganism of the present invention is produced by a CJXSu10401 (KCCM-10609), which is a mutant strain of CJXFT0301 (KCCM-10530). The shoulder/playing #磨CJXFT0301 (KCCM-10530)' is used as a parent strain in the present invention, and is disclosed in Korean Patent Application No. 10-2003-089714. The microorganism according to the present invention has the following main features and potency similar to those of the calcareous CJXFT0301 (KCCM-10530). i) demand for adenine ii) demand for guano sputum 9 129.8743 iii) sensitivity to lysosomes iv) resistance to proline analogues v) resistance to glutamate analogues vi Resistance to tryptophan analogs In addition to the above-described characteristics, the microbial sputum CJXSuHMO1 (KCCM-l〇6〇9) produced by the microorganism according to the present invention has resistance to sulfamethine and is therefore capable of being cultured. A higher concentration and a higher yield of 5, xanthanic acid are directly accumulated in the liquid. 5. The biosynthetic pathway of xanthine (XMP) is very complex and is a series of reactions involving a variety of amino acids and coenzymes. In particular, the XMP biosynthetic pathway from the phosphoribosyl pyrophosphate (PRPP) to the XMP system was gradually completed by 11 reactions. Furthermore, two molecules of glutamic acid, one molecule of glycine, one molecule of aspartic acid, one molecule of hc〇3· and two molecules of hydrazine, fluorenyltetrahydrofolate (THF_ch〇) ) is included in the reactions. Therefore, the addition of these starting materials which are included in the base of the purine can help the synthesis of purine bases. A sulphate di- sulfonamide antibiotic which is a structural derivative of p-aminobenzoic acid (paba). Sulfonamide dimerization blocks the action of dihydropteroate synthetase and thus prevents the formation of folate synthesis intermediate, dihydropteroic acid. Because of this effect, ~amine dimorphine can inhibit the synthesis of tetrahydrofolate in microorganisms. Four-gas folate plays a key role in the synthesis of sputum, thymine, DNA and similar molecules. (d) The decrease in the content of tetrahydrofolate inhibits the growth of U organisms. A strain having sulfonamide resistance, cjxSu1〇4〇1 (KCCM-1 0609)' is intended to increase the synthesis of dihydropteric acid to overcome the growth inhibition of sulfamethoxazole 1298743. This thus increases the total amount of tetrahydrofolate necessary for the synthesis of ruthenic acid. This increase in the content of tetrahydrofolate causes a large amount of synthesis of samaric acid, which results in a high concentration and a high yield of lycopene by a direct fermentation method as compared with the prior art. This finding has led to the production of the present invention. The microorganisms can be acupointed by X-rays, ultraviolet light, chemical mutagens (e.g., mercaptonitronitroguanine, diethyl sulfate, ethylamine, etc.) and the like. In order to obtain the microorganism according to the present invention, Li Guang #欢•#磨CJXF丁0301 (KCCM-1〇53〇) was used as a parent strain. In order to obtain a mutant vegetative propagation line which can survive in the presence of sulfonamide, the parent strain is a mutagen, methyl-specific γΝ-nitrosoguanamine 根据 according to the method commonly used in the art of Haier. NTG) was treated and screened in media supplemented with various concentrations of 'moon-female-one, which were labeled "Μ", which is described in Table 2. The microorganism of the present invention is screened from the mutant vegetative propagation line obtained by the above method. Sulfonamide was included in the medium used for the test of the present invention, and it was 5 mg/L. The pro-chicken 扃 扃 欢 捧 C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C Conversely, the strain selected to be resistant to sulfonamide was found to be CJXSiil0401 (KCCM-1〇6〇9) and was found to grow in even i mg/Lisulfonamide.

The selected mutant asexual reproduction line was named "Maternal shoulder #说#磨CJXSU1G401, which was deposited at the Korea Microbiology Center (K〇rean Cuhure W 11 1298743) on July 16, 2004 under the Budapest Treaty.

Microorganisms, KCCM; Hongjae 1-dong ^ Seodaemun-gu ^ Seoul, Republic of the K0rea), and was given accession number (Accessi〇n number) KCCM-10609. CJXSul〇4〇1 has also been deposited with the Food Industry Development Institute under the registration number Bcrc 910307. The novel mutant strain CjxSu1〇4〇i (KCCM-1 0609) isolated in the present invention has the biochemical properties as described in Table 1, and is capable of growing in a medium supplemented with 1 mg/L sulfonamide. The mutant strain of the present invention has the property of resistance to sulfamethine as described in Table 1, and in Table 1, the resistance of the mutant strain to the parent strain CJXFT〇3〇l to sulfonamide was compared. Table I Sulfonamide Digestion Concentration (mg/L) 0 0.1 0.2 0.3 0.5 1 2 5 CJXFT0301 + + + ++ — One CJXSu0401 (KCCM-1 0609) + + + + + + + + + + + + + + — — Note: + : growth; —: no growth; the strains were cultured at 30 ° C for 5 days. The method for producing 5,-xanthic acid using the novel strain of the present invention is as follows. The strain CJXSiil0401 (KCCM_10609) according to the present invention is cultured in a conventional medium containing a suitable carbon source, a nitrogen source, an amino acid, and a vitamin, under aerobic conditions, and factors such as temperature and pH. All are under control. Examples of carbon sources that may be utilized include carbohydrates such as glucose, fructose, and aseptically processed molasses (i.e., molasses that is converted to reducing sugar). Examples of suitable nitrogen sources include inorganic nitrogen sources such as urea, ammonium chloride, and ammonium sulfate, and organic nitrogen sources such as Peptone, NZ-amine, meat extraction 12 129.8743 (Meat extract), yeast extraction , c〇ni steep liquor, casein hydrolysate, fish or its decomposition products, and fat-free soy cake or its decomposition products. Examples of inorganic compounds that may be utilized include acid-removal, dipotassium silicate, sulphuric acid, ferrous sulfate, manganese sulfate, and calcium carbonate. The medium may be further supplemented with vitamins, Auxotrophic bases, and the like, depending on the intended purpose. The culture is carried out in an aerobic environment (Aer〇bic c〇n (jition), for example, with shaking or aeration-stirring, preferably at 20 to 40 ° C. During the culture, the medium is preferably The soil is kept close to neutral pH. The culture lasts for 5 to 6 days. 5'-xanthic acid, accumulated by direct fermentation, is analyzed according to the conventional method. 、, 果果, § using the distant relative strain CJXFT0301 by direct When fermenting 5,· = tannic acid, 52.2 g/L of 5,5 g/L was obtained. In contrast, the field used the dog strain of this Ming, shy, holding Huai C to Sui〇4 At 1 o'clock, 160.4 g/L of 5'_xanthic acid was obtained under the same environment. These results indicate that compared with the parental strain CJXFT〇3〇1, CJXSul〇4〇1 strain, obtained the sulfonamide two tillage Resilience, with enhanced 5,_xanthene production. 々 - Further understanding of the present invention can be obtained by the use of the T embodiment, which is set forth to illustrate and not to limit the invention. The microorganism culture system of the present invention comprises a nutrient medium using the soil described in Table 2 below. A medium named "ΜΓ", a basic culture (named "M2"), a medium containing sulfonamide (named 13 1298743) for seed culture in a flask (defined as 'M4') ), the main fermentation medium in the flask (named M5), the flask sterilization (Flask fermentati〇n) isolation sterilization medium (named "M6"), the medium used for the seed culture in the fermentation tank (named "M7"), the medium used for the second seed culture in the fermentation tank (named "M8") and the medium used for the main fermentation in the fermentation tank (named M9). However, the medium is not It is limited to the ingredients listed in Table 2, and all media suitable for the culture table are available.

Table II

Medium composition Ml 巧g/L Portuguese! Sugar, 1〇g/L peptone, 1〇g/JL yeast extract, 2·g/L gasification sodium, 3 g/L urea, 150 mg/L adenine 150 mg/L guano cockroach, pH 7.2 & M2 20 g/L glucosinolate, 1 g/L disc acid unloading, 1 g/L bismuth acid unloading, 2 g/L urea , 3 g / L of ammonium sulfate, 1 g / L of magnesium sulfate, 1 〇〇 mg / L of calcium chloride, 20 mg / L of ferrous sulfate, 1 〇 mg / L of manganese sulfate, 1 〇 mg / L-zinc sulfate, 30 pg/L biotin (Biotin), 0.1 mg/L thiamine HC1, 0.8 mg/L copper sulfate, 20 mg/L·adenine, 20 mg/L· Avian guanidine, pH 7.2 M3 medium Prepared M4 3 0 g/L glucose by adding 0.1, 0.2, 0.3, 0.5, 1, 2 and 5 mg/L sulfamethoxazole to the lowest nutrient medium (M2) 15 g/L protein gland, 15 g/L yeast extract, 2.5 g/L sodium chloride, 3 g/L urea, 150 mg/L adenine, 150 mg/L guano嘌呤, pH is 7.2 M5 60 g/L glucose, 10 g/L magnesium sulfate, 20 mg/L ferrous sulfate, 10 mg/L zinc sulfate, 1 mg/L manganese sulfate, 30 mg/ L adenine, 30 Mg/L guano, 1〇〇pg/L biotin, 1 mg/L i copper sulfate, 5 mg/L thiamine HC1, 10 mg/L calcium carbonate, pH 7.2”, M6 10 g/L of potassium monobasic acid, 1 〇g/L of acid-filled acid, 7 g/L of urea, 5 g/L of ammonium sulfate μ M7 3 0 g/L of glucose, 15 g/L Protein gland, 15 g/L yeast extract, 2.5 g/L sodium chloride, 3 g/L urea, 150 mg/L gland guanidine, 150 mg/L guanine, pH 7.2 1 M8 瞧----- 60 g/L glucose, 2 g/L monopotassium phosphate, 2 g/L dipotassium phosphate, :· g/L magnesium sulfate, 22 mg/L sulfuric acid Iron, 15 mg/L zinc sulfate, 10 mg/L manganese sulfate, 1 mg/L sulfuric acid steel, 100 mg/L gasification consumption, 150 ug/L biotin, 150 mg/L gland嘌呤, 150 mff/T 14 129.8743 g/L glucose, 32 g/L phosphoric acid, 25 g/L potassium hydroxide M9 stone six gland gas: 119 mg/L guano, 60 mg/ L guar ferrous iron, 42 mg/L zinc sulphate, 15 mg/L manganese sulphate, 2.4 mg/L sulphur bismuth copper, 22 mg/L alanine, 7.5 mg/L NCA, 0.4 Mg/L biotin, 15 g/L magnesium sulfate, 3 〇mg/L cysteine Acid, 30mg/L histidine, 149 mg/L calcium carbonate, 15 mg/L thiazide HC1, 0.7 5 dose, 27 ml/L corn steep liquor (CSL), 6 g/L Salmon feed, pH 7.3 Example 1: Establishment of a sulfonamide-resistant strain The mutant strain according to the present invention was established as follows. The abundance of the axe was used. The CJXFT0301 (KCCM-10530) was used as a parent strain and treated with a final concentration of 30 pg/ml2 N-methyl_N_nitronitrosoguanine, at 30. (: The mutation was induced for 60 minutes. Then, the parent strain was washed three times with 85% physiological saline, and appropriately diluted with a medium by adding 1 mg/L of sulfamethoxazole: Prepared by a basic medium (M2) containing 17% of Qiongyue sugar, and then the diluted parent strain is spread on a solid medium. Each of the emerging colonies is cultured in a nutrient medium (Ml), seed medium. (M4) for 24 hours, and then cultured in a mixed culture medium of fermentation medium (M5) and isolated sterilization medium (M6) for 4 days. In these asexual reproduction lines, the highest yield can be produced 5,_ Astragalus acid, which is accumulated in the culture liquid, is selected as the vegetative propagation line. The selected asexual reproduction line is named as "Yuyu holding itch mill CJXSU10401", which is in 2 years and 4 years. 16曰 was deposited in the Korean Microbiology Center according to the Budapest Treaty and was given accession number KCCm 10609. 'Example 2 · Fermentation in Erlenmeyer flask 15 129.8743 (Titer test) Test used strain: C JXSul0401 and CJXFT0301 medium for seed culture in flasks: M4 listed in Table 2 Medium for main fermentation in flasks: Isolation and sterilizing medium for flask fermentation listed in Table 2: M6 5 listed in Table 2 The seed culture medium of ml is aliquoted into the formula of i8 (five) melon and sterilized under high pressure according to the conventional method. Each strain is inoculated in sterile seed culture and cultured at 3 〇. 18 hours, with shaking at 180 rpm. This seed culture was used as a fermentation inoculum (Inoculum). The medium for the main fermentation and the isolated sterilization medium were separately sterilized under high pressure according to a conventional method, and respectively 29 ml and 10 ml were aliquoted into 500-ml shaken oulander flasks. Seed cultures of 丨ml were inoculated into the flasks and incubated for 90 hours. The rearing was only performed at 30 °C. It was shaken with 200 rpm. After that, it was measured in the liquid of the culture medium and accumulated in the culture liquid, 5,_xanthic acid. It was produced by the conventional strain CJXFT0301 (KCCM-10530). 5'_yellow citric acid was found to accumulate in the culture solution at a concentration of 28 6 g/L' while the scutellaria acid produced by the mutant strain CJXSul0401 (KCCM-10609) of the present invention accumulated at a concentration of 30-7 g/L. In the culture liquid, the cumulative concentration of 5-xanthic acid (χΜΡ) is given by 5,-ΧΜρ · Na3 · 7h2〇. Example 3: Fermentation force in the fermentation tank: the strain used: CJXSul0401 and CJXFT0301 Medium for the first-seed culture in the fermentation tank·· listed in Table 2, M7 1295743

Two-seed culture medium ··M8 listed in Table II Fermentation medium: M9 listed in Table 2 The main _ seed culture used in the fermentation tank for the fermentation tank: 50 ml first _ The seed culture medium was aliquoted into a 500 ml shaken zone Renlan's flask, and under the high pressure, the microorganism was reduced by μ min jl and cold. Each strain was inoculated into the sterile medium and incubated at 3 °C for 24 hours with shaking at 18 rpm. Second-seed culture: 2 liters of the second _ seed medium was aliquoted into 5 liters of experimental fermentation tank, under high pressure at i2i (: 2 〇 min,

And v but 5 〇 mi said that the first-seed culture was inoculated into the fermentation tank and incubated at 31 C for 24 days, with shaking at 900 rpm and oxygen at 0-5 vvm. The culture was adjusted to pH 7.3 with ammonia water (eight (3) old ammonia) during the fermentation. Fermentation: 8 liters of the medium for the main fermentation was aliquoted into 3 liters of the experimental fermentation tank, sterilized at 121 °c for 2 Torr at high pressure, and cooled. 1.5 L of the second-seed culture was inoculated into the fermentation tank and incubated at 33 ° C with stirring at 400 rpm and 1 vvm of vent gas. During the fermentation, when the amount of residual sugar is less than 1%, sterile glucose is added to the medium to adjust the amount of sugar in the medium for the main fermentation to 30%. The culture was adjusted to pH 7.3 with ammonia (Amm〇niagas) based on the fermentation period. The entire fermentation was carried out for 80 hours. Thereafter, the amount of 5, xanthanic acid produced and accumulated in the medium was measured. 5,-xanthic acid produced by the conventional strain CJXFT0301 (KCCM-10530) was found to accumulate in the culture liquid at a concentration of 152.2 g/L, and the mutant strain CJXSul0401 (KCCM-10609) of the present invention was produced. 5,-xanthic acid 17 I2R8743 ♦ temporarily accumulated in the culture liquid at a concentration of 160.4 g / L. 5. The cumulative concentration of xanthine (XMP) is given by 5'-XMP · Na3 · 7H20. Industrial Applicability As described above, the strain CJXSuHMO1 (KCCM-10609) according to the present invention can economically produce higher concentrations and higher yields of 5 xanthic acid than conventional strains by direct fermentation. [Simple diagram description] None [Main component symbol description] None

18

Claims (1)

Patent application scope f7 years/amendment 1 1. A microbial production fan/coin γ, i 才 才 〇 寄存 寄存 寄存 寄存 寄存 寄存 寄存 , , , , , , , , 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物 微生物The microorganism has resistance to sulfamethine and is capable of producing 5'-xanthic acid. A method for producing 5'-xanthic acid based on cultivating a microorganism according to the first aspect of the patent application to produce 5'-xanthic acid in a culture liquid. XI. Schema: