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    • 专利摘要:
    2,5-Diketo-D-gluconic acid is prepared in high yield and in high broth concentration by cultivating newly isolated microorganisms of genus Erwinia in an aqueous nutrient medium in the presence of D-glucose. The production is also possible by simple contact of said microorganisms or their processed products therefrom, with D-glucose.
    公开号:SU1190992A3
    申请号:SU813362894
    申请日:1981-08-13
    公开日:1985-11-07
    发明作者:Сонояма Такаясу；Яги Сигео；Кагеяма Бундзи；Танимото Масахиро
    申请人:Сионоги Энд Ко.,Лтд (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to technical microbiology, namely to a method for producing 2,5-diketo-1) -gluconic acid (2,5-DKG) using producer microorganisms belonging to new strains of microorganisms of the genus Erwinia.
    A known method for producing 2,5-diketo-B-glutenic acid by cultivating aerobic microorganisms of the genus Pseudomonas fluorescens on a nutrient medium containing D-glucose as a carbon source, nitrogen sources, phosphorus, mineral salts, and stimulants. . that 1. ,
    There is also known a method for producing 2,5-DKG, according to which microorganisms of the genus Acetobacter are used as a producer of the target product, which are cultivated under conditions of aeration of the medium on a nutrient medium containing D-glucose as a carbon source, a source of nitrogen, phosphorus, mineral salts and growth stimulator under conditions of aeration of the medium followed by release of the target product 2.
    According to this method, L-proline is additionally introduced into the nutrient, which contributes to an increase in the yield of the desired product. However, this additive, although it increases the yield of the product, does not accelerate the process and also complicates it. The latter lasts up to 64 hours, which naturally leads to high energy costs.
    The aim of the invention is to simplify and accelerate the fermentation process with a high yield of the target product.
    The goal is achieved by the fact that according to the method for producing 2,5-diketo-1) gluconic acid by cultivating its producers on a nutrient medium containing E-glucose as a carbon source, sources of nitrogen, phosphorus, mineral salts and a growth stimulator, under the condition x aeration of the medium followed by separation of the target product from the culture medium; the producers use the strain Erwinia citreus ATCC 31623 or the strains Erwinia punctata ATCC 31624, or Erwinia punctata. ATCC 31625, or Erwinia punctata ATCC 31626, or Erwinia punctata ATCC 31627, or shtaf1 Erwinia terreus ATCC 31628, or Erwinia terreu ATCC 31629, or Erwinia terreus ATCC 31630, or Erwinia terreus ATCC 31621.
    According to the proposed method, the concentration of O-glucose in the medium should be set from 5 to 20 vol.%.
    Thus, the proposed method can be carried out using the indicated strains of the genus Erwinia capable of selectively oxidizing I) -glucose and turning it into 2,5-DKG.
    These strains are listed in Table 1. They are stored at the Institute for Fermentation Research in Japan and at the American Crop Collection in Washington. ,
    These taxonomic studies of these microorganisms are presented in Table. 2..
    A. Observations.
    1. Cell form (canted broth agar at 28 ° C for 2k, 72
    and 168 hours).
     Single cells predominate numerically, the sticks indicated in the table of sizes with rounded ends for all strains. Sometimes in some cases, deformed cells of the indicated sizes are observed.
    2. Motility and flagellation (sloped broth agar at 20 and 25 ° C for 18-24 h).
    Motility is determined by the hanging drop method.
    Flagellation is confirmed under an optical microscope after staining by the Toda method or by means of a transmission electron microscope after cultivation by the method of cultivation in a supported membrane.
    Strains SHS-2003, 2004, 2005, 2006 and 2007 are mobile.
    Strain SHS-2008 mobile with monolateral flagellum.
    The strains SHS-2009, 2010 and 2011 are mobile with one or two lateral flagella. .
    3. Spores do not form any of these strains.
    4. Gram stain (beveled broth agar for 24, 72, 168 h). Negative for all of the following
    : strains. 5, Acid resistance. All strains are negative. B. Growth on various environments (tab. 3). 1. Colonies on broth agar, for 24, 48, 72 and 168 h. The initial colonies are all round, whole, smooth, transparent and oily. 2. Skewed broth agar at 28 s for 24–168 h (Table 4). The initial colonies are all filiform and oily. 3. The broth at 28 C for 24-68 hours. All strains have no odor (Table 5). 4. A column of bouillon gelatin at 20 ° C for 40 days. All strains do not liquefy. Growth along the axis of the column. 5. Litmus milk at 28 ° C for 40 days. Strain SHS-2003. Acidification begins on day 7. The poor coagulum begins approximately on the 18th day and ends on the 38th day. From the 18th to the 32nd day, the upper layer becomes pink and the lower layer becomes grayish brown, but on the 32nd day the whole layer is uniformly pink. No changes were observed in strains SHS-2004, 2005, 2006, 2007, 2008, 2010 and 2011, during the incubation period, 6.Slanted potatoes for 24-168 h. Colonies in all strains are filiform, oily and shiny (tab. 6). B. Physiological properties (based on observations at 28 ° C for 14 days, unless otherwise indicated). 1. Nitrite. All strains produce nitrite from nitrate. 2.Nitrate breathing. All have neither growth nor gas production in paraffin-sealed broth containing 1% KNO. 3. Test with methylrotom. All positive, with the exception of strain SHS-2010 (weakly positive). 4. Voges-Proskanera reaction (glucose). The strain SHS-2003 - weakly positive. The strains SHS-2004 2005, 2007, 2008, 2010 and 2011 - is positive. The strains SHS-2006 and 924 2009 -t are negative or weakly positive. 5.Indol. All strains - otrutsitna. 6. Hydrogen sulfide. Bacto peptone water-lead-acetate reactive paper is positive for everyone. TSI-arap. All strains are negative. Agar Klieglera. All strains are negative. 7. Ammonia. All strains do not produce. 8. Hydrolysis of starch. Negative for all strains. 9. Growth on citrate media. Wednesday Simmon, supplemented with a vitamin mixture. Growth - all strains. Wednesday chrrstensina. Growth in all strains. Growth on inorganic nitrogen sources. Ammonia (Wednesday - Huckera glucose, supplemented with a vitamin mixture). Growth at all. Nitrate (medium - Dimmika glucose, supplemented with a vitamin mixture). Growth is observed in strains SHS-2003, 2004, 2005, 2008, 2009, 2010 and 2011, strain SHS-2006 does not grow, and strain SHS-2007 grows very weakly, 11. Pigment (culture with a stroke on petri dishes). Blue pigment (yeast extract 1%, glucose 1%, CaCO, 2% at 28 s for 7 days). Everyone is negative. is negative. Pink, diffusible pigment (the medium is similar to that given), All strains are negative. Yellowish pigment (broth agar at 28c for 7 days). All strains are negative. 12. Ureaza. All strains are negative. 13. Catalase. All strains are negative. (4. Oxidase (sloped broth agar containing tetramethylphenylenediamine, for 18-24 h). Negative for all strains. 15. Temperature dependence (bactro-yeast extract 0.5%, bactopeptone 0.5% and glucose 0.5%, pH 7) is presented in Table 7.
    16. Dependence on pH (glycerin 1%, bactro-yeast. Your extract is 0.5%,
    bactopeptone 1%, chloride 5% three 0.5%, in a buffer of 0.1 M 3,3-dimethylglutaric acid, 28 ° C for 2-4 days) is presented in table. eight.
    17. The need for oxygen. All strains are optional.
    Rod culture, sealed with liquid paraffin. In this case, the glucose-HRV medium containing,%: bactom extract 1, bacto peptone 1, bact yeast extract 0.2, sodium chloride 0.5, bromocresol purple 0.004, and agar powder 1.5 (pH 7.0-7.2 ), poured into test tubes with a diameter of 18 mm and a height of about 8 s and sterilized at 121 C for 15 min. The bars were inoculated until the medium was completely solid. Then the tubes were sealed with sterile liquid paraffin (5-7 ml) to a depth of 4 cm, cultured at 28 ° C and observed after 7 days.
    With a significant growth along the axis of the rods from the top to the bottom of the medium for 2-3 days, the culture began to uniformly turn a pale yellow color, showing a clear difference in the color of each of the strains shown in the table compared to the control sealed, non-inoculated tube. Over time, the intensity of the yellow color increased.
    An anaerobic gas-cancer system (BB L).
    A petri dish with bacto-nutritious agar containing,%: bactom extract 1, bactopepton 1, sodium chloride 0.5 and agar powder 1.5, or a petri dish with agar I UR containing,%: glycerin 0.5; baktomodol extract 0.5; bactopeptone 0.3; KHjPO 0.1; - M. 0.02; agar powder 1.5; pH 7.0-7.2 Weak inoculation of the suspension suspension The test organism was cultivated: at 2P. C for 48 hours after the removal of oxygen by a two-hour reaction at 45 ° C in an incubator.
    All strains showed weak growth, weaker than Escherichia coli, taken for control.
    18 (Open field test. Enzymatic for all strains. Medium containing,%: Bactotrypton 1:, bactro-yeast extract 0.1; bromcresol
    purple 0.004; glucose (or lactose) 1% and agar powder 0.2; pi 7.0-7.2, poured into test tubes (18 mm diameter) to a depth of approximately 8 cm and sterilized at 121 ° C for 15 minutes. After the medium temperature reaches approximately 30-40 ° C, the inoculation for each organism from the strains in the table is duplicated. Then one tube from each pair was sealed with sterile liquid paraffin (5-7 ml) to a depth of approximately 4 cm, cultured at 28 ° C and monitored for 7 days.
    The acid is produced both under aerobic and anaerobic conditions from D-GLUCOSG (and also from lactose in the case of SHS-2003); gas does not produce. There is a distinct growth along the axis of the rods from top to bottom in both the sealed and the unsealed culture.
    The acid is not produced under anaerobic conditions as quickly as Escherichia cioli, taken for control.
    19. Production of acids and gases from hydrocarbons is shown in Table. 9 and 10.
    20. Methylen blue (broth, 18-24 h). All strains recover
    21.D-gluconic acid. All strains and use.
    22.2-keto-0-gluconic acid. Use all strains.
    23i Production of reduced compounds from sucrose. Positive for all strains excluding strain SHS-2003.
    24. Decarboxylation of various amino acids.
    D-glutamic acid. Negative for strains SHS-2003, 2008, 2009, 2010 and 2011; positive for strains SHS-2004, 2005, 2006 and 2007.
    L-lysine. Negative for all strains.
    L-arginine. Negative for all strains.
    L-ornithine. Negative for all gptammy.
    25. Lipase. Negative for all strains.
    26. Oxidation of gluconate. Positive in all strains.
    27. The decomposition of pectate. Negative for all strains. 28. Hydrolysis of casein. Oriditel on u.vseh strains. 29. Symplasmatum. SHS-2003, 2004, 2005, 2007, 2008 and 2009 is negative for strain-SW. SHS-2006, 2010 and 2011 are positive for strains. 30.D-Naza (bakto-P-Naza agar test). Negative for all strains. 31. Phenylalanine deaminase (phenylalanine / malonic acid media). The strains SHS-2003, 2008, 2009, 2010 and 2011 are negative or weakly positive; in strains SHS-2004, 2005 2006 and 2007 is negative. 32.KCN Suppression. Positive for all strains. 33. Growth in 5% sodium chloride broth. Positive in all strains. 34.Auxrtrofi (Grayand Tatunia Wednesday). Nicotinic acid or nicotinamide is required for all strains. 35. The use of some compounds (OS environment, 28 ° C, 20 and 44 h, shaking) is presented in Table. 1 36..Ubiquinone. The strains of SHS-2003 and 2008 are ubiquinone-8 (and ubiquinone-7). The strains SHS-2004, 2005, 2006, 2007, 2009, 2010 and 2011 are ubiquinone G. Origin. The strains SHS-2003, 2004, 2006, 2007, 2010 and 2011 are noble Mandarin. Strains SHS-2005. and 2009 - persimmon, virginska. Strain SHS-2008 - soil. 111 in the table. 1 is determined by comparing the mentioned taxonomic properties of the corresponding strains in Table. 2 with limestone. 1. Distribution within, within the family. Based on the results of the observation, it can be concluded that all the strains in the tables we are short sticks of gram-negative and facultative anaerobes that do not form spores and show catalase activity. but / do not possess oxidase activity, they are assigned to the family Enterobacterialceae. 2. Distribution within the genus All the strains listed in the tables are attributed to the genus Erurieia based on taxonomic properties, in particular they produce acids from HR-methyl glucoside and sucrose (with the exception of strain SHS-2003, which does not produce acid from sucrose, but uses the latter in as a sole carbon source), but not from adonite, dulcite or melezitoza, do not use benzoate, oxalate and propionate, are not able to hydrolyze starch, are not capable of decarboxylated glutamic acid, arginine, lysine and ortin. The production of strains SHS-2004, -2005 and 2007, which decarboxylate glutamic acid) and do not show urease or lipase activity. 3. Distribution within the species. Although the SHS-2003 strain is considered close to Erwinia stewartu of the herbicola group based on the absence of flagellation, it has other taxonomic properties that are very different from the properties of Erwinia stewartu in need of nicotinic acid or nicotinamide for growth, in the production of hydrogen sulfide from cysteine, in recovery nitrates to nitrites, in non-production of acid from sucrose (although it cannot produce acid from sucrose, however, it uses sucrose as the sole carbon source), arabinose, raffinose or sorbitol, in dosing of acid from fat, cellbiose and glycerol, not using terrate as the sole carbon source. In addition, since this strain did not coincide with any other known species of the genus, it should be attributed to the new species proposed by the invention Erwinia Citreus. Although the strains SHS-2004, 2005, 2006 and 2007 are considered close to Erwinia stewartu in terms of no flagellation, they have taxonomic properties that are very different from the properties of Erwinia stewartu in need of nicotine or nicotinamide for growth, in the production of hydrogen sulphide from cysteine, reduction of nitrates to nitrites, decarboxylation of glutamic acid. In addition, they do not produce acids from arabinose, maniitol, lactose, or sorbitol, but produce acids from salicin and cellobiose and do not use tartrate 9 as the sole carbon source .. In addition, these strains are different from the described strain ZN8-20b3. that glutamic acid is decarboxylated, lacto acetate and lactate are hardly used as a single carbon source, do not produce acids from mannitol and lactose, and do not have activity for litmus milk. Since these strains did not coincide with any other known species of the genus, they should be attributed to jHOBOMy species called Erwinia pun tata .:. Of these strains, strains SHS-2004 and 2006 do not produce acid from. raffinose and do not use mannitol, acetate or lactate, - But the SHS-2004 strain differs from the SHS-2006 strain in that it produces acetoin from glucose, uses nitrate as the nitrogen source, produces acid from glycerol and uses formate as the sole carbon source. The SHS-2005 strain, like the SHS-2006 strain, is almost incapable of using mannitol, acetate and lactate as the sole carbon source; however, the former differs from the latter in that it produces acid from glycerol, acetoin from glucose, forms fluorescent pigments in the medium King B almost does not produce raffinose acid and uses formate as the sole carbon source. In addition, the SHS-2007 strain and the SKS-2006 strain are almost incapable of using mannitol, acetate and lactate as the sole carbon source, but the former differs from the latter in that it produces acid from raffinose, the acetoin from glucose, uses formate as the only carbon source and has a wide range of growth temperatures from 4.0 to 47.5 ° C. Based on the indicated observation results, all strains of SHS-2004, 2005, 2006 and 2007 are attributed to Erwini punctata and. each of them represents a variant with respect to the others. Although the SHS-2008 and 2010 strains are close to En rinia tracheiphila or 9210. From the point of view of their mobility with a monolateral flagellum, Erwinia guereina, but their taxonomic properties are very different from the properties of Erwihia tracheiphila in the following: growth at a temperature above 36 ° C, abundant growth on broth agar, show mucoid growth, restore nitrates to nitrites, produce fragments of nitric acid, extracts , xylose, melibiose and mannose and use lactate as the sole carbon source. In addition, there are differences between their taxonomic properties and the properties of Erwinia guerina in that they oxidize gluconic acid, reduce nitrates to nitrites,. produce acids from melibiose and cellobiose, do not produce acids from mannitol, uJ-methyl glucoside, esuklin and sorbitol, do not produce gas on a glucose-peptone medium. In addition, since these strains do not coincide with any other known species of the genus, it is advisable to refer them to the Erwinia species. Strain SHS-2010 shows a significant match with. with the SHS-2008 strain, with the exception of some differences, which consist in the fact that it does not produce acid from glycerol, SHS-2008 produces its acid from ribose, and SHS-2010 almost does not produce it. In addition, he has a weakly positive reaction in the test with methylrot and he forms fluorescent pigments on the medium. . Strain SHS-2009 is close to Erwinia tracheipVdla, E. guerce / .na and E. herbicola from the point of view of motility with a monolateral flagellum. However, there is a noticeable difference between the taxonomic properties of this strain and the properties of Erwinia tracheiphila in the fact that abundant growth is observed on broth agar, growth at a temperature above 36 C. Cro. in addition, it exhibits mucoid growth, reduces nitrates to nitrites, and produces acids from salicin, xylose, melibioses, cellobiose, glycerin, and mannose. Found also a noticeable difference from the properties of Erwinia guereina in the oxidation of gluconate, the reduction of nitrates to nitrites, the weak formation of acetoin and glucose, the production of acids from xylose, melibiose and cellobiose. In addition, TorojOH does not produce acids from mannitol, oi-methylglucoeid, esuklin, ribose, or sorbitol, does not use tertrate as the sole carbon source, and does not produce gas on a glucose-peptone medium. The difference between the properties of the SHS-2010 strain and the properties of Erwinia herbicola. The var herbicola consists in its need for nicotinic acid or nicotinamide for growth, in the weak formation of acetoin from glucose, it is not. liquefying gelatin, produces acids from melibiose, cellobiose and glycerin, produces acids from arabinose, mannitol, maltose, dextrin, rhamnose, ribose or sorbitol. Although there are still some differences when comparing this strain. with a previously defined strain of SHS-2008 in a weak formation of acetoin from glucose, in the non-production of acid from ribose, however, the first strain coincides with the latter for the other predominant properties and, therefore, is identified as a variant of the previously defined strain. Strain SHS-2011 is close to Erwinia tracheiphila or Erwinia. amylavara 1c in terms of mobility with monosateral flagellum. When comparing the properties of this strain with the properties of Erwinia tracheiphila, the difference is in its moderate growth in broth agar, growth at a higher temperature. In addition, it exhibits mucoid growth, restores nitrates to nitrites, produces acids from salicin, xylase, melibioses, dellobiozymes, and uses lactate in the amount of a single carbon source. When comparing the properties of this strain with the properties of Erwinia amylovola, the differences consist in the formation of hydrogen sulphide from cysteine, growth at a temperature and above, reduction of nitrates to nitrites. In addition, it does not dilute gelatin, produces acids from salicin, xylose, melibiose, cellobiose and mannose, does not produce acid from ribose. When compared with the SHS-2008 shtack, this strain coincides with the latter in properties, but does not produce acid from ribose or glycerin and is identified as a variant of the previously defined strain. Certain microorganisms grow abundantly in an aqueous nutrient medium containing D-glucose as a major carbon source, corn infusion as a nitrogen source, and a small amount of inorganic salts. If they are cultivated under aerobic conditions, they grow on medium with a very high concentration of D-glucose with sufficient stability to produce 2,5-diketo-B-gluconic acid (2,5-DKG) with a good yield compared with known microorganisms, used to make this product. The concentration of D-glucose in the broth can reach, if necessary, AO vol.%, Although from an economic point of view it is advisable to maintain a D-glucose concentration in the range of 15-25 vol%, more preferably, to obtain 2,5-diketo-B-gluconic acid. 20% by volume The temperature of the fermentation medium is 15-35 ° C. Preferably, 20-30 ° C can be set up. the interval is 5.5-7.5, preferably 6.0-7.0. The pH values of the medium can be maintained during fermentation in the required A range, 0-5.5 by introducing inorganic salts, for example calcium carbonate, with a buffering effect, into the original medium or by introducing bases, for example sodium hydroxide, into the medium as the fermentation process proceeds. . The inoculated fermentation medium should be continuously stirred with a stirrer (approximately 17AAb / mi) with aeration in the amount of approximately 600 N. ml / min. Fermentation ends after 17-31 hours from the beginning of the process, when the conversion of D-glucose to 2,5-DKG reaches a value that corresponds to a yield of approximately 90%. The accumulating 2,5-DKG or its salts can be isolated from the culture liquid in the form of crystals after treatment by any method, for example, by adjusting the pH, or the medium can be successfully used as such as a nutrient medium at the next stage. For example, a medium containing 2,5-DKG can be directly applied to produce 2-keto-1.-gulonic acid. Thus, the latter can be obtained with a large yield using a simplified technology.
    The proposed method can also be practically carried out by simply contacting any of the products obtained by treating the cells of these microorganisms with any of the substrates containing D-glucose.
    Example 1. For sowing, a medium (aqueous solution) containing vol.% Is used:
    D-glucose (hydra. Tirovna, content of 91%) 1.0. Real maize (CSL) 5.0 Potassium dihydrogen phosphate () 0.1 Mg sulfate (MgS04-7H20) 0.02 Cald carbonate (CaCO) 0.5
    The medium is adjusted to pH 6.8-7.0 with a 10% aqueous solution of caustic soda, divided into 50 ml portions and placed into sterile 500 ml conical flasks.
    Each portion of the medium in the flask is inoculated with a complete loop of one of the strains listed in Table. 3, and shake (71 mm stroke, 270 beats per minute) at a temperature of about 8 hours. Cultivation ends when the optical density (OD) of the medium reaches about 8 (end point) ..
    A culture for planting is thus obtained. i.
    The fermentation medium (water solution) is prepared with the following composition,
    vol.%: ...
    20.0 3.0
    0.1
    6.3 50
    The pH of the fermentation medium is adjusted to 6.8-7.0, divided into 455 ml portions and placed in 1 liter fermenters. To each of the fermenters, add 45 ml of the indicated medium for seeding. Then fermentation is carried out under the following conditions:
    Temperature, C 28 Stirring, rpm 1740
    Aeration, N. ml / min. 600 Duration, hours 17-31 The resulting product is chromatographed on a filter paper carrier by means of a capillary lift and the spot is determined using a densitometry method. The carrier is Touye Roshi No. 50 and the developing solvent, phenol: formic acid: water, taken in a ratio of 75: 4: 25.
    For dyeing, sprinkle a solution of water-saturated n-butanol (100 ml) containing aniline (0.93 g) and phthalic acid (1.66 g), and conduct subsequent processing at 105 ° C for 2 minutes to show color . The results are presented in table. 12.
    In addition, thin layer chromatography was also carried out with TLC allumisheet cellolose and with the described developing solvent system. and coloring technique. In this case, the determination is made by comparing the chromatograms with those obtained for authentic samples.
    The fermentation is complete, the pink spot of the 2-keto-B-glcconic acid disappears from the indicated paper or from the thin-layer chromatogram. The results obtained after incubation of the strains are presented in table. 13
    Example 2. Production using a cell suspension and a crude enzyme extract. To obtain microbial cells, I use a medium (aqueous solution) of the following composition:% by volume:
    Cn ,,, 1
    Na2S04-7H20O., 02
    СаСО50,6
    The medium is adjusted to pH 7.0, divided into 80 ml portions, and placed in 500 ml conical flasks.
    Each of these media is inoculated with the corresponding strain m (Table 4 according to the method described in Example 1 and shaken (71 mm stroke, 270 beats per minute) for 16 hours.
    After shaking, the microbial cells are separated by centrifuging the culture medium, twice
    15
    washed with saline and divided into two parts.
    One of these parts is suspended again with saline and a suspension of cells is obtained. The second part is suspended in a 1/50 M Tris-HCl buffer (pH 7.5), pulverized with ultrasound, centrifugation removes the insoluble residue, and a crude enzyme extract is obtained in the form of a floating layer.
    Incubations are then carried out with the cell suspension and with the crude enzyme extract, respectively.
    When incubated with a cell suspension, a mixture is prepared from a 0.1 M buffer of 3,3-dimethylglutaric acid (pH 5.0) containing approximately 5% by volume of D-glucose, in which the cell concentration is adjusted so that the OD at 660 M W approximately 10. The mixture is divided into portions of 10 ml, placed in 23 mm tubes (diameter) x 196 mm (height) and incubated at 28 ° C for 3 hours.
    The mixture is then centrifuged to form a floating layer, which
    9099216
    analyzed by paper chromatography.
    The results obtained after incubation with the cell suspension, as well as the concentration of D-glucose in the mixture before incubation, are presented in Table. 14.
    When incubated with the crude enzyme extract, the crude enzyme extract is added to the mixture prepared as described above so that its concentration, expressed as the amount of protein, is 0.25 mg / L. After shaking in the same manner as described, the mixture is treated with two drops of 10% trichloroacetic acid solution to remove the protein portion and chromatographed on filter paper. The results obtained after incubation with a crude enzyme extract are presented in table. 15.
    Thus, the proposed method provides a high yield of 25 2-keto-B-gluconic acid in 17-31 hours of fermentation, which speeds up and simplifies its preparation.
    Table 1
    table 2
    2009
    Pale beige
    Brilliant schA
    2010 Same
    Also
    2011
    Pale red yellow
    Smooth, but gradually changes to a rough and embossed
    Convex, but gradually changes to the hiked
    Convex, but gradually changes to flat. Sticky but gradually changing to oily,.
    f a b n p 4
    Table
    Abundant
    Icy red yellow
    Bezhev
    Moderate
    Pale beige Bezhev
    Plentiful
    Moderate
    Table 7
    Table 9
    +
    +. + Note. Note. Produces acid without gas (); produces neither acid nor gas (-); weakly produces acid without gas (±); Uses as the sole carbon source (+); poorly used or not used as the sole carbon source (-); does not use (-). TABLE AND
    Table 15
    权利要求:
    Claims (2)
    [1]
    1. METHOD FOR PRODUCING
    [2]
    2,5-DICETO-0-GLUCONIC ACID by cultivating its producers on a nutrient medium containing Ώ-glucose as a carbon source, sources of nitrogen, phosphorus, mineral salts and a growth stimulator, under conditions of aeration of the medium with subsequent isolation of the target product, which differs in order to simplify and accelerate the fermentation process with a high yield of the target product, Erwinia citreus ATCC 31623 strain or Erwinia punctata ATCC 31624 strains, or Erwinia punctata ATCC 31625, or Erwinia punctata ATCC 31626, or Erwinia punctata ATCC are used as producers. , or w Amma Erwinia terreus ATCC 31628 or Erwinia terreus ATCC 31629 or Erwinia terreus ATCC 31630 or Erwinia terreus ATCC 31631.
    2. The method of pop. 1, with the fact that the concentration of D-glucose in the medium is set from 5 to 20 vol.%.
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    同族专利:
    公开号 | 公开日
    IE51496B1|1987-01-07|
    YU43032B|1989-02-28|
    DE3167468D1|1985-01-10|
    DK149963C|1987-05-04|
    EP0046284B1|1984-11-28|
    YU198281A|1983-12-31|
    ES8204762A1|1982-05-16|
    GB2083028B|1984-09-19|
    GB2083028A|1982-03-17|
    KR830006428A|1983-09-24|
    IE811847L|1982-02-14|
    JPS6041596B2|1985-09-18|
    EP0046284A2|1982-02-24|
    DK361281A|1982-02-15|
    HU188073B|1986-03-28|
    AU546761B2|1985-09-19|
    JPS5736991A|1982-02-27|
    CA1168999A|1984-06-12|
    EP0046284A3|1982-04-21|
    BG41824A3|1987-08-14|
    DK149963B|1986-11-03|
    MX7115E|1987-06-29|
    AU7420481A|1982-02-18|
    KR840001257B1|1984-09-01|
    US4879229A|1989-11-07|
    US5134077A|1992-07-28|
    CS224624B2|1984-01-16|
    ES504732A0|1982-05-16|
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    US7256027B1|1999-06-15|2007-08-14|Rutgers, The State University Of New Jersey|Enzymes for the production of 2-keto-L-gulonic acid|
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    US6727277B1|2002-11-12|2004-04-27|Kansas State University Research Foundation|Compounds affecting cholesterol absorption|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP55112406A|JPS6041596B2|1980-08-14|1980-08-14|
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