KR970002249B1 - MICROORGANISM PRODUCING 5óÑ-XANTHYL ACID - Google Patents

Abstract

The microorganism (KFCC 10740) producing 5'-xanthylic acid is claimed. This microorganism which belongs to a variant of Brevibacterium ammoniagenes 10352 is leaky auxotrophic to adenine and guanine in the growth, contains an inactivated 5'-xanthylic acid aminase and has the high susceptibility to lysozyme, an cell-wall hydrolysing enzyme.

Description

Microorganisms Producing 5'-Xanthyl Acid

The present invention relates to a microorganism itself producing 5'-xanthyl acid (hereinafter abbreviated as XMP), wherein XMP not only has a taste as an intermediate of nucleic acid metabolism, but also 5'-inosinic acid (IMP) and 5'- It is also important as a raw material for manufacturing guanylic acid (GMP). In particular, the most widely used method of producing 5'-guanylic acid, which has high taste and high commercial value, is the most economical process of producing XMP by microbial fermentation and converting it to 5'-guanylic acid enzymatically. The demand for XMP is as important as the demand for 5'-guanylic acid.

Conventional XMP production methods include 1) chemical synthesis, 2) deamination of 5'-guanylic acid produced by decomposing ribonucleic acid in yeast, and 3) adding xanthine as a precursor in fermentation broth. Method, 4) fermentation method by microbial mutant strain, 5) manufacturing method by adding antibiotics (Japanese Patent No. 42-1477, small 44-20390), 6) manufacturing method by adding surfactant (Japanese Patent No. 42-3835, Small 42 -3838) are known. Among them, the present invention is industrially advantageous for the production of XMP by microbial mutants, and the present inventors have improved the Brevibacterium ammonia Genes ATCC6872 to give various complex traits in which XMP can be produced to the maximum and possessed by conventional strains. We have developed a breakthrough variant that far surpasses the productivity of XMP due to its simple properties.

More specifically, a strain of Brevibacterium ammonia genes (ATCC6872) is an inactive strain of adenine and guanine leaky auxotroph-type xanthyl acid aminase, resistant to guanosine analogs, and lysozyme, a cell wall degrading enzyme. It is a microorganism with very high sensitivity and microorganisms that can use saccharide raw materials and accumulate aerobically in nutrient medium with limited nitrogen source and accumulate XMP directly in culture medium with high yield and high concentration. Deposited to the Association. (Accession No .: KFCC-10740)

Thus, the inventors have found that adenine, guanine semiconducting forms, and XMP, which partially block the biosynthetic pathway from 5'-inosinic acid to 5'-adenylic acid and 5'-guanylic acid, allow microorganisms to produce large amounts of XMP directly. Intracellular 5'-guanylic acid of 5'-inosinic acid dehydrogenase enzyme involved in biosynthetic pathway from X 'to inosinic acid to XMP aminase enzyme in order to prevent metabolic flow In order to release the feedback regulation by the guanine purine analogs, it was modified to be resistant to thioguanine. Furthermore, the present invention was completed by lysozgme, a cell wall degrading enzyme that is expected to be partially lacking in cell wall synthesis so that extracellular secretion is desired when a large amount of intracellular XMP is accumulated. .

In the following, the separation method of the present invention is described in more detail, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), which is an ultraviolet irradiation or chemically modified strain organic agent as a parent strain of Brevibacterium ammonia gene (ATCC 6872) )-, Diethyl sulfate, ethyl sulfate, etc., 2% glucose, 0.1% potassium monophosphate, 0.1% potassium diphosphate, 0.1% magnesium sulfate, 100mg / 1 calcium chloride, 20mg / 1m iron sulfate, 10mg manganese sulfate / 1, zinc sulfate 10mg / 1, biotin 100㎍ / 1, copper sulfate 0.8mg / 1, thiamin hydrochloride 5mg / 1m, urea 0.2% and grows very slowly in the minimum batch (media 1) adjusted to pH 7.3 Adenine and guanine were added to the minimum medium (medium 1) at concentrations of 50-200 mg / l each, and adenine and guanine-added minimal medium (medium 2) separated the adenine and guanine stimulating growth-promoting cultures. XMP aminase activity inactivated It was selected for two weeks. At this time, the penicillin concentration method was used to increase the yield of mutant strains.

XMP aminase inactive strains of the adenine and guanine semiconductive constituents selected by the above method were named as trait 1 and mutated by the above method, and then 150 mg / 1 of adenine and guanine were added to the medium (medium 1). The mutant strains that were resistant to Thiog uanine and maintained trait 1 (type 2) were selected by Thioguanine concentration of the minimum medium added with Thioguanine up to 10-100 ㎍ / ml (Medium 3). After mutated by 1% peptone, 1% yeast extract, 0.25% sodium chloride 0.25% adenine and 150mg / l of guanine was added to the solid medium (medium 4) to separate each colony (Colony) after pure separation Lysozyme susceptible to lysozyme that maintained trait 2 by two-speaking (Toothpiocking) to lysozyme susceptibility test medium (medium 5) added to the solid medium (medium 4) lysozyme concentration up to 5㎍ / ml-100㎍ / ml The migration was screened and named XFCC10740.

Biochemical characteristics of the modified strain of the present invention obtained and separated according to the above-described method are as described in Table 1 below.

^* +: Growth-: Not growing

^* +: Growth-: Not growing

Fermentation medium that can be used in the present invention is an industrially inexpensive saccharide raw material (glucose, fructose and polysaccharide hydrolysates thereof), nitrogen sources (organic and inorganic nitrogen sources) and inorganic salts necessary for the growth of microorganisms, trace elements, Any medium can be used if the medium is properly added.

The culture method was incubated for 3 to 4 days aerobicly while maintaining at 30 ℃ ~ 35 ℃, PH 6 ~ 8 as described in the Example.

The present invention is explained in more detail in the following examples.

Example 1

XMP of parent strain, conventional strain and variant strain of the present invention in an Erlenmeyer flask

Productivity Comparison

Dispensing and sterilizing 5 ml of seed media adjusted to pH 7.2 consisting of glucose 3%, peptone 1.5%, yeast extract 1.5%, sodium chloride 0.25%, adenine 150 mg / l, guanine 150 mg / l, urea 0.2% Then, the strains of the parent strain ATCC 6872, the conventional strain KFCC 10004, and the modified strain KFCC 10740 of the present invention were inoculated and shaken and cultured at 150 rpm for 12-14 hours at 30 ° C.

Glucose 6%, 1% phosphate 1, 2 potassium each, 1% magnesium sulfate, calcium chloride 100mg / l iron sulfate 20mg / l, manganese sulfate 10mg / l, zinc sulfate 10mg / l, biotin 100µg / l, adenine 3mg / l, guanine 30mg / l, copper sulfate 1mg / l, thiamine hydrochloride 5mg / l, urea 0.7%, fermented medium adjusted to pH 7.2 is dispensed in 30ml 500ml shaking Erlenmeyer flask and sterilized at 121 ℃ for 20 minutes. After cooling, the culture completed solution obtained from the seed culture was inoculated in 5 ml each and incubated at 180 rpm for 60-70 hours at 30 ° C.

After completion of culture, the accumulation amount of 5'-xanthyl acid in medium was 3.2 g / l for ATCC 6872 strain, 12.8 g / l for strain KFCC 10004, and 18.4 g / l for strain KFCC 10740 of the present invention (XMP accumulation). The concentration is expressed as 5'-sodium xitrate 7H ₂ O).

Example 2

Comparison of XMP Productivity of Conventional Strains and Variants of the Invention in Fermenter

The seed medium of Example 1 was dispensed into a 500 ml shaking Erlenmeyer flask with a volume of 40 ml, and after sterilization, the seed strains of KFCC 10004 and the modified strain KFCC 10740 of the present invention were respectively inoculated and shaken at 180 rpm for 12-14 hours at 30 ° C. (Species culture).

Glucose 18%, Phosphate 1, 2 potassium each 2%, Adenine 150mg / l, Guanine 150mg / l, Iron sulfate 20mg / l, Zinc sulfate 15mg / l, Manganese sulfate 10mg / l, Copper sulfate 1mg / l, Biotin 150µg fermentation medium consisting of / l, magnesium sulfate 1%, calcium chloride 100mg / l, thiamine hydrochloride 5mg / l, zinc sulfate 15mg / l, adjusted to pH 7.3, and dispensed 1.8 liters each into a 5 liter test fermenter and at 121 ° C. After sterilizing and cooling for 20 minutes, inoculate 180 ml of the seed culture, and incubating at 1,2.5 liters of air every minute and rotating at a temperature of 30 ℃ -35 ℃ for 1 minute after rotation, the residual sugar concentration in the culture reached 1-2%. Sterilized glucose was supplied to control culture of the total sugar added to the fermentation medium to 24%. The pH of the culture was adjusted to 7.0-7.4 with ammonia water and incubated for 60-70 hours. After completion of culture, the accumulation amount of 5'-xanthyl acid in the medium was 71.7 g / l for the KFCC 10004 strain of the conventional strain and 94.1 g / l for the variant KFCC 10740 strain of the present invention (XMP accumulation concentration was 5'- sodium xanthate 7H 0).

Claims (1)

Brevibacterium ammonia genes strain, 5'- highly susceptible to lysozyme, an inactive strain of 5'-xanthyl acid aminase, resistant to guanosine analogs, resistant to guanosine analogs KFCC 10740, a microorganism that produces xanthyl acid.