JPH059062B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-proline by fermentation. In the method for producing L-proline by fermentation, microorganisms capable of producing L-proline are cultured under aerobic conditions in a medium containing a carbon source, a nitrogen source, inorganic salts, etc. A method of producing and accumulating it in a culture solution has been used. When producing L-proline industrially, it is necessary to increase the amount of L-proline accumulated in the culture medium and improve the yield. It is effective to increase the sugar concentration as high as possible, and a high concentration medium with a sugar concentration of 6 to 14 g/dl is usually used. In order to produce L-proline industrially, it is necessary to further improve the fermentation yield, but even if the sugar concentration of the medium is further increased for this purpose, the yield does not improve and the growth of microorganisms increases. is significantly suppressed, and the yield actually decreases. In addition, in fermentation methods, the so-called feeding system, in which sugar solution is continuously or intermittently supplied to the culture solution, is effective in improving yield; however, we attempted the feeding system in L-proline fermentation. However, it was not possible to improve the yield. The present inventors conducted research aimed at improving the yield using the feed method, and found that if the concentration of ammonium sulfate in the medium was set in the range of 0.17 to 0.27 mol, the fermentation yield could be greatly increased by applying the feed method. I discovered that I can improve. The present invention has been completed based on this invention. The method of the present invention will be explained below. The microorganisms capable of producing L-proline used in the present invention belong to the genus Brevibacterium, Corynebacterium, Microbacterium, etc. and have the ability to produce L-proline. An example is Brevibacterium lactofermetum AJ11225 FERM- which is an L-proline producing bacterium resistant to DL-3.4-dehydroproline.
P4370, Brevibacterium flavum AJ11226
FERM-P4371, Corynebacterium glutamicum AJ11227 or Microbacterium ammoniaphyllum AJ11228 FERM-P4373, etc., or L
-L-proline-producing bacteria with isoleucine auxotrophy, sulfaguanidine resistance, monofluoroacetic acid, ketomalonic acid, or fluoromalonic acid, and high citrate synthase activity, such as JP-A-57-2691 Brevibacterium flavum AJ11512 FERM−P5332 described in the publication No.
AJ11513 FERM−P5333, AJ11514 FERM−
P5334, Corynebacterium glutamicum
AJ11522 FERM−P5342, AJ11523 FERM−
P5343 etc. are used. The medium used in the present invention has no particular characteristics other than containing 0.17 to 0.27 mol of ammonium sulfate.
A medium containing a conventional carbon source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins, is used. The concentration of ammonium sulfate
If it is lower than 0.17 mol or higher than 0.27 mol, even if the carbon source is supplied by the feed method, L
- The yield of proline is not improved and the object of the invention is not achieved. As a nitrogen source, in addition to the above-mentioned ammonium sulfate, ammonia gas, urea, etc. are used auxiliary. As the carbon source, saccharides such as glucose, sucrose, fructose, and maltose, vine starch saccharification liquid containing these saccharides, cane molasses, sugar beet molasses, Hites, etc. are also used. As inorganic ions, phosphate ions, magnesium ions, calcium ions, iron ions, manganese ions, etc. are added as appropriate. The concentration of the carbon source in the starting medium is preferably about 6 to 10 g/dl, and there is no need to increase it any higher; culturing L-proline-producing bacteria in a medium containing such a concentration of carbon source promotes the growth of the microorganism. , the equilibrium phase is reached in a shorter time and L-proline production begins. After the growth of the microorganism reaches an equilibrium phase, a carbon source is continuously or intermittently supplied to the culture solution, and the culture is carried out while maintaining the carbon source concentration in the range of 0.5 to 6.0 g/dl. At this time, good results cannot be obtained if the concentration of the carbon source exceeds 6.0 g/dl because microbial growth is suppressed. When cultured while maintaining the carbon source concentration in the range of 0.5 to 6.0 g/dl, the microorganisms proliferate, albeit slightly, and the production of L-proline is accordingly promoted, producing L-proline at a high yield. I can do it. Note that the supply of the carbon source is continued until the consumption rate of the carbon source is substantially reduced. Cultivation of microorganisms is usually carried out under aerobic conditions at a pH of 4 to 10 and a temperature of 20 to 40°C. The pH of the culture solution is adjusted to a predetermined value within the above range using aqueous ammonia, ammonia gas, or alkali metal hydroxide. The culture is continued until the supply of carbon source is stopped and the carbon source in the culture solution is no longer assimilated. Examples will be described below. Example 1 A medium (medium A) with the composition shown in Table 1 containing 6.0 g/dl of glucose and a medium (medium B) containing 13.5 g/dl of glucose were prepared, and after adjusting the pH to 7.0,
Dispense 300ml each into a 1.0 volume fermenter and heat at 115℃.
Heat for 20 minutes to sterilize.

[Table] Brevibacterium flavum AJ11512 FERM-P5332 (isoleucine auxotrophy, sulfaguanidine resistance, monofluoroacetic acid resistant) was inoculated with 10 ml of culture solution, and agitation culture was started at 31.5°C. In the case of medium A, after the growth of microorganisms reaches an equilibrium state (after the start of culture)
20 to 25 hours), 60 g/dl glucose solution was intermittently supplied, and the glucose concentration in the culture solution was maintained at 2.0 to 2.0 hours.
Culture was carried out while maintaining the amount within 6.0 g and adjusting the pH of the culture solution within the range of 6.0 to 7.5 using aqueous ammonia. In the case of medium B, culture was carried out while controlling only the pH without feeding glucose, and the culture was completed in 72 hours. L-proline in each of the obtained culture solutions was quantified, and the amount accumulated in the culture solution and the yield relative to sugar were determined. The results are shown in Table 2.

[Table] As shown in Table 2, ammonium sulfate concentration
When the feed method is carried out using 0.17 to 0.27 mol of the medium, the yield is as high as 37.1 to 41.5%, which is about a 6% improvement in yield compared to the case where the feed method is not carried out. The culture solution obtained by culturing in medium A containing 3.0 g of ammonium sulfate was centrifuged to remove bacterial cells, and
ml of supernatant was obtained. Next, ion exchange chromatography was performed using an anion exchange resin column to separate L-proline, and 15.6 g of L-proline was concentrated and crystallized.
-Proline crystals were obtained.

Claims (1)

[Claims] 1 A microorganism capable of producing L-proline is cultured in a medium containing sugars as a carbon source and 0.17 to 0.27 mol of ammonium sulfate, and after the growth of the microorganism reaches an equilibrium phase, the carbon source is continuously or intermittently supplied. The carbon source concentration in the culture solution is maintained in the range of 0.5 to 6.0 g/dl while culturing is carried out to produce and accumulate L-proline in the culture solution, and the L-proline is collected from the culture solution. L- by a fermentation method characterized by
Production method of proline.