JP3163362B2 - Method for producing L-proline - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing L-proline by a fermentation method.

[0002]

2. Description of the Related Art Heretofore, as a method for producing L-proline by a fermentation method, a method using an isoleucine-requiring strain or an arginine-requiring strain of the genus Brevibacterium or Corynebacterium (Japanese Patent Publication No. 43-11751). ), A method using a sulfaguanidine-resistant strain (Japanese Patent Publication No. 51-40158), a method using a DL-3,4-dehydroproline-resistant strain (Japanese Patent Publication No. 57-22319), and a citrate synthase activity-enhancing strain. A known method (JP-B-62-36679) or a method using a histidine- or methionine-requiring strain of the genus Bacillus or Escherichia (JP-B-44-1198) is known.

[0003]

SUMMARY OF THE INVENTION The object of the present invention is to provide an L-
An object of the present invention is to provide a method for producing L-proline by a fermentation method which is industrially advantageous by improving the production yield of proline.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to develop a method for producing L-proline by a fermentation method which is cheaper and more efficient than the conventional method. When culturing microorganisms having a L-proline in a liquid medium and producing and accumulating L-proline in the culture medium, it is expected that the culturing of the liquid medium with the addition of gluconic acid will dramatically improve the production yield of L-proline. They have found and completed the present invention.

[0005] In other words, the present invention provides gluconic acid in an amount of 0.0
A microorganism having an ability to produce L-proline is cultured in a liquid medium containing at least 1 g / dl, and L-proline produced and accumulated in the culture solution is cultured.
L- by a fermentation method characterized by collecting proline
The present invention provides a method for producing proline.

[0006] The microorganism used in the present invention belongs to the genus Brevibacterium, Corynebacterium, Microbacterium and the like, and any strain having L-proline producing ability can be used. As a specific example, L-3,4-dehydroproline-resistant L-
Brevibacterium lactofermentum AJ11225 (FERM P-437) which is a proline producing bacterium
0), Brevibacterium flavum AJ11226
(FERM P-4371), Corynebacterium glutamicum AJ11227 (FERM P-437)
2), Microbacterium ammonia film AJ
11228 (FERM P-4373) and Brevibacterium flavum AJ11512 (FE
RM P-5332), AJ11513 (FERM P
-5333), AJ11514 (FERM P-533)
4) Corynebacterium glutamicum AJ11522 (FERM), which is an L-proline-producing bacterium having L-isoleucine requirement and high citrate synthase activity
P-5342), AJ11523 (FERM P-53)
43) etc. are used.

[0007] The medium used in the present invention is 0.01 g / d.
A normal liquid medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic trace nutrients such as amino acids and vitamins as appropriate, other than containing 1 or more gluconic acids, is used. Examples of the carbon source include sugars such as glucose, sucrose, fructose and maltose, starch saccharified solutions containing these sugars, sweet potato molasses, sugar beet molasses, Hitest molasses, and organic acids such as acetic acid, and alcohols such as ethanol and glycerin. Kinds are also used. Nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea,
Nitrate and the like are used, and in addition, oil cakes, soybean hydrolysate, other amino acids, and peptides such as corn steep liquor, yeast extract, peptone, and the like are used as an auxiliary source of organic nitrogen. As the inorganic ions, phosphate ions, magnesium ions, calcium ions, iron ions, manganese ions, and the like are appropriately added.

[0008] Culture of microorganisms is usually carried out at pH 4 to 10,
The reaction is performed under aerobic conditions while controlling the temperature in the range of 20 to 40 ° C. In order to adjust the pH of the culture solution, an inorganic or organic acidic or alkaline substance, urea, calcium carbonate, or ammonia gas can be used.
Thus, when the culture is performed for about 20 to 100 hours, a remarkable amount of L-proline is produced and accumulated in the culture solution.

[0009] L-proline is collected from the culture solution by a combination of an ion exchange resin method and other known methods.

[0010]

The present invention will be described in more detail with reference to the following examples.

Example 1 Gluconic acid of each concentration was added to the compositions shown in Table 1 and 20 ml of a liquid medium adjusted to pH 7.2 was dispensed into a 500 ml shake flask and sterilized by heating. Brevibacterium flavum AJ1 previously grown on a natural medium
One platinum loop of 1512 cells was inoculated and shake-cultured at 30 ° C. for 72 hours. As a control, culturing was carried out in a medium without gluconic acid. Table 2 shows the results of measuring the amount of L-proline produced and accumulated in the culture solution after the completion of the culture. As is clear from this, the amount of L-proline accumulated was significantly improved by culturing with the addition of gluconic acid.

[0012]

[Table 1]

[0013]

[Table 2]

Example 2 A medium having the composition shown in Table 3 and a gluconic acid 0.05 added to the composition shown in Table 3 were used.
After preparing a medium to which g / dl was added and adjusting the pH to 7.2, 20 ml of each medium was dispensed into a 500 ml shake flask and sterilized by heating. Brevibacterium flavum AJ11512, Corynebacterium glutamicum AJ11522 or Microbacterium ammonia filum AJ112 previously grown on a natural medium.
Twenty-eight bacterial cells were inoculated with a loopful of platinum and cultured in the same manner as in Example 1. The results of measuring the amount of L-proline produced and accumulated in the culture solution were as shown in Table 4.

[0015]

[Table 3]

[0016]

[Table 4]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification symbol FI (C12P 13/24 C12R 1:15) (58) Investigated field (Int.Cl. ⁷ , DB name) C12P 13/24 BIOSIS ( DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] To 1. A liquid medium containing gluconic acid, Brevibacterium
Bacterium flavum, Brevibacterium lacto
Fermentum, microbacterium ammonia
L- belonging to rum or Corynebacterium glutamicum
A method for producing L-proline by fermentation, comprising culturing a microorganism having proline-producing ability and collecting L-proline produced and accumulated in a culture solution.