JP2971158B2 - Method for producing L-malic acid polymer by microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing L-malic acid polymer (poly-L-Malic acid, hereinafter referred to as PMLA) by microorganisms.

[0002] PMLA is a biodegradable and absorbable high molecular compound, which is a bioabsorbable suture system, an osteosynthesis material, an artificial tendon,
The use in the fields of medicine and medical care is greatly expected as a polymer carrier for artificial blood vessels and drug delivery.

[0003]

2. Description of the Related Art PMLA is a chemical synthesis method using monobenzyl or monomethyl malate as a raw material [Reports
of the Faculty Engineering, Tottori University, 8 ,
124 (1977)], a chemical synthesis method by ring-opening polymerization of benzylmalolactonate [U.S. Pat. No. 4,265,247], and a direct thermal condensation method [Polymer Transactions, 44 , 701 (1987)].
Production methods by chemical synthesis methods have been reported.

In the production of PMLA using microorganisms, Penicillium cyclium (Penicillium cyclium) is used.
opium) [Agricultural B
iological Chemistry 33 (4), 459 (1969)] has been reported.

[0005]

However, the production by the chemical synthesis method has disadvantages that not only the raw materials are expensive, but also that the yield is low due to the necessity of many steps.

[0006] On the other hand, in the production by the solid culture method using penicillium cyclopium, the yield is low.
Further, it is difficult to say that this method is a practical method because purification requires many steps. Therefore, there has been a demand for the development of a method for producing a high-yield material from a less expensive raw material.

[0007]

The present invention provides a method for producing PMLA, which comprises culturing a PMLA-producing bacterium belonging to the genus Aureobasedium and collecting PMLA from the culture.

[0008] Microorganisms belonging to the genus Aureobasidium include microorganisms conventionally classified into the genus Pullularia and later re-classified as the genus Aureobasidium, and are generally referred to as black yeasts. (black yeast) [Mycopathologia et Mycologia Applicat
a, 12 , 1 (1959), and 17 , 1 (1962)]. Therefore, PM
Any black yeast capable of producing LA is also included in the scope of the present invention. Specific examples of such microorganisms include, for example, the following.

[0009] Oureobacedium pullulans (Aureo
basidium pullulans) IFO 4464, IFO 4
465, IFO 4875, IFO 7775, ATCC 7305, Aureobasidium mansonii IFO 6421, IF
O 8194, IFO 9233, ATCC 14
249, Oureobacedium microstickum (A
ureobasidium microstictum) IFO 32066, I
FO 32067, IFO 32068, IFO
32069, Aureobasidium
Sp. A-91 strain and mutants thereof.

[0010] Among the above strains, Aureobasedium S
p. The A-91 strain is a strain newly isolated from the soil by the present inventors, and its bacteriological properties are as follows. A. Growth condition on medium a) Microscopic findings (after cultivation on YM agar medium at 25 ° C for 5 days) Size of vegetative cells: 3-6 x 3-20 µm Shape of vegetative cells: bacterial system and yeast-like single cell, B) Agar slant (potato glucose agar medium) Growth: good Glossy: no Color: When cultured for 3 days or more, it becomes a dark cream, black colony from white cream. c) Liquid culture YM liquid medium: good growth B. Ascospores of forming potato glucose agar medium: formation without corn meal agar: formation without YM agar: V ₈ agar without forming: not formed C. Physiological properties Oxygen requirement: aerobic Growth temperature: 5 to 35 ° C. Growth pH: 2 to 9 KNO ₃ availability (Wickerham synthetic medium): Yes (NH ₄ ) ₂ SO ₄ availability (Wickerham synthetic medium) :
Yes Decomposition of urea: Yes Liquefaction of gelatin: No Generation of organic acids: Yes Requirement of vitamins (Wickerham synthetic medium): No Assimilable carbon source (Wickerham synthetic medium) Glucose, sucrose, maltose, arabinose, xylose, mannose, fructose, trehalose, glycerose, mannitol, starch, ethanol, citric acid, isocitric acid, succinic acid, DL-malic acid, Fumaric acid.

The taxonomic position of this strain having the above mycological properties is described as "The Genera of FungiSporulating in pure
culture "(3rd edition) JA Von Arx ed. Lubrecht & cra
As a result of an examination by the company (1981), it was found that the strain was a new strain belonging to the genus Aureobasedium. It was named A-91 strain.

This strain was sent to the Research Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology, "Microorganism Accession No.
No.).

The method for producing an L-malic acid polymer of the present invention will be described below.

The above-mentioned bacterium of the genus Oleobacedium can be cultured in a usual manner using a medium containing nutrients such as a carbon source, a nitrogen source and inorganic salts.

As a carbon source for the medium, for example, a saccharide raw material, particularly glucose, is suitably used, and the concentration of the added carbon is 1 to 5.
30% by weight, preferably 1-20% by weight is suitable.
As the nitrogen source, nitrogen-containing organic or inorganic substances usually used in culturing microorganisms, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, organic acid ammonium salts such as ammonium succinate, urea, etc., alone or in combination. Can be used,
As the inorganic salt, for example, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like can be used. In addition, if necessary for the growth of the fungus, nutrients such as yeast extract, corn steep liquor, peptone, meat extract, casamino acid, and various vitamins can be added to the medium and used.

The cultivation is carried out under aerobic conditions such as shaking and aeration and stirring. The culturing temperature is generally 20 to 40 ° C., preferably 2 to 40 ° C.
2-3 ° C is preferred. The culture pH is 2 to 10, preferably 3 to 9, and more preferably 3 to 7.

The culture period is not particularly limited, but is usually performed for 1 to 10 days.

The separation and purification of PMLA produced in the culture solution by the above-described culture method are performed according to a method known per se, for example, an ion exchange resin treatment method, a precipitation method such as acetone, methanol, ethanol, and the like. , N-propanol, isopropanol, methyl ethyl ketone, or the like, and an organic solvent precipitation method or the like can be appropriately combined.

The physicochemical properties of PMLA produced by the method described above are as follows. (1) The molecular weight measured by the GPC method is about 2,000
It is in the range of ５０50,000. (2) Only L-malic acid is produced by the hydrolysis treatment with a 1N sulfuric acid solution.

[0020]

According to the present invention, PMLA-producing bacteria belonging to the genus Aureobasedium are cultured to produce P
MLA can be obtained.

[0021]

Next, the present invention will be described more specifically with reference to examples. However, the following examples are given only as an aid to gain a specific understanding of the present invention, and the present invention is not limited thereto.

Example 1 Medium (Glucose 12%, KH ₂ PO ₄ 0.05%, M
gSO ₄ · 7H ₂ O0.05%, yeast extract 0.02
%; No pH adjustment) was dispensed into a 500 ml Erlenmeyer flask, sterilized (121 ° C., 15 minutes), and then 1 ml of a sterilized 6% ammonium nitrate solution was added. The culture was performed by adding 1 g of calcium carbonate to the prepared medium, inoculating the microorganisms shown in Table 1, and shaking the culture at 25 ° C for 3 days.
This was a preculture. Main culture is 2,000 ml of the above medium
Into a 5,000 ml Erlenmeyer flask and sterilized (121
(15 ° C., 15 minutes), then add 40 ml of 6% ammonium nitrate and 60 g of dry and sterilized calcium carbonate, add 2% (v / v) of the preculture and shake for 8 days at 25 ° C. Culture was performed.

After completion of the culture, 200 ml of the culture was centrifuged (8,000 rpm, 15 minutes, room temperature), and 200 ml of methanol was gradually added to 100 ml of the obtained supernatant to precipitate the product. The precipitate was centrifuged (8,000
The mixture was separated at 0 rpm for 10 minutes at 4 ° C.), washed with a mixed solution of methanol and water (2: 1), dried in a desiccator under reduced pressure, and weighed. The results are shown in Table 1.

Table 1 ─────────────────────────────── * ○ Strain name Amount produced (mg) 表────────────────────────── Aureobasidium pullulans IFO 4464 50 IFO 7757 4200 IFO 4875 500 ───────────── Aureobasidium mansonii IFO 6421 150 IFO 9233 3800 ATCC 14249 50 ────────── Aureobasidium microstictum IFO 32066 920 IFO 32068 450 IFO 32069 1200 ───────────────────────────── ── Aureobasidium SP. A-91 8300 ○ ○ * The product is weighed as calcium salt

Example 2 For the recovered product of Example 1, the molecular weight was measured using G
As a result of analysis by the PC method (column: TSK gel G3000 PW _XL , mobile phase: 0.05 M phosphate buffer (pH 7.0), 25 ° C., detector: UV (210 nm) and RI),
The molecular weight varies depending on the strain and culture conditions, and
It was 50,000.

50 mg of the product was dissolved in 1N H ₂ SO ₄ solution 1
After dissolving in 0 ml, the solution was hydrolyzed in boiling water at 100 ° C. for 3 hours. The treated solution was subjected to paper chromatography (developing solvent, ethyl acetate: acetic acid: water = 50: 12: 1).
As a result of analysis according to 0), a single spot corresponding to the Rf value of malic acid was observed. Further, as a result of analyzing the organic acid by high performance liquid chromatography (LC-5A, manufactured by Shimadzu), a single peak indicating the retention time of malic acid was obtained. For example, Aureo Basidium Manthony
basidium mansonii ) High-performance liquid chromatography of acid hydrolyzate on the product recovered from the culture of IFO 9233 [column: HPX-87H (BIO-RA
D), column temperature: 60 ° C., solvent: 0.01 MH ₂ SO
₄ , flow rate: 0.7 ml / min, detector: UV (210 nm)] As a result of analysis, it showed the same retention time as the standard substance L-malic acid.

Further, the obtained hydrolyzate was analyzed using an L-malic acid measurement kit (manufactured by Boehringer Mannheim), and as a result, it was found to be the L-form.

From the above results, it was found that this product was obtained by polymerization of L-malic acid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 645)

Claims (3)

(57) [Claims] An L-malic acid polymer-producing bacterium belonging to the genus Oleobacedium is cultured, and the L-malic acid polymer is collected from the culture. Manufacturing method. 2. An L-malic acid polymer-producing bacterium belonging to the genus Oureobacedium, which is an Oleobacedium sp.
The method according to claim 1, which is an A-91 strain. 3. Oleobacedium sp. Having an ability to produce L-malic acid polymer. A-91 strain.