JPH07284395A - Production of poly-l-malic acid - Google Patents

Abstract

PURPOSE:To obtain the subject polymer being an in vivo decomposing and absorbing polymer compound in high yield by reacting a poly-L-malic aid- producing yeast cell belonging to the genus Aureobasidium or its immobilized material with a saccharide raw material under specific conditions by a method directly using the yeast cell in stationary state as an enzymatic catalyst. CONSTITUTION:This method for producing poly-L-malic acid comprises reacting a poly-L-malic acid-producing yeast cell belonging to the genus Aureobasidium, e.g. Aureobasidium sp. A-91 (FERM P-11966) or its immobilized material with an aqueous solution containing a saccharide raw material and iron ion by a method directly using the yeast cell in stationary state as an enzymatic catalyst.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a polymer of L-malic acid (Poly-L-Malic Acid, hereinafter sometimes abbreviated as "PMLA"), and more specifically, it is obtained by culturing. The microbial cells are recovered and the microbial cells are allowed to act on an aqueous solution of a composition in which the microbial cells cannot grow, so that the quiescent bacterial cells are used as they are as an enzyme catalyst (hereinafter, this method is referred to as “rest cell method”). Sometimes) L-malic acid polymer.

[0002] PMLA is a biodegradable and absorbable polymer compound, which is a bioabsorbable suture, an osteosynthesis material, an artificial tendon,
It is expected to find many applications in the fields of medicine and medicine as a polymeric carrier for artificial blood vessels and drug delivery.

[0003]

2. Description of the Related Art PMLA is a chemical synthesis method using malic acid monobenzyl or monomethyl ester as a raw material [Reports
of the Faculty Engineering, Tottori University, 8,
124 (1977)], a chemical synthesis method of ring-opening polymerization of benzyl malolactonate (US Pat. No. 4,265,247), a direct thermal condensation method [Polymer Papers, 44, 701 (1987)].
A production method by a chemical synthesis method such as the above has been reported.

In the production of PMLA using microorganisms, Penicillium cyclium is used.
Example of production by solid culture using opium [Agricultural
Biological Chemistry, 33, 459 (1969)], a fermentation method using a microorganism of the genus Aureobasidium (Japanese Unexamined Patent Publication No. 4-111385), and an enzymatic method (Japanese Unexamined Patent Publication No. 4-341189). ing.

[0005]

However, in the production by the chemical synthesis method, there are drawbacks that not only the raw materials are expensive, but also the yield is low because many steps are required. On the other hand, the production by the solid culture method using the above-mentioned penicillium-cyclopium has a low yield and requires multiple steps for purification, so it cannot be said to be a practical method. Further, in the fermentation method and the enzyme method using a bacterium of the genus Aureobasedium, the yield is not sufficient for industrial production, and it has been desired to develop a method for producing a higher yield at a lower cost.

[0006]

The present inventors have found that PMLA
As a result of diligent examination of a method for producing a high yield of L., L-malic acid polymer-producing bacteria belonging to the genus Aureobasedium, at least containing sugar raw materials such as glucose and iron ions The present inventors have found that PMLA can be produced in a high yield by acting on an aqueous solution of a composition which is not obtained to cause an enzymatic reaction, and have completed the present invention.

That is, according to the present invention, the L-malic acid polymer-producing bacterium belonging to the genus Aureobasedium or an immobilized product thereof is used as at least one of the essential components for microbial growth containing at least a sugar raw material and iron ion. A method for producing an L-malic acid polymer, which comprises allowing an L-malic acid polymer to be produced and accumulated by acting on an aqueous solution containing no component, and collecting the L-malic acid polymer from the aqueous solution.

The method of the present invention will be described in more detail below. The microorganisms belonging to the genus Aureobasedium used in the method of the present invention also include microorganisms conventionally classified into the genus Pullularia and later reclassified into the genus Aureobasedium. These microorganisms are also collectively called black yeast [Mycopathologia et
Mycologia Applicata, 12, 1 (1959), same 17, 1 (196
2)]. Therefore, as long as it is a black yeast capable of producing PMLA, all of these microorganisms are included in the range of L-malic acid polymer-producing bacteria belonging to the genus Aureobasedium used in the present invention.

Specific examples of the above microorganisms include the following. Aureobasidium pullulans IFO446
4, the same IFO4465, the same IFO4875, the same IFO7
757, ATCC7305, Aureobasedium
Man Sony (Aureobasidium mansonii) IFO642
1, the same IFO8194, the same IFO9233, the same ATCC
14249, Aureobasidium microstictum IFO32066,
Same IFO 32067, Same IFO 32068, Same IFO 3
2069, Aureobasedium sp. A-91.

Among the above strains, aureobasedium
Species (Aureobasidium sp.) A-91 is entrusted by the Institute of Biotechnology, Institute of Industrial Science and Technology, Contract number: FERM
Deposited as P-11966. The above-mentioned L-malic acid polymer-producing bacterium belonging to the genus Aureobasedium is cultivated according to a commonly known method known per se using a medium containing nutrients such as a carbon source, a nitrogen source and an inorganic salt. be able to.

As the carbon source of the medium, for example, sugar raw materials such as glucose, fructose, sucrose and maltose, and among them, glucose is particularly preferably used, and the addition concentration thereof is 1 to 30% by weight, preferably 1 ~ 2
0 wt% is suitable. As the nitrogen source, a nitrogen-containing organic or inorganic substance usually used in culturing microorganisms,
For example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, organic acid ammonium salts such as ammonium succinate, urea and the like can be used alone or in combination, and as the inorganic salt, for example, potassium monohydrogen phosphate, phosphorus Potassium dihydrogen acid, magnesium sulfate and the like can be used.

In addition to the above, yeast extract, corn stay liquor, peptone, meat extract,
Nutrients such as casamino acids and various vitamins can be added to the medium. Culturing is performed under aerobic conditions such as shaking and aeration stirring, and the culturing temperature can be usually 20 to 40 ° C, preferably 22 to 35 ° C. Culture pH is usually 2
10, preferably 3 to 9, more preferably 3 to 7.

When carrying out the method of the present invention, cells can be collected from the culture obtained by culturing as described above, washed with water or an appropriate buffer, and then used as it is. Alternatively, the cells are immobilized by a method known per se,
It can be used as an immobilized product. Examples of the method for immobilizing microbial cells include a method using a polymer monomer such as acrylamide and a method of insolubilizing using a suitable carrier such as alginate or carrageenan.

The bacterium or the immobilized product thus obtained is an aqueous solution containing at least a sugar raw material and iron ions and not containing at least one component essential for microbial growth (hereinafter referred to as "aqueous reaction solution"). Sometimes, the L-malic acid polymer can be produced and accumulated in the aqueous reaction solution. The aqueous reaction liquid contains a sugar raw material and iron ions and lacks at least one component such as a nitrogen source, which is an essential component for cell growth, phosphoric acid, vitamins, etc., and the cells cannot be substantially grown. There is no particular limitation as long as it is an aqueous solution, and it is usually water containing a sugar raw material and iron ions, or a buffer solution such as phosphoric acid or tris hydrochloric acid.

The sugar raw material added to the aqueous reaction solution is not particularly limited as long as it can be assimilated by the microorganism used in the present invention, and examples thereof include glucose, fructose, sucrose, maltose and the like. Mention may be made, of which glucose is preferably used. The concentration of the sugar raw material added is not particularly limited, but is generally 0.
A range of 1 to 30% by weight, preferably 0.2 to 15% by weight is suitable.

As the iron ions added to the aqueous reaction solution, there are commonly used inorganic or organic salts of iron, such as iron sulfate, iron chloride, iron nitrate, iron citrate, iron oxalate and iron fumarate. Can be mentioned. The addition concentration is not particularly limited, but in general, the iron ion concentration is 0.01 to
A range of 50 mM, preferably 0.1 to 3 mM is suitable.

The resting cell reaction according to the present invention can be carried out generally at a temperature of about 20 to 50 ° C, preferably about 25 to 40 ° C, usually for about 10 to 170 hours. Further, the pH of the reaction solution is appropriately in the range of 2 to 10, preferably 3 to 9, and more preferably 3 to 7.

The PMLA produced and accumulated in the aqueous reaction solution by the above-mentioned resting cell method is collected from the reaction solution according to a commonly used separation / purification method known per se, for example, an ion exchange resin treatment method. , Precipitation methods such as acetone,
It can be carried out by appropriately combining an organic solvent precipitation method such as methanol, ethanol, n-propanol, and methyl ethyl ketone.

P produced by the method of the present invention described above
The physical properties of MLA are as follows. (1) The molecular weight measured by the GPC method (gel filtration method) is in the range of about 2,000 to 50,000. (2) Only L-malic acid is produced by the hydrolysis treatment with a 1N sulfuric acid solution.

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, PMLA can be obtained in a higher yield than the conventional fermentation method and enzyme method.

[0021]

EXAMPLES Next, the present invention will be described more specifically with reference to Reference Examples and Examples. However, the example below
The present invention is given only as an aid to gain specific recognition, and the present invention is not limited thereto.

Reference Example Medium (Glucose 8%, NH ₄ Cl 0.2%, KH _{2
PO 4 0.05%, MgSO 4 ·} 7H 2 O 0.05
%, Yeast extract 0.02%) was dispensed into a 500 ml Erlenmeyer flask and sterilized (121 ° C., 15 minutes). After adding 1 g of dry heat-sterilized calcium carbonate to the medium, Aureobasidium species (Aureobasid)
ium sp.) A-91 (FERM P-11966) was inoculated and shake-cultured at 25 ° C. for 3 days to obtain a preculture. Next, add 100 ml of the above medium to 1000 ml
After pipetting into an Erlenmeyer flask and sterilizing (121 ° C., 15 minutes), 3 g of dry heat sterilized calcium carbonate was added, 20 ml of the preculture was added, and shaking culture was carried out at 25 ° C. for 3 days. .

After the culture was completed, the culture was centrifuged (8,000
The cells were collected at 0 rpm for 15 minutes at room temperature) to collect the cells. The obtained bacterial cells were washed with a washing solution (100 mM phosphate buffer, pH 7.
It was washed once in 0).

Example 1 A certain amount of the bacterial cells obtained in the above Reference Example was added to a reaction solution (glucose 8%, 100 mM phosphate buffer solution) containing ferrous sulfate at each iron ion concentration shown in Table 1. After suspending and adjusting the cell concentration (turbidity A 580 nm = 30), 50 ml thereof was dispensed together with 1.5 g of calcium carbonate into a 300 ml Erlenmeyer flask and reacted at 30 ° C. for 3 days.

After the reaction is completed, the reaction product is centrifuged (8,
1 m of supernatant sterilized by 000 rpm, 15 minutes, room temperature)
1 ml of 2N H ₂ SO ₄ was added to 1 and hydrolyzed at 100 ° C. for 5 hours. The amount of PMLA produced was measured by high performance liquid chromatography (LC-5A manufactured by Shimadzu).
The organic acid (malic acid) is analyzed by [column: HP
X-87H (BIO-RAD), column temperature: 60 ° C,
Mobile phase: 0.01 MH ₂ SO ₄ , flow rate: 0.7 ml / min,
Detector: UV (210 nm)], and quantification was performed as the amount of malic acid.

The results are shown in Table 1. Free malic acid in the reaction solution supernatant before the acid hydrolysis treatment was similarly analyzed by high performance liquid chromatography, but no peak corresponding to malic acid was detected. From this, it was confirmed that all malic acid produced by acid hydrolysis is derived from PMLA.

[0027]

[Table 1] Table 1 Iron ion concentration (mM) PMLA amount (mg / ml) 0 10 0.5 22 22 1.0 23 2.0 2.0 24 3.0 24

Claims (2)

[Claims] 1. An L-malic acid polymer-producing bacterium belonging to the genus Aureobasedium or an immobilized product thereof, which contains at least a sugar raw material and iron ions and contains at least one component essential for cell growth. Not acting on aqueous solution L
-Malic acid polymer is produced and accumulated, and L-from the aqueous solution is accumulated.
A method for producing an L-malic acid polymer, which comprises collecting a malic acid polymer. 2. The L-malic acid polymer-producing bacterium belonging to the genus Aureobasidium is Aureobasidium sp. A-91.
The method described.