JPH037588A - Production of l-malic acid using fumarase - Google Patents

Abstract

PURPOSE:To obtain L-malic acid at low cost in high efficiency by making a specific cultured product act on fumaric acid in the presence of saponin to carry out reaction so as not to involve succinic acid as a byproduct. CONSTITUTION:A strain such as Thermus aquaticus ATCC 25105 strain is put to culture in a medium containing e.g. glucose to obtain a microorganism cultured product (A) capable of converting fumaric acid to L-malic acid. Thence, (B) fumaric acid and (C) saponin obtained by extraction from soybean or Saponaria officinalis are added to the component A to make a reaction to obtain a reaction product (D). The component D is then filtered and purified, thus producing the objective L-malic acid using fumarase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-malic acid using microorganisms, and more specifically, a culture of a microorganism capable of converting fumaric acid to malic acid is brought into contact with fumaric acid in the presence of sabonin. The present invention relates to a method for producing L-malic acid from fumaric acid without producing by-product systems such as cono-lyric acid while increasing the ability of the microorganism to produce L-malic acid.

(b) Industrial application field Provides a technology for producing high-purity malic acid that does not contain foreign organic acid succinic acid as a food additive by using the technology of the present invention from fumaric acid, which is supplied at low cost by the synthetic chemical industry. do.

(b) Prior Art Conventionally, as a method for producing L-malic acid from fumaric acid, a method typified by Japanese Patent Publication No. 37-4511 is known. Furthermore, it is well known that a simple reaction between microorganisms and fumaric acid produces impurity by-products, such as conical acid.

For the purpose of simplifying the purification process, improved methods such as a method using a bioreactor containing bile acids or their salts, which do not involve by-products such as succinic acid (Tokuko Kokō 5)
2-31952), and a method of activating immobilized microorganisms with a surfactant (see Japanese Patent Publications No. 52-8396 and Japanese Patent Publication No. 50-69289). These methods have not been able to efficiently manage the desired purpose, and the method proposed to solve this problem, namely, the bile acids used to inhibit by-product reactions, are extracted from animal organs. ,
There are many problems in practical use in terms of resource quantity, cost, stability, etc.

(c) Problems to be Solved by the Invention The present inventors aim to reduce the cost of the above-mentioned conventional technology by efficiently producing fumaric acid from L-apple without involving any by-product reactions that are easy to put into practical use. The aim is to develop a new technology to convert it into acid.

(d) Means for Solving the Problems The present inventors searched for resources in relatively inexpensive natural organic compounds, and as a result of extensive testing and investigation of their inhibitory effects on the reaction system that produces succinic acid from fumaric acid, they found that It was discovered that the original sabonin has an inexpensive and effective effect. The fact that these sabonins have an inhibitory effect on the by-product of succinic acid in the biological reaction of producing L-malic acid from fumaric acid by microorganisms has not been previously known, and this invention is the first such fact.

In the present invention, it is essential to have plant-derived sabonin present in the reaction system, and these are recently readily available as many commercially available food additives or medicines. As the sabonins used in the practice of the present invention, triterpenoid glycosides such as Soyasaponin obtained from soybeans and Quillaja sabonin extracted from Qullaja 5aponaria are inexpensive and exhibit effective effects. In addition, although there are practical problems in terms of cost, sabonins extracted from so-called Chinese herbal plants such as licorice (Glycyrhizae rad)
ix) Extract glycyridine (Gly-cyrrhizi)
n), Panax ginseng (Ginssen)
sabonin (S), an extracted glycoside of Mishimasaiko
A similar effect is also observed in drugs such as Aikisaponin). Therefore, in order to prevent the production of succinic acid by-products, it is possible to appropriately select from the sabonin group.

Microorganisms involved in the production of L-malic acid from fumaric acid include, for example, Micrococcus spp., Agromobacterium spp., Corynebacterium spp., and Brevibacterium spp., which have strong fumarase activity and are described in Japanese Patent Publication No. 44-1191. Strains such as these can be selected as appropriate from those available from strain repositories such as the American Type Culture Collection (ATCC) and the Fermentation Research Institute. Furthermore, by screening strains with strong fumarase activity from nature according to the method described in the same specification, practical strains can now be easily obtained. In addition, recently, the present inventors screened ATCC under high temperature conditions and found that the Thermus Rm strain, which performs the reaction at a high temperature, was
It has also been confirmed that it can prevent side reactions like strains such as 25105. The conversion of fumaric acid to malic acid by microorganisms can be easily carried out by conventional culture methods, but effective results can also be obtained by enzymatic reaction using immobilized microorganisms in a bioreactor. The culture and reaction conditions in this case follow known temperatures and pH, except for adding sabonins to the culture and reaction solution. The amount of the plant antioxidant active substance added to the culture and reaction solution varies depending on the type and purity of the plant component used, but in most cases, the main component is 0.
．． Add in a range of 0.05 to 0.5 g/dl. This active substance is relatively inexpensive even if it is a commercially available gourade, but it is sufficiently effective if it is partially purified and extracted from plants using the methods used to develop each active agent.

Embodiments of the present invention will be described below with reference to Examples, but these are merely illustrative and do not limit the present invention in any way.

Example 1゜Glucose 1.0g/<II, urea 0.2g/di, phosphoric acid-potassium 0, 2g/di, cornstarch liquor 0
．． 5 g/di, fumaric acid neutralized with calcium carbonate 10 g/di, and antifoaming agent Adekanol LG-109 (Asahi Denka: trade name) 0.5 ml/di.
was placed in a 500 m Erlenmeyer flask, and Brevibacterium ammoniagenes was added to this.
rium ammoniagenes) IAM1645
The bacterial strain was inoculated and cultured under rotary aeration at pH 7.0 and temperature 30'C. Chiranin P-2 10 hours after the start of culture
0 (Product name Koshabon no Kisuponin: manufactured by Maruzen Kasei) 1
The solution was added at a concentration of 20 mg/dQ, and the culture was continued for an additional 30 hours. Most of the fumaric acid in the culture solution was converted to L-malic acid and recovered as calcium salt, but no succinic acid was observed in the calcium salt obtained. The amount of L-malic acid produced in 100 ml of the fermentation-finished liquid was 11.30 g. Furthermore, when culturing was performed in parallel without adding Sunfood in the target test, the amount of succinic acid produced in this culture solution was 0.58/dl, which clearly indicates that polyphenol antioxidants It was also confirmed that side reactions were prevented.

Example 2゜ Thermus aquaticus (Thermus aquaticus) was used as the seed fungus.
aquaticus) ATCC25105 strain was used.

As a medium, urea 0.2g/di, KH2PO-0.2
g/dl, magnesium sulfate 50 mg/dl, cornstarch liquor Ig/dl, calcium fumarate 0.5 g/dL glucose 2, pH 7,
It was adjusted to 0. Fermentation was carried out at 30°C for 24 hours in a 2Of2 jar fermenter containing the above medium 10c. 200 g of maric acid, 181 g of calcium carbonate, and 2.0 g of Soybean Savonin Pure (trade name: manufactured by Inabata Kogyo) were added to the obtained culture solution, and the mixture was incubated at 35°C for 24 hours.
The reaction was carried out under stirring conditions of 360 r, p, m for hours, and the entire amount was left in a refrigerator overnight and filtered to obtain 255 g of crude crystals of calcium I,-malate. No by-product succinic acid was confirmed in this crystal.

Example 3 Microbacterium ammoniaph as a fermentation strain
Ilum) ATCC 13345 strain was cultured according to Example 2, and in the reaction solution composition, sabonin was formulated from soap tree (trade name: Tashiro Kogyo 180 mg/di, cetyl bilimcinium chloride 10 mg/dl). The reaction was carried out under the same conditions except that the obtained calcium I,-malate was 265
g, and no by-product succinic acid was observed using conventional analytical methods.

Example 4 The fumarase source Brebibacterium ammoniagenes (Brebibacterium ammoniagenes) obtained by culturing in Example 1
rium ammoniagenes)lFO1645
After washing the bacterial cells twice with physiological saline, 25 g and 800 mg of Iconine were kneaded into an aqueous solution of carboxylated chitosan to make a stock solution, which was then dropped into a mixed aqueous solution of 3% cobalt chloride through a nozzle with a pore size of 0.3 mm. Speed 3ml
/min to obtain immobilized bacterial cells. The entire amount of the immobilized bacterial cells was set in a 10Q capacity immobilized bioreactor. 10 g/12 fumaric acid pl (7,0 neutralized solution) containing 0.02% cetyl pyridinium chloride was continuously added at a flow rate of 10 mQ/min using a pump to react to L-malic acid. 14 g of calcium carbonate was added to the resulting reaction solution 1f2, and the total volume was concentrated to 173 ml, and the mixture was cooled overnight. 10.8 g of crystallized calcium chloride was isolated from the bones. Raw acid was in a range that could not be confirmed by quantitative analysis.

(Effects of the Invention) By controlling the enzymatic reaction by fumarase in the presence of the plant-derived glycoside sabonin, L-malic acid can be produced without producing succinic acid as a by-product.

Claims (2)

[Claims] (1) A method for producing L-malic acid using fumarase, which does not involve the by-product of succinic acid, by allowing a microbial culture capable of converting fumaric acid to L-malic acid to act on fumaric acid in the presence of sabonin. . (2) A method for producing L-malic acid in which a microbial culture and fumaric acid are reacted in the presence of sabonin extracted from soybeans or soap trees.