JPH02182193A - Production of 4-hydroxy-2-butynoic acid - Google Patents

Abstract

PURPOSE:To selectively obtain the subject compound useful as a starting substance, etc., for asperdiol which is an antitumor agent by culturing a microorganism belonging to the genus Rhinocladiella in a culture medium containing 2- butyne-1,4-diol. CONSTITUTION:A microorganism [e.g. Rhinocladiella atrovirens KY-201 (FERM P-10463)] belonging to the genus Rhinocladiella is cultured in a culture medium containing 2-butyne-1,4-diol at 20-35 deg.C for 10hr-7 days normally by a shaking culture method, etc., to afford the objective compound.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to a method for producing 4-hydroxy-2-butyric acid from 2-butyne-1,4-diol using a microorganism belonging to the genus Rhinocladia. 4-Hydroxy-2-butyric acid contains a hydroxyl group, a carboxyl group, and a triple bond in its molecule, so it is useful as a raw material for the synthesis of organic compounds.For example, it is used as a starting material for the antitumor drug asperdiol (Journal of Organic
Chemistry, 48°4785-4786, 1983
) is used as.

[Conventional technology]

Conventionally, methods for producing 4-hydroxy-2-butyric acid have been known, including the following chemical method and method using microorganisms. (1) A method of oxidizing 2-butyne-1,4-diol with chromic acid under acidic sulfuric acid using acetone aqueous solution as a solvent (USSR Patent No. 514806; Chemical Abstracts, 85.123360e, (1976)). (2) Oxidize 2-butyne-1,4-diol using non-spore-forming soil bacteria with polar flagella to produce 4-
Method for producing hydroxy-2-butyric acid (Fermentation Engineering Journal, 49(3), 202-205° (1971)
). The chemical production method (1) has problems in the treatment of wastewater containing highly toxic chromium, and countermeasures against pollution are required if it is to be implemented industrially. The method (2) using microorganisms is not suitable for industrial implementation because the yield of 4-hydroxy-2-butyric acid is low at less than 10%, the selectivity is low, and neutral compounds other than the target product are produced as by-products. is inappropriate. Furthermore, the microorganisms used in method (2) are only described as soil bacteria and have not been clearly identified mycologically.

[Problem to be solved by the invention]

An object of the present invention is to provide a method for fermentative production of 4-hydroxy-2-butyric acid with excellent yield and selectivity.

[Means and effects for solving the problems] In order to solve the above problems, the present inventors searched for microorganisms other than the bacteria known in the conventional method (2), and found that certain filamentous fungi
The present inventors have discovered that 4-droxy-2-butyric acid can be efficiently produced using butyne-1,4-diol as a substrate, and have completed the present invention. That is, the present invention uses 2-butyne-1,4-diol as a microorganism belonging to the genus Rhinocladiella that produces 4-hydroxy-2-butyric acid using 2-butyne-1,4-diol as a substrate.
Cultured in a medium containing diol, 4-hi)-roxy2-
The present invention relates to a method for producing 4-hydroxy-2-butyric acid, which comprises producing butyric acid and collecting it. The microorganisms belonging to the genus Rhinocladia used in the present invention include Rhinocladiella atrovirens K.
Y-801 (Rh1nocladiella atr
. virens K Y -801) strain [FERM P-10463]. Rhinocladiella atrovirens obtained by the present inventors■
The mycological properties of GuY-801 (Feikoken Bibori No. 10463) are shown below. Microscopic findings and culture findings on potato glucose agar media The growth is slow on potato glucose agar media, and exhibits a dark gray-olive woolly or felt-like appearance, with an olive-black underside. The colorless to brown hyphae have septa and may be present singly or in bundles. It branches from the hyphae or aerial hyphae to produce short conidiophores, and symbogeo-shaped conidia are formed at the tips of the conidiophores. Conidiophores are usually around 2.2 to 3 μm wide, but about 600 μm wide.
In some cases, it reaches μm. Some have a slight bulge in the middle. It may have a single septum in the middle, or it may branch to form a conidiophore. In many cases, several conidia are formed directly from the hyphae through short protrusions. Conidia are unicellular, obovate, synovoid, 1-4 x 4.5-
6μm, colorless to light olive or light brown, formed singly or in clusters. Because conidia form in clumps,
The typical symbogeotype was not clearly recognized, and it was thought that it was a modification of the genus Phalophora, but by observing the slide cultures over time,
It turned out to be a genus Rhinocladiella. Rhinocladiella atrovirens Nanfeld (Rh1nocladiel la
atrovirens NANNFELD). In the production of 4-hydroxy-2-butyric acid by the method of the present invention, a microorganism belonging to the genus Rhinocladiella that produces 4-hydroxy-2-butyric acid using 2-butyne-1,4-diol as a substrate is used to produce 2-butyne-1,4-diol. - It is carried out by culturing in a medium containing diol to produce 4-hydroxy-2-butyric acid. The method of carrying out the present invention includes a so-called fermentation method in which an inoculum of the microorganism of the present invention is added and cultured in a medium containing 2-butyne-1,4 diol and a nutrient source that the microorganism of the present invention can utilize. After the microorganism of the present invention is grown and cultured in a normal medium, the microorganisms obtained by centrifugation etc. are added and cultured in a medium containing 2-butyne-1,4-diol as the sole carbon source. There is a so-called enzymatic method. The culture concentration of 2-butyne-1,4-diol is usually 0.
It is carried out in the range of 5 to 10 g/ρ, preferably 2 to 5 g/ff. The culture method is not particularly limited as long as it is an aerobic culture method used for general microorganisms, but usually a shaking culture method or an aerated agitation culture method using a liquid medium is used. As a medium used for preculture in the enzymatic method,
Any nutrient source may be used as long as it contains a nutrient source that can be assimilated by Rhinocladiella microorganisms that produce 4-hydroxy-2-butyric acid using 2-butyne-1,4-diol as a substrate. That is, carbon sources include pentoses such as arabinose, glucose, mannose, fructose,
Hekitoses such as galactose, trisaccharides such as sucrose and maltose, starch decomposition products, sugar alcohols,
Polyhydric alcohols such as glycerin and 1,4-butanediol, polypeptone, peptone, meat extract, malt extract, yeast extract, various amino acids, organic acids, etc. are used. Further, as the nitrogen source, an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, or an organic nitrogen source such as polypeptone, peptone, meat extract, etc. is used. In addition, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate may be added. Culture conditions are not particularly limited, but shaking culture or aerated agitation culture is usually performed at 20 to 35°C for 10 hours to 7 days. In one embodiment of the enzymatic method, a microorganism of the genus Rhinocladia that produces 4-hydroxy-2-butyric acid is grown and cultured using 2-butyne-1,4 diol as a substrate in a medium containing the above nutrients, and then 2-butyne-1
, 4-diol aqueous solution and cultured with shaking or stirring with aeration. In addition, in the case of the fermentation method, 4-hydroxy-2-diol is used as a substrate using 2-butyne-1,4-diol.
It is sufficient to directly add a substrate to the microorganisms of the genus Rhinocladiella that produce butic acid. Furthermore, a microorganism of the genus Rhinocladia that produces 4-hydroxy-2-butyric acid using 2-butyne-1,4-diol as a substrate was immobilized on a carrier such as sodium alkinate, carrageenan, or urethane foam. It may also be used as a bacterial cell. In this way, in order to separate and collect 4-hydroxy-2-butyric acid that does not accumulate in the culture solution, the bacterial cells are separated from the culture solution by centrifugation, and then the centrifuged supernatant is directly concentrated under reduced pressure. However, the present invention is not limited to these methods. Hereinafter, the present invention will be explained in detail with reference to Examples.

【Example】

Example 1 Rhinocladiella atrovirens KY801 (Feikoken Bacteria No. 10463) was cultured by the method shown below. Glucose 2g, ammonium chloride 0.2g, potassium dihydrogen phosphate 0.02g, magnesium sulfate heptahydrate 0
．． 02g, potassium chloride O,OO2g, ferrous sulfate.
Heptahydrate 0.001g, manganese sulfate tetrahydrate 0.000
2 g of zinc sulfate heptahydrate, 0.0007 g of copper sulfate, and a trace amount of vitamin B1.hydrochloride, tap water was added to adjust the pH to 14 (pH 6.5), and this was used as a culture medium. 100 ml of this medium was weighed into a 500 ml volume Sakaro flask and sterilized in an autoclave at 120° C. for 20 minutes. Rhinocladiella atrovirens KY8 was added to this medium.
01 (Feikoken Bacteria No. 10463) was inoculated with a platinum loop,
The cells were cultured with shaking at 30°C in the dark for 4 days. After culturing, collect the bacteria using a 0.2 μm membrane filter to obtain 0.1 g (dry weight) of bacterial cells. This is 0.
10 ml of 2% aqueous solution of 2-phydine-1,4-diol
and cultured with shaking at 30° C., 112 rpm, in the dark for 2 days. The bacterial cells and supernatant were separated by centrifugation, and 4-hydroxy 2-butyric acid in the centrifuged supernatant was quantified by high-performance liquid chromatography. As a result, the production yield was 98.
%, and no butyndioic acid was produced. The infrared absorption spectrum, 13C nuclear magnetic resonance spectrum, and mass spectrum of the compound isolated and purified by direct dehydration and concentration of the centrifugal supernatant under reduced pressure all agreed with the structure of 4-hydroxy-2-butyric acid. IR(neat) max 3225cm-'(OH). 2237 cm-' (C=C). 1701cm-' (COOH) February 22, 1999

Claims (1)

[Claims] (1) Using 2-butyne-1,4-diol as a substrate, 4-
The method is characterized by culturing microorganisms belonging to the genus Rhinocladia that produce hydroxy-2-butyric acid in a medium containing 2-butyne-1,4-diol, producing 4-hydroxy-2-butyric acid, and collecting the same. A method for producing 4-hydroxy-2-butyric acid.