JPH03280888A - Production of one terminal monocarboxylic acid by microorganism - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the subject hardly available carboxylic acid having an odd number of carbon atoms at ordinary temperature under ordinary pressure by culturing a microorganism having specific properties in a culture medium containing a chain hydrocarbon. CONSTITUTION:A microorganism (e.g. FERM P-11391), capable of assimilating a chain hydrocarbon, having one terminal oxidizing ability and belonging to the genus Exophiala, Candida, Debaryomyces or Rhodotorula is cultured in a culture medium containing the chain hydrocarbon to preferably convert the chain hydrocarbon with microbial cells of the resultant microorganism or treated microbial cells. Thereby, the objective carboxylic acid is produced.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for producing a monocarboxylic acid at one end or a salt thereof from a chain hydrocarbon using microorganisms. Acids are widely used as raw materials for surfactants, stabilizers, and various esters in the fields of oil chemistry, petrochemistry, medicine, agrochemicals, cosmetics, food, and the like.

[Conventional technology]

Conventionally, when producing monocarboxylic acids at one end, particularly higher monocarboxylic acids at one end, a method using natural animal and vegetable fats and oils such as beef tallow and coconut oil as raw materials has been mainly carried out. However, these natural oil and fat raw materials have problems such as fluctuations in price and limited supply. Furthermore, single-end monocarboxylic acids obtained from natural fats and oils are generally a mixture of multiple types of single-end monocarboxylic acids, and the number of carbon atoms is also limited to an even number. In addition, although methods for synthetically producing them from petroleum-based raw materials are known, they have not yet been widely implemented industrially.

[Problem to be solved by the invention]

In recent years, it has become clear that certain monocarboxylic acids at one end have biological activity, and by applying biotechnology that utilizes specific high selectivity of microorganisms, only certain monocarboxylic acids at one end have biological activity. There is a growing demand for manufacturing. In particular, non-natural monocarboxylic acids with an odd number of carbon atoms are attracting attention as biologically active monocarboxylic acids at one end, and methods for producing monocarboxylic acids at one end with an odd number of carbon atoms are attracting attention. development is desired.

[Means to solve problems]

In order to solve these technical problems, the present invention searches for microorganisms in nature that have the ability to assimilate chain hydrocarbons and produce monocarboxylic acids at one end, and produce monocarboxylic acids at one end. As a result of careful consideration of the method,
A plurality of microorganisms having the ability to oxidize one end belonging to the genus Exifiala, Candida, Debaryomyces, or Rhodotorula were discovered, and the present invention was accomplished.

In particular, all of these strains have the ability to produce not only monocarboxylic acids at one end with an even number of carbon atoms, but also non-natural monocarboxylic acids at one end with an odd number of carbon atoms. It is worth noting that.

The microorganism used in the present invention is KO-
101 strain, KO-105 strain, KO-107 strain, and KO-109 strain, which have the mycological properties shown in Table 1 below.

(Margins below this page) When the above mycological properties are classified based on the bacterial classification book for yeast bacteria, "The Yeasts, 3rd edition," strain KO-101 is classified as a member of the genus Exophiala.
iala), and the KO-105 strain is of the genus Candida (C
andida), and the KO-107 strain belongs to the genus Debaryomyces (
Debaryomyces + and 0-109 strains were identified as microorganisms belonging to the genus Rhodotorula.

These microorganisms have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the following numbers.

Bacterial strain Deposit number KO-101 Microtechnical Research Institute No. 11391 KO-105 Microtechnology Research Institute No. 11390 KO-107 Microtechnology Research Institute No. 11
No. 389 KO-109 Microtechnical Research Institute No. 11388 The present invention provides, in addition to the use of microorganisms having single-terminal oxidation ability belonging to the genus Exophiala, genus Candida, genus Debaryomyces, or genus Rhodotorula. For example, this includes the use of so-called mutant strains that have been subjected to known mutation treatments such as X-ray irradiation, ultraviolet irradiation, and treatment with mutating agents such as N-methyl-N-nitrosoguanidine. The hydrocarbon may have an even or odd number of carbon atoms, and may be saturated or unsaturated. Further, branched hydrocarbons can also be used.

The one-terminal monocarboxylic acid in the present invention is produced using a chain hydrocarbon as a carbon source, a nitrogen source, and an inorganic salt.

The microorganism of the present invention is cultured aerobically using a medium containing appropriate vitamins and other nutrients.

The chain hydrocarbon used in the present invention is, for example, chain paraffin, and the chain hydrocarbon may be produced by chemical synthesis and/or obtained by fractional distillation of petroleum. , may be used as a single species depending on the purpose of producing one-terminal monocarboxylic acid,
It may also be used as a mixture.

The nitrogen source used in the culture medium of the present invention may be an inorganic nitrogen source such as ammonium sulfate, ammonium nitrate, urea, or yeast extract.

It may be an organic nitrogen source such as meat extract, peptone, etc.

The pH of the medium is usually 4.5-9. Preferably the temperature is 6 to 8°C, usually 20 to 35°C2, preferably 25 to 32°C.

Cultivation can be determined as appropriate depending on the growth status of the microorganism, the purpose of producing the monocarboxylic acid at one end, etc., but is usually carried out under aerobic conditions for 1 to 14 days.

Furthermore, in the production of the monocarboxylic acid at one end in the present invention, microorganisms cultured using the above-mentioned medium are subjected to centrifugation, for example.
The microorganisms are collected by a method such as filtration, suspended in water or a buffer such as a phosphate buffer (e.g., pH 6 to 8), and a chain hydrocarbon is added to the suspension to coexist under aerobic conditions. This can be carried out by a reaction using so-called resting bacterial cells, or by using a treated bacterial cell material containing enzymes, such as crushed bacterial cells.

Furthermore, the above reaction can also be carried out by immobilizing the bacterial cells or enzymes according to a conventional immobilization method using, for example, acrylamide gel or calcium alginate.

As described above, according to the present invention, one-terminated monocarboxylic acid can be produced, but modifications that are normally carried out according to the spirit of the present invention are included within the scope of the present invention.

Next, the monocarboxylic acid product of one terminal oxidation in the present invention was detected by gas chromatography. The gas chromatographic filler used must be selected depending on the target monocarboxylic acid, but for example,
For the detection of tridecanoic acid, pentadecanoic acid, and heptadecanoic acid, which are one-end oxidation products corresponding to the linear hydrocarbon tridecane, pentadecane, and heptadecane in 3.15 and 17, respectively, use EFAP as the liquid phase, for example. It can be easily detected by using a fused cylinder force capillary column with an inner diameter of 0.53+Ily* and measuring at a gas chromatography column temperature of, for example, 195°C.

〔Effect of the invention〕

The present invention utilizes microorganisms belonging to the genus Exophiala, Candida, Debaryomyces, or Rhodotorula, which assimilate chain hydrocarbons and have the ability to oxidize one end. It relates to a method for producing carboxylic acids, which can not only be carried out at room temperature and pressure, but also capable of producing monocarboxylic acids at one end, which are difficult to obtain from nature, such as monocarboxylic acids with an odd number of carbon atoms. It has industrially useful effects.

〔Example〕

The present invention will be explained below with reference to examples, but it goes without saying that these examples do not limit the scope of the present invention.

Example 1 Tridecane (13 carbon atoms), a chain hydrocarbon, was used as a starting material.
), pentadecane (15 carbon atoms), and heptadecane (17 carbon atoms) in the medium shown in Table 2 below.
391), Candida sp. KO-105 strain (FEI Bacteria No. 11390), Debaryomyces sp. KO-107 strain (FEK Bibliography No. 11388), or Rhodotorula sp. 3 each
After culturing with shaking at 0°C for 7 days, the culture solution after 7 days was extracted with acidic ethyl using a conventional method and analyzed by gas chromatography, and it was confirmed that the following one-terminal monocarboxylic acids were produced by each microorganism. . The results are summarized below.

KO-101 stock KO-105 stock KO-107 stock KO
-109 elliptolic acid + ++
+ Pentadecanoic acid + +++ Hebutadecranic acid + +++ Furthermore,
A typical gas chromatography detection chart is shown in Fig. 1. FIG. 1 shows the acidic ether fraction analyzed by gas chromatography from a 10-day culture solution of Candida sp. In FIG. 1, the horizontal axis indicates retention time. The solid line indicates the production of monocarboxylic acid at one end, which is an oxidation product extracted from the culture solution. The column temperature was kept constant at 195°C. Incidentally, even if the same method is used to analyze the culture at the start, the peak of monocarboxylic acid at one end, which is an oxidation product, cannot be detected at all.

2nd surface chain hydrocarbon” 0°5% (NH4) zsOa 0.1%KJP
On 0.2%KH, P0.
0.1%Mg5Oa 4Hz0 10
0ppm100pp ・2Hz0 10pp
mFeSO4-7H2010pp+n ZnSO410ppm nZnS0410pp Yeast extract 30ppm Meat extract 30ppm Peptone 30ppm Book Equal weight mixture of tridecane, pentadecane and hebutadecane Main chapter pH 6,8 Example 2 113
91), Candida sp. KO-105 strain (Feikoken Bacteria No. 11390), Debaryomyces sp. KO-107 strain (
Microtechnical Research Institute No. 11389), or Rhodotorula spp. K
Four strains of O-109 strain (Feikoken Bibori No. 11388) were cultured with shaking using the medium (IOM) of Example 1 above, and the bacterial bodies of each strain were centrifuged from the culture. The bacteria were collected using the buffer solution 10 shown in Table 3 below. d to prepare a resting cell suspension of four types of cultured cells.

To this was added 5000 ppm of an equal weight mixture of chain hydrocarbons consisting of tridecane, pentadecane, and heptadecane, and the mixture was reacted at 30°C with shaking for 24 hours.

During this time, the reaction solution was sampled and extracted with acidic ether using a conventional method, and it was confirmed that a monocarboxylic acid at one end was produced. The results are summarized below.

(Margins below this page) 0 101 shares 0 105 shares KO-107 strain 109 shares Tridecanoic acid bentatecanic acid Hebuta fb acid + + ten + + ten + + + KH2PO.

K, HPO4 MgSO4・7H20 CaC1z ・2) 1z.

aCI FeSOn 4HzO NazMoOn Na2venge 04 nSO4 calcium pantothenate Inositol Table 3 0.2g/1 1.6g#! 0.2g/1 0.1g/1 0.1g/1 10mg/42 0.5mg/1 0.5mg/j! 0.5mg/1 400μgノ! 2000 μgel Niacin p-7 minobenzoic acid Pyridoxine hydrochloride Thiamine hydrochloride ri f flap pin biotin Vitamin B12 400μg/f 200μg71 400μge1 400μg/42 400μg/1 2μg72 0.5μg/f

[Brief explanation of drawings]

FIG. 1 is a diagram showing a detection chart of gas chromatography analysis.

Claims (1)

[Scope of Claims] 1. Microorganisms belonging to the genus Exophiala, Candida, Debaryomyces, or Rhodotorula that assimilate chain hydrocarbons and have the ability to oxidize one end in a medium containing chain hydrocarbons. 1. A method for producing a monocarboxylic acid at one end or a salt thereof, which comprises culturing to produce a monocarboxylic acid at one end. 2. Microorganisms belonging to the genus Exophiala, Candida, Debaryomyces, or Rhodotorula that assimilate chain hydrocarbons and have the ability to oxidize one end are cultured in a medium containing chain hydrocarbons. 1. A method for producing a monocarboxylic acid at one end or a salt thereof, the method comprising converting a chain hydrocarbon into a monocarboxylic acid at one end using microorganism cells or a treated product of the microorganism. 3. The method for producing a monocarboxylic acid at one end or a salt thereof according to any one of claims 1 to 2, wherein the monocarboxylic acid at one end has an odd number of carbon atoms.