JPH0343090A - Production of n-alkanol - Google Patents

Abstract

PURPOSE:To selectively obtain 1-12C n-alkanol useful as a synthetic intermediate for various chemicals by reacting an n-alkane with an enzyme in the presence of a cell containing methane monoxygenase poisoned with cyclopropane in an aqueous medium. CONSTITUTION:In reacting an n-alkane with an enzyme in the presence of a cell containing methane monoxygenase, in an aqueous medium, methanol dehydrogenase is previously poisoned with cyclopropane. Methylosinuse trichospporium OB3b (FERM P-4981) may be cited as the enzyme.

Description

[Detailed description of the invention] Industrial use! The present invention relates to a method for selectively producing n-alkanols having 1 to 12 carbon atoms, which are useful as synthetic intermediates for various chemical products, by microbiological oxidation of n-alkanes.

Conventional techniques and their problems Microbiological methods (fermentation methods) are known as methods for oxidizing alkanes under mild conditions, but a method for selectively producing lower alkanols directly from alkanes has not been established. Not yet.

The types of alcohol that can be obtained by fermentation methods are extremely limited, while the production of alcohol by synthetic methods requires many reaction steps.

Methane oxygenase (hereinafter abbreviated as MMO) is known to effectively act on methane, the most inactive substrate, to produce methanol.

The method for completely isolating MMO is complicated, and for its industrial purposes, microbial cells harboring MMO are usually used as they are.

Bacterial cells that contain MMO also contain alcohol dehydrogenase, etc. For example, in the case of methane, the methanol produced by oxidation changes to formaldehyde, which is then normally oxidized to carbon dioxide via formic acid. be.

Hethylococcus capusulatus
, )fethylosinustricospori
Although MMO has been isolated and purified from methane-assimilating bacteria such as um OB 3 b, the isolated MMO is extremely unstable and is inappropriate to be used as it is for methane oxidation.

On the other hand, since the microbial cells containing MMO are stable, by using these microbial cells as they are, they can be used for a long time.

However, when the bacterial cells are used as they are, methanol is further oxidized to carbon dioxide due to the action of methanol dehydrogenase (MD) present within the bacterial cells.

Alkanes other than methane are not oxidized to carbon dioxide gas, but various oxidation products such as aldehydes, ketones, and epoxides are produced, and it is difficult to stop the reaction at the alcohol stage.

・Means for solving the problem As a result of intensive search for a method to selectively inhibit only the action of alcohol dehydrogenase and make only MMO work effectively, the inventor found that after culturing the bacterial cells, they were cultured in cyclopropane gas in advance. The inventors discovered that the objective could be achieved by treating the liquid and completed the present invention.

That is, the present invention is characterized in that methanol dehydrogenase is poisoned in advance with cyclopropane when n-alkanes and oxygen are reacted in an aqueous medium in the presence of methane monooxygenase-carrying bacterial cells.
-Provides a method for producing alkanol.

The details of the mechanism by which cyclopropane selectively inhibits alcohol dehydrogenase are not clear, but the cyclopropyl alcohol produced when cyclopropane is oxidized by MMO is adsorbed to the active site of alcohol dehydrogenase and becomes a coenzyme. It is presumed that it reacts with a certain pyrroloquinone quinoline and causes deactivation.

Methane monooxygenase used in the method of the present invention is a methane-assimilating bacterium that can utilize methane as a carbon and energy source [generally methylomonas
rophs). ] is known to exist widely.

The enzyme source used in the present invention is not particularly limited, and for example, Methylomonas metanica
hanica), Methylococcus capsulatus (Bass strain) [Hethylococcus caps]
ulatus (Bath> ), Methylomonastricosborium (OB 3 b ) (Hethylos
inus trichosporium), Methylosinus nis p. CRL31 (Hethylosinu
s Sp, CRL 31), etc., and for example, Methylocinus trichosporium 0B3b (Fiber Science and Technology Research Institute No. 4981) is preserved at Tottori University Research Institute.

As a medium for culturing methane monooxygenase-producing bacteria, either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and, if necessary, a small amount of nutrients.

Cultivation is carried out in the presence of methane under aerobic conditions such as shaking culture or aerated agitation culture. Culture temperature is 20-40
The temperature range is usually about 30°C. The 0th mouth of the culture solution is preferably in a neutral range.

Methane monooxygenase can be extracted from cultured bacterial cells using known methods such as 1. J, old ggins et al.

J, General Microbiology 12
5. 63-72 (1981).

However, in the method of the present invention, methane oxygenase does not necessarily have to be an extracted pure enzyme.

That is, a culture of methane monooxygenase-producing bacteria,
Live bacterial cells collected from a culture by methods such as centrifugation, dried bacterial cells, processed bacterial cells obtained by grinding, autolysis, ultrasonication, etc., and extractions from these bacterial cells. Crude products of enzymes obtained from extracts of these products can also be used.

Of course, the above-mentioned enzyme or enzyme-containing crude product may be insolubilized (immobilized 1 mmobillized).

The method of the present invention involves contacting n-alkane as a reaction substrate with air in a sterilely prepared buffer solution at around 0, and then inoculating a small amount of the above-mentioned bacterial cells or an enzyme-containing solution. It is done.

The reaction can be carried out under mild conditions, ie, under normal pressure and at a temperature around 30°C.

The contact between methane or n-alkane and air may be carried out batchwise or continuously.

The progress of the reaction is analyzed by gas chromatography, and the reaction is stopped when the selectivity of the target n-alcohol reaches an optimum value, and the n-alcohol is separated and recovered by a conventional method.

Example [Culture of methane monooxygenase producing bacteria] An inorganic medium containing the following components was prepared.

Ingredients: 2 HPO to the amount dissolved in 11 water
40,79 ni mouth 2 PO40,54g MCJSO4・7 days 20 1.0gN mouth 4
C, Q o, 5gFeSO
4-7 120 4
myCa(, Q -2 days 20 0.2
yZnSO4-7020100μ9 MnC,! ! -4 days 20 30μ9
13 BO2300μ 9 CoCu ・6020 200μCuC
fJ2 ・20,0 10μ9N i (
jl 2 ・6020 20μ3Na2
MnO4-202030 μy Metylosinus
Trichosporium 0B3b was inoculated, methane gas and air were introduced at a volume ratio of 1:1, and cultured with shaking at 30'C for 20 hours.

[Poisoning of Bacterial Bodies with Cyclopropane After culturing Comethylotophus, the bacterial cells collected by centrifugation were washed with a 0.1M phosphate buffer solution (proa, O) and suspended in the same buffer.

Next, the air was replaced with cyclopropane, and the bacterial cell suspension was vigorously stirred at 30°C while in contact with cyclopropane. Thereafter, cyclopropane was removed from the system by bubbling helium gas and used for the next reaction.

Example 1 [Methane oxidation] 0.1M phosphate buffer (7.0
) 2.0d, bacterial cell suspension 6-Bm in terms of dry weight
After adding 490 μmol of sodium formate and sealing the container, remove the air from the container and add 1.5 rn of cyclopropane.
The mixture was stirred at 30° C. for 5 minutes. Thereafter, cyclopropane was removed by bubbling helium gas for 2 minutes.

Next, 1×10 −4 mol of methane and ax “to” mol of oxygen were charged into a reaction vessel and a reaction was carried out for a predetermined period of time.

After 2 hours, 2×10' mol of methanol had been produced.

Comparative Example 1 The reaction was carried out in the same manner as in Example 1 except that the cyclopropane treatment was not carried out. Comparative results showed that only 0.4 x 10'mol of methanol was produced after 1 hour, and could not be detected after 2 hours. Ta.

Example 2 [Oxidation of ethane 1] 1 x 10' 1 lot of ethane was charged instead of methane and the reaction was carried out for a predetermined period of time in the same manner as in Example 1.
15 x 10'mol of ethanol was produced in 50 minutes. There was very little acetaldehyde by-product.

Comparative Example 2 A reaction was carried out in the same manner as in Example 2 except that the cyclopropane treatment was not carried out, and a comparison was made. In 50 minutes, ethanol and acetaldehyde were co-concentrated, 5 x 1
Q'''6mol producedLTcf'j, ethanol disappears in 75 minutes and acetaldehyde becomes 12X10'mol.
l generation site.

Example 3 [Oxidation of Propane] A reaction was carried out in the same manner as in Example 1, using ix'to-4mof propane instead of methane.

After 100 minutes, 1×10' mol of 1-propanol was produced, and no propionaldehyde was detected.

Comparative Example 3 A reaction was carried out in the same manner as in Example 3 except that the cyclopropane treatment was not performed. After 100 minutes, 5x 10' mol of 2-propatool and 2x acetone
10' mol was produced.

Example 4 [Oxidation of Butane] A reaction was carried out in the same manner as in Example 1 except that 1×lO−4 mol of butane was charged in place of methane.

0 after 2 hours. The formation of 1-butanol of I X1Q' mo + was detected, and no 1-butyraldehyde was detected.

Comparative Example 4 A reaction was carried out in the same manner as in Example 4 except that the cyclopropane treatment was not performed. After 2 hours, 2-butanol and 2-butanone were produced in an equal amount of 5×10' mol.

Claims (1)

[Claims] 1. A method for producing n-alkanol, which comprises reacting n-alkane and oxygen in an aqueous medium in the presence of methane monooxygenase-carrying microbial cells poisoned with cyclopropane.