JPH09282A - Stabilization of monooxygenase activity - Google Patents

Abstract

PURPOSE: To inhibit the reduction in monooxygenase activity with the passage of time, caused in conversion reaction by adding a specific aliphatic compound as a stabilizer for monooxygenase activity to the oxidation system for an aromatic compound utilizing a microorganism having the monooxygenase activity. CONSTITUTION: A microorganism in Mycobacterium, catalyzing the reaction to endow oxygen to an aromatic compound, is utilized and an aliphatic compound of 3-10 carbon atoms having the action to induce the monooxygenase activity, for example, propane, butane, pentane, acetone, methyl ethyl ketone, methyl propyl ketone, methyl butyl. ketone, isopropanol, 2-butanol or 2-pentanol is added to the reaction system to carried out the oxidation reaction.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for stabilizing monooxygenase activity. More specifically, the present invention relates to a method for suppressing the time-dependent decrease in monooxygenase activity that occurs during a substance conversion reaction using a microorganism having a monooxygenase activity that catalyzes a reaction that imparts oxygen to an aromatic compound. is there.

[0002]

BACKGROUND ART Monooxygenase is also called a monoatomic oxygenase, and a typical one is hydroxylase. It is a type of oxygenase that catalyzes the reaction of introducing monoatomic molecular oxygen into a substrate. Utilizing this activity, production of useful substances by various substance conversions is known. Methane-utilizing bacteria are known as microorganisms having a monooxygenase activity (JP-A-54-157,895, JP-A-57-65,187, JP-A-58-4).
7,496, JP-A-58-193,691 and JP-B-62-47,517). It is also known that microorganisms that have completely different mycological properties from methanotrophic bacteria and that assimilate alkanes with two or more carbon atoms also have monooxygenase activity that oxidizes various compounds. (JP-A-60-210,991, JP-A-60-210,992 and JP-A-63)
-133,980), and further, JP-A-59-1.
No. 7,980 describes the epoxidation of alkenes, the oxidation of alkanes, the oxidation of alicyclic compounds and the oxidation of aromatic compounds using cells grown in ethane, propane and butane.

Oxidation reactions of various compounds can be carried out using the cells having these monooxygenase activities. However, when the compound that oxidizes (reaction substrate) does not have the effect of inducing the activity of monooxygenase, the reaction rate is short in the short time from the start of the reaction with any of the microorganisms listed above. Although it is constant, the instability of monooxygenase is a problem in that if the reaction is early, the reaction rate starts to decrease within 1 hour and the reaction eventually stops.

In order to solve the above problems, a method of recovering the activity by recovering the cells once the activity is once reduced, inoculating them again into a medium capable of inducing monooxygenase activity, and treating them for a certain period of time, Methods have been devised, such as a method of sequentially exchanging the cells with high activity. However, the former method of recovering the activity that has been once lost causes problems such as the fact that culture that is substantially equivalent to bacterial cell culture is required, and that the operation becomes complicated and extra equipment and labor are required. Also,
The latter method, in which cells with high activity are sequentially exchanged, is economically disadvantageous because the cells prepared over a very long period of time compared to the reaction duration are used and discarded. There is a drawback that.

Therefore, it is considered that this problem can be fundamentally solved only by developing a method for suppressing the decrease of the monooxygenase activity of the bacterial cells and prolonging the reaction duration. Therefore, a method capable of efficiently oxidizing aromatic compounds by suppressing the decrease in monooxygenase activity has not been proposed yet, and development of such a method is still strongly demanded.

[0006]

Therefore, the present invention is directed to the activity of the monooxygenase activity over time in a substance conversion reaction using a microorganism having a monooxygenase activity which catalyzes the reaction of imparting oxygen to an aromatic compound. It is an object of the present invention to provide a method for efficiently and simply suppressing the decrease.

[0007]

The present inventors have discovered a novel microorganism belonging to the genus Mycobacterium which has the ability to convert an aromatic compound into a phenol derivative or the like. When this microorganism is subjected to the oxidation reaction of an aromatic compound, like the conventionally known microorganism having monooxygenase activity, the initial reaction rate is maintained for only 10 hours, and thereafter the reaction rate gradually decreases. It caused the problem of being lost. Therefore, in order to solve the above problems, the present inventors have elucidated the cause and activity of a decrease in monooxygenase activity that occurs during a conversion reaction for obtaining a phenol derivative or the like from an aromatic compound using a reaction by monooxygenase. We have conducted intensive studies on the development of a method for improving the stability of the.

[0008] As a result of the investigation, it is found that the activity decreases with time not only due to the inactivating action of the enzyme by the reaction substrate and the reaction product as reported previously but also due to the physiological action of the bacterial cell. For the first time, it was clarified that it was due to the phenomenon of being told. That is, the decrease in activity is such that the inducible substrate, which is a substance capable of inducing monooxygenase activity in the environment in which the cells are placed, disappears or is reduced to a concentration at which the effect is not exerted. He identified what happened. Furthermore, it was strongly suggested that the degradation of monooxygenase by bacterial cells may be one of the causes of the decreased activity. At this time, it is considered that the protease that is a proteolytic enzyme decomposes the monooxygenase, but since the activity does not decrease in the environment where a sufficient amount of the inducing substrate for the monooxygenase exists, the protease is a monooxygenase. It was considered to be an inducible enzyme that is biosynthesized under the condition that the concentration of the inducing substrate of E. coli was lower than the effective concentration. Therefore, it was considered that the reduction of the monooxygenase activity could be suppressed and therefore the activity could be maintained by taking measures to make this inducing substrate exist in the reaction system at a concentration higher than the effective concentration.

Based on these research results, 3-1 which has an effect of inducing monooxygenase activity as an inducing substrate
The inventors have devised a method for preventing a decrease in monooxygenase activity by adding an aliphatic compound having 0 carbon atoms in a predetermined concentration or more. In this way, we found a method that can effectively suppress the decrease in monooxygenase activity,
We succeeded in improving the stability of the activity, which
The present invention has been completed.

That is, the above-mentioned object has the effect of inducing a monooxygenase activity and has a carbon number of 3 in a substance conversion reaction system utilizing a microorganism having a monooxygenase activity which catalyzes a reaction of imparting oxygen to an aromatic compound. It is achieved by a method for stabilizing monooxygenase activity, which is characterized in that the above reaction is carried out by adding an aliphatic compound of 10 to 10.

In the present invention, the above aliphatic compound is propane,
It shows a method for stabilizing the monooxygenase activity which is at least one selected from the group consisting of butane, pentane, acetone, methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, isopropanol, 2-butanol and 2-pentanol. . The present invention also shows a method for stabilizing the monooxygenase activity in which the above-mentioned aliphatic compound is present at a concentration in the range of 10% by weight to 200% by weight with respect to the initial concentration of the aromatic compound in the reaction solution. is there. The present invention further provides that the compound produced by applying oxygen to the aromatic compound is a phenol derivative having a hydroxyl group in the aromatic ring, an aromatic carboxylic acid having a carboxyalkyl group in the aromatic ring and an aromatic ring. It shows a method for stabilizing the monooxygenase activity which is selected from the group consisting of aromatic ketones having an alkyl ketone group.

The present invention further provides that the microorganism having a monooxygenase activity is Mycobacterium.
The method for stabilizing monooxygenase activity according to any one of the above, which is a microorganism belonging to the genus.

[0013]

The method for stabilizing the monooxygenase activity of the present invention comprises the steps of carrying out an oxidation reaction of a desired compound using a microbial cell having a monooxygenase activity which catalyzes a reaction of imparting oxygen to an aromatic compound, The reaction system is characterized in that an aliphatic compound having an effect of inducing monooxygenase activity and having a carbon number of 3 to 10 is present as an inducing substrate in a predetermined concentration or more and is cultured.

Hereinafter, the present invention will be described in detail.

An embodiment of the method for imparting oxygen to an aromatic compound while stabilizing the monooxygenase activity according to the present invention will be described below.

[0016] Mycobacterium sp. NS12523 strain (Mycobacterium sp.
NS12523) 1 platinum loop is inoculated into 5 ml of a medium having the medium composition shown in Table 1 (hereinafter referred to as A medium) to which an appropriate carbon source is added, and the seed medium is prepared. Then, this seed culture is inoculated into a 500 ml Sakaguchi flask containing 100 ml of the medium having the same composition as above, and cultured until the growth reaches the stationary phase to prepare a culture containing a fixed amount of cells. Centrifugation (1,000-10,000 xg, 5
~ 20 minutes) to recover the bacterial cells, and further added a carbon source having an effect of enhancing the monooxygenase activity.
Active cells having a high monooxygenase activity are prepared by inoculating and culturing the cells in a 500 ml Sakaguchi flask containing 100 ml of the medium. By centrifuging the culture solution (1,000 to 10,000 × g, 5 to 20 minutes), only the active cells are recovered from the culture solution, and if necessary, with water, buffer solution or physiological saline. Wash the cells. The active cells thus recovered were treated with a phosphate buffer solution (containing 0.2 g / liter of magnesium sulfate, 5
0 mM KH ₂ PO ₄ solution; pH with sodium hydroxide
The cell concentration is 0.1 to 50 g wet weight / 1
00 ml, preferably 0.5-20 g wet weight / 100 m
The suspension is adjusted to 1 to prepare a bacterial cell suspension as a catalyst in the intended substance conversion reaction. 50 ml of this bacterial cell suspension was placed in a 500 ml flask, an aliphatic compound was added as an induction substrate, a substrate and an energy source were added, and the mixture was shaken under aerobic conditions at 20 to 35 ° C. for a desired time (50 to 50 ° C.). At 300 rpm), the substrate is oxidized by the monooxygenase to obtain the desired product (phenol derivative, aromatic aldehyde, aromatic alcohol, aromatic carboxylic acid, aromatic ketone, etc.).

[0017]

[Table 1]

In the present invention, an aromatic compound is used as the substrate used in the oxidation reaction by monooxygenase, although it varies depending on the kind of the target product. Specific examples thereof include phenol, acetanilide, benzonitrile, phenylacetonitrile, phenylalanine, salicylic acid, o-cresol, toluene, benzyl alcohol, p-xylene and p-cresol. In addition, phenol, acetanilide, benzonitrile, phenylacetonitrile, phenylalanine,
When salicylic acid, o-cresol, toluene, benzyl alcohol, p-xylene and p-cresol are used as substrates, hydroquinone, p-acetaminophenol, p-cyanophenol, p-hydroxyphenylacetonitrile, tyrosine, gentisic acid, respectively. ,
Methylhydroquinone, benzoic acid, benzoic acid, p-toluic acid and p-hydroxybenzoic acid are produced by the oxidation reaction of monooxygenase. The concentration of the substrate added is 100 to 50,000 ppm, preferably 500 to 30,000 ppm.

In order to carry out the oxidation reaction in the present invention more efficiently, it is preferable to add a substance serving as an energy source to the reaction solution. The energy source refers to a substance used to supply NADH and NADPH which are coenzymes necessary for the oxidation reaction.

In the present invention, the supply of an energy source for supplying reduced coenzymes NADH and NADPH necessary for the above-mentioned oxidation reaction, and the monooxygenase-inducing substrate and the monooxygenase activity decrease with time. An aliphatic compound having 3 to 10 carbon atoms is added to the above oxidation reaction system in order to supply the substance having the suppressing effect.

The above-mentioned aliphatic compound is not particularly limited as long as it has the effect of inducing monooxygenase activity, is a compound having 3 to 10 carbon atoms, and can be used for obtaining energy by microorganisms. . Specific examples thereof include alkanes such as propane, butane and pentane, ketones such as acetone, methyl ethyl ketone, methyl propyl ketone and methyl butyl ketone, and isopropanol, 2-butanol and 2-butanol.
Examples thereof include alcohols such as pentanol, and of these, propane, butane, acetone, methyl ethyl ketone and 2-butanol are preferably used. These aliphatic compounds may be used alone or in the form of a mixture of two or more kinds.

The concentration of the above-mentioned aliphatic compound added needs to be at least a concentration sufficient to improve the stability of the monooxygenase activity. Furthermore, since the aliphatic compound used in the present invention is also a substrate for monooxygenase, if added at a too high concentration, it may inhibit the desired oxidation reaction, so it is added within a range that does not adversely affect the reaction. Is desirable. Therefore, the addition concentration of the aliphatic compound is usually, for example, preferably within a range of 50 to 5,000 ppm and at a concentration that gives the best result in each oxidation reaction system.

Further, the aliphatic compound used in the present invention does not affect the stability of the monooxygenase activity, but may be used in combination as a substance that can be used as an energy source by the bacterial cells. At this time, the addition concentration of the aliphatic compound may cause competitive inhibition with the substrate to be oxidized because the aliphatic compound also serves as a substrate for monooxygenase. It must be within the concentration range
Furthermore, the amount of monooxygenase added may vary depending on the type of substrate to be oxidized and the affinity for the added aliphatic compound. For example, the addition concentration of the aliphatic compound is 10% by weight with respect to the initial concentration of the aromatic compound in the reaction solution.
Above, preferably adjusted to be present at a concentration in the range of 10 to 200% by weight. More specifically, when using methyl ethyl ketone as the aliphatic compound, the addition concentration of methyl ethyl ketone is 10% by weight or more, preferably 10 to 10% by weight based on the initial concentration of the aromatic compound in the reaction solution.
It is adjusted to be present at a concentration in the range of 200% by weight, more preferably 50-150% by weight. When acetone is used as the aliphatic compound, the addition concentration of acetone is 10% by weight or more, preferably 10 to 200% by weight, based on the initial concentration of the aromatic compound in the reaction solution.
More preferably, it is set so that it is present at a concentration in the range of 50 to 150% by weight.

In the oxidation reaction by monooxygenase in the present invention, molecular oxygen is required as an oxygen supply source.
Therefore, it is necessary to supply oxygen to the reaction solution consisting of the bacterial cell suspension, the substrate and the energy source in sufficient quantity. Examples of the method of supplying oxygen to the reaction solution include stirring, shaking, and aeration and aeration operations of air or oxygen in an aeration and agitation tank. The conditions for supplying oxygen by aeration stirring or shaking are not particularly limited, and are appropriately determined depending on the size and shape of the device used and the amount of cell suspension.

The oxidation reaction by the monooxygenase in the present invention is carried out within a pH range suitable for the properties of the substrate and the substance to be produced. In the present invention, the above pH
The range is 5 to 9, preferably 6 to 8. From this, the liquid for suspending the active cells that can be used in the present invention can be used as long as it is within the above-mentioned pH range, but as a typical example, a phosphate buffer, a dimethyl glutarate buffer and Tris. A hydrochloric acid buffer solution and the like can be mentioned. Also,
In addition to the above buffer solution, a saline solution such as physiological saline may be used. Furthermore, if the pH does not fluctuate significantly during the reaction, or if the pH can be controlled,
A buffer does not necessarily have to be used.

In the above-mentioned embodiment, the cells were separated by centrifugation, but the separation of the cells is not limited to this method, and can be carried out by a general method for separating cells such as filtration and sedimentation separation. Done.

[0027] In the above embodiment, the Mycobacterium sp. NS12523 strain (Mycobacteri
um sp. NS12523) was used as a microorganism, but the microorganism that can be used in the present invention is not limited to the above-mentioned microorganism, and is not particularly limited as long as it has a monooxygenase activity, but specifically, the genus Methylocinus ( M
ethylosinus), Methylococcus genus (Methylococcus), Rhodococcus genus (Rhodococcus), Mycobacterium genus (M
ycobacterium), Nocardia (Nocardia), Pseudomonas (Pseudomonas), and the like. Of these, Mycobacteri
um), Nocardia and Rhodococcus (R
hodococcus) is preferably used, and more preferably Mycobacterium sp. NS125.
Twenty-three strains (Mycobacterium sp. NS12523) are used. In addition, mycobacterium SP NS12523 strain
(Mycobacterium sp. NS12523) is a genus of Mycobacterium having the ability to oxidize an aromatic compound discovered by the present inventors and convert it to a phenol derivative or the like.
rium) is a novel microorganism.

The Mycobacterium sp. NS12523 strain (Mycobacteri
The mycological properties of um sp. NS12523) are shown below.

(A) Morphological properties Cell shape and size: A bacillus having an irregular morphology and a size of about 1 μm × 3 to 6 μm Presence / absence of cell polymorphism: Length changes Motility: None Spores: None Anti-acidity: Negative to positive (varies depending on culture time) (b) Culture properties Meat agar plate culture (30 ° C): Wavy and dry peripheral edge To form white colonies. The size of the colony is about 3 to 10 mm in diameter when cultured at 30 ° C. for 3 days. Broth broth: Grows on the surface. Diameter 0.3 ~
A cell aggregate of about 1 mm is formed. Broth gelatin stab culture: No liquefaction of gelatin Litmus milk: Alkaline (c) Physiological properties Gram stainability: Positive Nitrate reduction: + Denitrification reaction: − MR test: − VP test: − Indole formation: − Hydrogen sulfide Production (TSI medium):-Starch hydrolysis: + Utilization of citric acid: Simmons medium: -Christensen medium: + Use of inorganic nitrogen source: + Pigment formation: King A (King) A) medium: -King B medium: -SCD agar medium: -Meat agar medium: -Urease: Christensen medium: + Oxidase: -Catalase: + Growth range (SCD medium): Viable pH 4.5-8.9 Viable temperature 14.0-47.5 ° C Attitude toward oxygen: Aerobic OF test (Hugh Leifson method): F type The reaction is weak) (d) presence or absence of generation of acid and gas from saccharides below

[0030]

[Table 2]

(E) Other characteristic physiological properties Degradability of salicylic acid: + Degradability of benzoic acid: + Resistance of penicillin G: + Mycobacterium, a mycobacterium known by thin-layer chromatography for mycolic acid Vaccae I
When compared with the FO14118 strain (Mycobacterium vaccae IFO14118), Mycobacterium vaccae I
Multiple mycolic acid spots were observed at positions close to the Rf values of the FO14118 strain and mycolic acid spots.

About this strain, Vergi's Manual of Systematic Bacteriology, Volume 2
(Bergey´s Manual of Systematic Bacteriology 2nd)
As a result of a search with reference to (1986), this strain was found to be a Mycobacterium smegmatis of the genus Mycobacterium.
It was found to have similar characteristics to atis). However, type culture, mycobacterium
Smegmatis IFO 3082, 3153, 3154
And 13167 strains were compared with this strain, they were different from this strain in that none of the type cultures grew in a completely synthetic medium containing methyl ethyl ketone as a carbon source. Furthermore, none of the type cultures had monooxygenase activity or had very low activity. From these results, since this strain has different properties from the conventionally known strains, this strain was designated as Mycobacterium SP NS12523 (Mycobacte strain).
rium sp. NS12523) (hereinafter, simply “NS12523 strain”)
It is named).

This NS12523 strain was produced on May 3, 1995.
It was deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science, dated 0, and the deposit number is FERM P-14958.

In the present invention, the medium used for culturing the microorganism may be a solid or liquid medium, and
Either a synthetic medium or a natural medium may be used as long as it is a medium containing a carbon source that can be assimilated by the strain used, an appropriate amount of nitrogen source, inorganic salts and other nutrients.

The carbon source that can be used in the culture of the strain of the present invention is not particularly limited as long as the strain can grow well and express a monooxygenase activity, and glucose, propane, butane, pentane, acetone. , Methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, isopropanol, 2-butanol, 2-pentanol propane, ethanol, propanol, 1-
Examples include butanol, acetic acid, sucrose and the like.
Of these, glucose, considering the growth of the strain,
Sucrose, acetic acid, acetone, methyl ethyl ketone, ethanol, propanol, 1-butanol and 2
-Butanol and the like are preferably used as the carbon source, and in consideration of enhancing the monooxygenase activity, propane, butane, acetone, methyl ethyl ketone, 2-butanol and the like are preferably used. At this time, the amount of the carbon source added is 0.01 to 5% by weight, preferably 0.1 to 3% by weight. These carbon sources can be used alone or in the form of a mixture of two or more.

Nitrogen sources usable in the culture of the strain of the present invention include meat extract, peptone, bactopeptone,
Polypeptone, yeast extract, soybean hydrolyzate, soybean powder, milk casein, casamino acid, various amino acids and organic nitrogen compounds such as corn steep liquor, and ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate and ammonium chloride, potassium nitrate and sodium nitrate, etc. Inorganic nitrogen compounds such as nitrates and urea. These nitrogen sources can also be used alone or in the form of a mixture of two or more.

As the inorganic salt which can be used in the present invention, those used for culturing general bacteria can be used. For example, phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc. ,
One or two selected from hydrochloride, sulfate and acetate
More than one species can be used. Of these, in order to obtain a high monooxygenase activity, it is preferable to add an iron salt as an inorganic salt at a relatively high concentration. The addition concentration of the iron salt is equivalent to iron in 1 liter of the culture solution. ,
It is 0.3-60 mg, preferably 1-30 mg.

Here, one composition example of the medium other than the A medium which can be preferably used when the NS12523 strain is used in the present invention is shown in Table 3 below, and this medium is hereinafter referred to as B medium. When the NS12523 strain is cultured in a medium prepared by adding the above-mentioned predetermined carbon source to these A medium and B medium, the NS12523 strain grows while forming small aggregates with a diameter of 1 mm or less. Then
When the culture solution is allowed to stand, it settles rapidly and only the bacterial cells can be easily recovered, and therefore it can be preferably used for culturing the microorganism in the present invention.

[0039]

[Table 3]

In the present invention, as a completely synthetic medium for obtaining a high monooxygenase activity, a medium obtained by adding the above-mentioned carbon source as an inducing substrate to A medium is preferably used, and an organic state for further promoting growth is used. As the nitrogen source medium, a medium obtained by adding the above carbon source to the composition of B medium is preferably used.

In the present invention, the culturing of the microorganism is not limited to the culturing described in detail in the above embodiment, and the culturing is carried out by a known culturing method capable of appropriately culturing the microorganism to be used. Generally, the culture of the microorganism according to the present invention is carried out under aerobic conditions, and the culture conditions at that time are appropriately selected depending on the type of the microorganism used, the composition of the medium and the culture method, and the strain used is It is not particularly limited as long as it can grow and express monooxygenase activity.
Usually, the culture temperature is 20 to 45 ° C., preferably 30 to
The temperature of the medium is 40 ° C., and the pH of the medium suitable for culture is 4.
It is 5-9, preferably 6-8.

Further, according to the present invention, when the carbon source as the inducing substrate disappears in the medium, the monooxygenase activity is reduced. Therefore, in order to obtain cells having high monooxygenase activity, the carbon source as the inducing substrate is obtained. It is important to finish the culture of the microorganism at the stage when the bacterium remains in the medium. However, if you simply want to obtain the desired amount of cells,
Regardless of the decrease in monooxygenase activity, cell growth may be continued until the stationary phase is reached. For this reason, as shown in the above-described embodiment, the culture step for obtaining a certain amount of cells and the inducing substrate added to the cells obtained in such a culture step to carry out short-time culture It is preferable to carry out the culturing of the microorganism by dividing it into culture steps for the purpose of obtaining cells having high oxygenase activity. However, the inducing substrate concentration is controlled to be constant without carrying out the two-step culture as described above. Both the growth of the bacterial cells and the expression of the monooxygenase activity may be carried out in one step using a culturing apparatus capable of carrying out the above-mentioned procedure to obtain the active bacterial cells.

The culturing time varies depending on the type of microorganism used, culturing temperature and pH, culturing conditions such as pH and initial concentration of microbial cells, and the culturing method. For example, in the culturing stage for obtaining a certain amount of microbial cells, NS12523 is used. The strain was inoculated into 5 ml of A medium containing 1 platinum loop and 1% by weight of methyl methyl ketone as a carbon source and cultured at 30 ° C. for 3 days to prepare a seed culture solution. It is 2 to 5 days when inoculated into a 500 ml Sakaguchi flask containing 100 ml and shake culture (140 rpm) at 30 ° C.
In addition, in the culturing step for obtaining the active bacterium, 500 ml volume containing 100 ml of fresh A medium added with 1% by weight of methyl ethyl ketone as the inducing substrate was obtained from the above-mentioned culturing step in which the monooxygenase activity was not sufficiently high. Sakaguchi flask and shake culture (140rpm) at 30 ℃
When it is carried out, it is 6 to 72 hours. By such a method, it is possible to prepare cells having a monooxygenase activity that can be subjected to the desired oxidation reaction.

The active substance thus obtained can be used to carry out a desired substance conversion reaction.

[0045]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 The NS12523 strain was cultured in B medium supplemented with 1% glucose as a carbon source at 30 ° C. for 72 hours. Since the strain cultured in this manner has almost no monooxygenase activity, the following comparative examination of the activity-inducing effect was carried out using this strain.

The NS12523 strain cultured under the above conditions was washed with 50 mM phosphate buffer (pH: 7.0) and suspended in the same phosphate buffer so that the cell concentration was 5 g wet weight / 100 ml. did. Then, 25 ml of the suspension thus obtained was placed in a 500 ml volume Sakaguchi flask and the inducing substrate shown in Table 4 and the concentration (0.5%) shown in the same table.
Alternatively, the induction treatment was started by adding to the same volume of phosphate buffer containing 1%). At this time, 24 at 30 ℃
Induction treatment was performed by shaking (140 rpm) for a period of time. At this time, the same treatment was performed except that a 50 mM phosphate buffer solution containing no induction substrate was used as a comparison target.

After the induction treatment for a predetermined time, only the bacterial cells were recovered from the culture solution by centrifugation (6,000 × g, 5 minutes), and each bacterial cell was adjusted to the same concentration in the same phosphate buffer solution. 5 g
The suspension was wet weight / 100 ml. 50 ml of each cell suspension was placed in a 500 ml flask, and phenol was added as a substrate and glucose was added as an energy source to 1,000 ppm, respectively, and the mixture was added at 30 ° C.
The oxidation reaction was performed by reciprocating shaking (60 rpm) for 3 hours. After a lapse of a predetermined time, the reaction solution was sampled from each flask and the concentration of hydroquinone produced was measured by high performance liquid chromatography (HPLC) analysis. Further, the dry cell weight was measured, the enzyme activity per unit cell weight was calculated, and the enzyme activities were compared. The results are shown in Table 4.

[0049]

[Table 4]

From Table 4, the enzyme activity after the induction treatment of monooxygenase activity was increased when acetone, methyl ethyl ketone and 2-butanol were used as induction substrates, irrespective of the concentration of the induction substrate, as compared with when no addition was made. Cage,
From this result, acetone, methyl ethyl ketone and 2-
It was revealed that all butanols have an activity-inducing effect. It was also found that, of the inducing substrates used, acetone is particularly preferably used for improving the activity.

Example 2 Mycobacterium SP NS12523 strain (Mycob) grown on a broth agar medium (manufactured by Difco)
Acterium sp. NS12523) was inoculated into a test tube containing one platinum loop and 5 ml of A medium containing 1% by weight of methylethylketone as a carbon source, and cultured at 30 ° C for 3 days to prepare a seed culture solution. Then, this seed culture was inoculated into a 500 ml Sakaguchi flask containing 100 ml of a medium having the same composition as above, and further cultured at 30 ° C. for 3 days,
A culture solution was prepared. The cells were collected from the thus obtained culture solution by centrifugation (6,000 × g, 5 minutes),
500 ml containing 100 ml of A medium newly prepared by adding methyl ethyl ketone at a concentration of 1% by weight as a carbon source
The strain was inoculated into a Sakaguchi flask, and cultured at 30 ° C. for a further 24 hours to prepare active cells having high monooxygenase activity. The culture solution thus obtained was centrifuged (6,
000 × g, 5 minutes) to recover only the active cells from the culture solution, and add the cells to a phosphate buffer solution at a cell concentration of 5
The suspension was suspended at a wet weight of 100 g / 100 ml.

50 ml of the cell suspension was dispensed into two 500 ml flasks, and 1,000 p was added to one of the flasks as a stabilizer for monooxygenase activity.
pm of acetone was added and the other flask had no stabilizer of monooxygenase activity. Next, add phenol as a substrate and glucose as an energy source so as to be 1,000 ppm each,
The oxidation reaction was performed by reciprocal shaking (60 rpm) at 30 ° C. At this time, since the concentration of each substance decreases as the oxidation reaction progresses, the concentration of each substance is measured by high performance liquid chromatography (HPLC) timely, and each substance is added successively so that the concentration does not fall below 500 ppm. While continuing the reaction for 70 hours.

The reaction solution was sampled from each flask over time from the start to the end of the reaction by HPLC.
The concentration of hydroquinone produced by the analysis was quantified. The results are shown in FIG.

From FIG. 1, by adding acetone having a monooxygenase activity-inducing effect, the reaction duration was extended and the reaction product (hydroquinone) concentration increased as compared with the reaction carried out without addition of acetone. Was remarkably recognized. From this result, by adding a substrate for inducing monooxygenase activity to the oxidation reaction system,
It was shown that the stability of monooxygenase activity was significantly improved.

[0055]

INDUSTRIAL APPLICABILITY As described above, the method for stabilizing monooxygenase activity of the present invention comprises a carbon having an effect of inducing a monooxygenase activity in an aromatic compound oxidation reaction system utilizing a microorganism having a monooxygenase activity. It is characterized in that an aliphatic compound of the formula 3 to 10 is added. Therefore, by using the method of the present invention, in a substance conversion reaction using a microorganism having a monooxygenase activity, by allowing a certain amount of an inducing substrate to be present in the reaction system, it is possible to efficiently decrease the monooxygenase activity over time. It can be easily suppressed, and further, the duration of the substance conversion reaction by monooxygenase can be remarkably extended.

Further, the method for stabilizing the monooxygenase activity of the present invention is a method which is extremely simply carried out by allowing a certain amount of the inducing substrate to be present.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the reaction time and the production amount of hydroquinone in the oxidation reaction using an inducing substrate according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location (C12N 1/38 C12R 1:32) (72) Inventor Koichi Sakano Kannondai, Tsukuba, Ibaraki 1-chome, 25-share, 12-share company, Nippon Shokubai

Claims (5)

[Claims] 1. A substance conversion reaction system utilizing a microorganism having a monooxygenase activity which catalyzes a reaction of imparting oxygen to an aromatic compound, which has an effect of inducing a monooxygenase activity and has 3 to 10 carbon atoms. A method for stabilizing monooxygenase activity, which comprises adding an aliphatic compound to carry out the reaction. 2. The aliphatic compound is propane, butane, pentane, acetone, methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, isopropanol, 2
-The method for stabilizing monooxygenase activity according to claim 1, which is at least one selected from the group consisting of butanol and 2-pentanol. 3. The aliphatic compound is 10% by weight to 200% by weight based on the initial concentration of the aromatic compound in the reaction solution.
The method for stabilizing monooxygenase activity according to claim 1 or 2, which is present at a concentration in the range of. 4. A compound produced by applying oxygen to the aromatic compound is a phenol derivative having a hydroxyl group on the aromatic ring, an aromatic carboxylic acid having a carboxyalkyl group on the aromatic ring, and an alkyl on the aromatic ring. The method for stabilizing monooxygenase activity according to claim 1, which is selected from the group consisting of aromatic ketones having a ketone group. 5. The method for stabilizing monooxygenase activity according to claim 1, wherein the microorganism having monooxygenase activity is a microorganism belonging to the genus Mycobacterium.