JPH09281A - Stabilization of monooxygenase activity - Google Patents

Abstract

PURPOSE: To inhibit the reduction in monooxygenase activity with time caused in conversion reaction by adding a protein synthesis inhibitor to the oxidation system for an aromatic compound utilizing a microorganism having the monooxygenase activity. CONSTITUTION: A protein synthesis inhibitor, for example, an antibiotic substance such as Chloramphenicol and/or a compound having serine protease activity inhibitor, cysteine protease activity inhibitor or a protease activity inhibitor such as phenylmethylsulfonyl fluoride are added to the oxidation reaction system for an aromatic compound utilizing a microorganism having the monooxygenase activity.

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 decomposition of monooxygenase can be prevented and therefore the activity can be maintained by taking measures to eliminate the influence of this protease.

Based on these research results, two methods for excluding protease activity were identified, namely, a method of inhibiting the biosynthesis of protease by bacterial cells by adding a protein synthesis inhibitor and a protease activity inhibitor. A method of inhibiting the action of protease by adding it has been devised. In this way, the inventors have found a method capable of effectively suppressing the decrease in monooxygenase activity, and succeeded in improving the stability of activity, thereby completing the present invention.

That is, the above object is characterized in that at least one selected from the group consisting of protein synthesis inhibitors and protease activity inhibitors is added to an aromatic compound oxidation reaction system utilizing a microorganism having monooxygenase activity. And a method for stabilizing monooxygenase activity.

The present invention shows a method for stabilizing the monooxygenase activity in which the above protein synthesis inhibitor is an antibiotic. The present invention also shows a method for stabilizing the monooxygenase activity in which the protein synthesis inhibitor is chloramphenicol or a derivative thereof. The present invention also provides at least one monooxygenase activity selected from the group consisting of a compound having an effect of inhibiting the serine protease activity, a compound having an effect of inhibiting the cysteine protease activity, and a mixture thereof, wherein the protease activity inhibitor is a monooxygenase activity. It shows the stabilization method of. The present invention also shows a method for stabilizing the monooxygenase activity in which the protease activity inhibitor is phenylmethylsulfonyl fluoride. The present invention further provides the method for stabilizing monooxygenase activity according to any one of the above, wherein the microorganism having monooxygenase activity is a microorganism belonging to the genus Mycobacterium.

[0012]

The method for stabilizing the monooxygenase activity of the present invention comprises using active cells having high monooxygenase activity obtained by culturing cells of a microorganism having monooxygenase activity in the presence of an inducing substrate. When carrying out the oxidation reaction of the compound, the reaction system contains a protein synthesis inhibitor,
It is characterized in that at least one selected from the group consisting of a protease activity inhibitor and a mixture thereof is added.

Hereinafter, the present invention will be described in detail.

One embodiment of the method for stabilizing the monooxygenase activity of the present invention will be described below. Mycobacterium sp. NS1 grown on broth agar
2523 strain (Mycobacterium sp. NS12523)
A seed culture solution is prepared by inoculating and culturing in 5 ml of a medium having an appropriate carbon source added to a medium having the medium composition shown in Table 1 (hereinafter referred to as A medium). 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. Cells were collected from the thus-prepared culture broth by centrifugation (1,000 to 10,000 × g, 5 to 20 minutes), and a monooxygenase activity-inducing substrate was added as a carbon source A. 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. Centrifuge the culture solution (1,000-1
The cells are washed with water, a buffer solution, physiological saline or the like, if necessary, to recover only the active cells from the culture solution. The active cells thus collected were treated with a phosphate buffer (containing 0.2 g / liter of magnesium sulfate, 50 mM KH ₂ PO ₄
The solution is suspended in sodium hydroxide to adjust the cell concentration to 0.1 to 50 g wet weight / 100 ml, preferably 0.5 to 20 g wet weight / 100 ml, for the purpose. A cell suspension is prepared as a catalyst in the substance conversion reaction. 50 ml of this cell suspension is 500 m
Put in a 1-liter flask, add protein synthesis inhibitor, then add substrate and energy source, 20-35 ° C.
By shaking (50-300 rpm) under aerobic conditions for a desired time, the substrate is oxidized by the monooxygenase to obtain the desired product (phenol derivative, aromatic aldehyde, aromatic alcohol, aromatic carboxylic acid and Aromatic ketone).

[0015]

[Table 1]

In the present invention, an aromatic compound is used as the substrate used for 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, as an energy source used for the oxidation reaction by monooxygenase, NADH and NAD which are coenzymes are reduced by reducing the coenzymes NAD and NADP with energy obtained by the strain decomposing the substance.
It is not particularly limited as long as it can generate PH, but specifically, sugars such as glucose and sucrose,
Examples thereof include organic acids such as acetic acid, alcohols such as methanol, ethanol, propanol and butanol, and carbon sources such as ketones such as acetone and methyl ethyl ketone. Of these, glucose, acetic acid and ethanol are preferably used because they are efficiently decomposed by microorganisms and do not inhibit the reaction by monooxygenase regardless of the concentration added. If the substrate used in the conversion reaction is converted to a phenol derivative and at the same time a part of it is decomposed and assimilated by microorganisms, it is considered that the cells can acquire energy by it. The supply of carbon source is not particularly required. The concentration of the energy source added is 0.1 to 10 times, preferably 0.2 to 5 times the molar concentration of the reaction substrate.

The protein synthesis inhibitor used in the present invention is not particularly limited as long as the stability of the monooxygenase activity can be improved by adding it, and it has an action of inhibiting protein synthesis. It is preferably an antibiotic. Representative examples of these are chloramphenicol, erythromycin, spiramycin, lincomycin, vernamycin, blasticidin S, thiostreptomycin, sporangiomycin, streptomycin, dihydrostreptomycin, dihydrospectinomycin, pactamycin, edein, tetracycline and these. Among these, chloramphenicol and chloramphenicol derivatives such as chloramphenicol succinate and chloramphenicol morpholine acetate are preferably used, and chloramphenicol is particularly preferable. The concentration of the protein synthesis inhibitor added is usually 1 to 1,50 per 1 liter of the reaction solution.
It is 0 μM, preferably 10 to 750 μM, and more preferably 50 to 500 μM.

In the oxidation reaction by the monooxygenase of the present invention, molecular oxygen is required as an oxygen 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 embodiment, the microbial cells were separated by centrifugation, but the microbial cells are not limited to this method, and can be separated by a general microbial cell separation method such as filtration and sedimentation separation. Done.

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 used in the above-mentioned embodiment (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 polymorphism of cells: 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

[0026]

[Table 2]

(E) Other characteristic physiological properties Degradability of salicylic acid: + Degradability of benzoic acid: + Resistance of penicillin G: + Mycobacterium, which is a known microorganism 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 either 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. Glucose, propane, butane, acetone, methyl ethyl ketone , 2-butanol, ethanol, propanol,
1-butanol, acetic acid, sucrose and the like can be mentioned. Of these, considering the growth of the strain, glucose, acetic acid, acetone, methyl ethyl ketone, ethanol, propanol, 1-butanol, 2-butanol and the like are preferably used as a carbon source, and considering that it enhances monooxygenase activity. , Propane, butane, acetone, methyl ethyl ketone, 2-butanol and the like are preferably used as the inducer of monooxygenase activity. At this time, the amount of the carbon source added was 0.
It is from 0.01 to 5% by weight, preferably from 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 that can be used 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 commonly used for culturing bacteria can be used. For example, phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc can be used. ,
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.

[0035]

[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 culturing method. For example, in the culturing step 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 ethyl ketone as a carbon source and cultured at 30 ° C. for 3 days to prepare a seed culture solution, and this seed culture solution was added to 100 ml of the same medium as above. It is 2 to 5 days when inoculated into a 500 ml Sakaguchi flask put in and shake culture (140 rpm) at 30 ° C.
In addition, in the culture stage for obtaining the active cells, 500 ml volume containing 100 ml of fresh A medium containing 1% by weight of methyl ethyl ketone as the inducing substrate was obtained from the above-mentioned culture 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.

Further, in the above embodiment, the protein synthesis inhibitor was used for the purpose of stabilizing the monooxygenase activity, but a protease activity inhibitor may be added instead of the protein synthesis inhibitor. At this time, the protease activity inhibitor that can be used is not particularly limited as long as it inhibits the induced or synthesized protease activity, but a compound that inhibits serine protease and / or cysteine protease activity is preferable. Examples of the protease activity inhibitor used in the present invention include chloromethyl ketone, diisopropylfluorophosphoric acid, phenylmethylsulfonylfluoride, 4-toluolsulfonylfluoride, L-1-
Tosylamide-2-phenyl (ethyl) chloromethylketone, N-p-tosyl-L-lysine chloromethylketone and serine protease inhibitors such as peptide aldehydes and L-trans-epoxysuccinyl-leucylamide (4-guanide) butane, peptides Examples include cysteine protease inhibitors such as aldehydes and peptidyl diazomethyl ketones, and of these, phenylmethylsulfonyl fluoride is particularly preferably used.
Furthermore, the concentration of the protease activity inhibitor added is usually 0.1 to 100 μM per 1 liter of the reaction solution,
Preferably 0.5 to 50 μM, more preferably 1 to 25
μM.

Further, for the purpose of stabilizing the monooxygenase activity according to the present invention, a combination of the above protein synthesis inhibitor and protease activity inhibitor may be added. In this case, the types and ranges of the protein synthesis inhibitor and the protease activity inhibitor used are the types and ranges described above, respectively. In addition, addition of protein synthesis inhibitor and protease activity inhibitor,
It is carried out at any ratio of the concentration within the above range. Specifically, the ratio of the protein synthesis inhibitor and the protease activity inhibitor added is 0.1: 1 to 1: 0.1 by weight. is there.

[0043]

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

Examples 1 to 4 and Comparative Example 1 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),
The cells were inoculated into a 500 ml Sakaguchi flask containing 100 ml of a fresh medium having the same composition as described above, and the cells were further cultured at 30 ° C. for 24 hours to prepare active cells having high monooxygenase activity. By centrifuging the culture solution thus obtained (6,000 × g, 5 minutes), only the active cells were recovered from the culture solution, and the cells were added to the phosphate buffer at a concentration of 5 g. The suspension was wet weight / 100 ml.

10 ml of this cell suspension was dispensed into 5 flasks each having a volume of 100 ml, and 4 flasks out of which were used as protein synthesis inhibitors and / or protease activity inhibitors, respectively, each containing 150 μM chloramphenicol. (Black rhombus in FIG. 1) (Example 1), 150 μm
M chloramphenicol + 0.5 μM phenylmethylsulfonyl fluoride (black triangle in FIG. 1) (Example 2), 150 μM chloramphenicol + 5 μM phenylmethylsulfonyl fluoride (white square in FIG. 1) (Example 3) and 5 μM phenylmethylsulfonyl fluoride (white diamond in FIG. 1) (Example 4) was added. As a comparative control, no inhibitor was added to the remaining flasks (black squares in FIG. 1).
(Comparative Example 1). After adding each inhibitor at a predetermined concentration, add phenol as a substrate and glucose as an energy source to 1,000 ppm,
The oxidation reaction was carried out by shaking back and forth (60 rpm) at 30 ° C. for 40 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. The results are shown in FIG.

From FIG. 1, protein synthesis inhibitors and / or
Alternatively, it is shown that the production amount of hydroquinone obtained by adding the protease activity inhibitor is higher than the production amount when the reaction is performed without addition. In addition, since the production amount of hydroquinone increased up to 40 hours in the system to which the protein synthesis inhibitor and / or the protease activity inhibitor was added, the monooxygenase activity was maintained at least until 40 hours after the start of the reaction. On the other hand, since the production of hydroquinone almost stopped in 18 hours in the system without addition, the reaction duration may be extended by adding the protein synthesis inhibitor and / or the protease activity inhibitor. Clearly shown.

[0047]

INDUSTRIAL APPLICABILITY As described above, the method for stabilizing monooxygenase activity of the present invention comprises a protein synthesis inhibitor, a protease activity inhibitor and a protease activity inhibitor in an aromatic compound oxidation reaction system utilizing a microorganism having monooxygenase activity. It is characterized in that at least one selected from the group consisting of these mixtures is added. Therefore,
By using the method of the present invention, in a substance conversion reaction using a microorganism having monooxygenase activity,
Eliminating the influence of protease, which is one of the causes of impairing the stability of monooxygenase activity, it is possible to efficiently and simply suppress the decrease in monooxygenase activity over time, and to significantly increase the duration of substance conversion reaction by monooxygenase. It can be extended.

The method for stabilizing the monooxygenase activity of the present invention is a method that is conveniently carried out by adding at least one selected from the group consisting of protein synthesis inhibitors, protease activity inhibitors and mixtures thereof. is there.

[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 the monooxygenase 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 (6)

[Claims] 1. At least one selected from the group consisting of a protein synthesis inhibitor and a protease activity inhibitor is added to an aromatic compound oxidation reaction system utilizing a microorganism having a monooxygenase activity. A method for stabilizing oxygenase activity. 2. The method for stabilizing monooxygenase activity according to claim 1, wherein the protein synthesis inhibitor is an antibiotic. 3. The method for stabilizing monooxygenase activity according to claim 2, wherein the protein synthesis inhibitor is chloramphenicol or a derivative thereof. 4. The protease activity inhibitor is at least one selected from the group consisting of a compound having an effect of inhibiting serine protease activity, a compound having an effect of inhibiting cysteine protease activity, and a mixture thereof. 4. The method for stabilizing monooxygenase activity according to any one of 1 to 3. 5. The method for stabilizing monooxygenase activity according to claim 4, wherein the protease activity inhibitor is phenylmethylsulfonyl fluoride. 6. The method for stabilizing monooxygenase activity according to claim 1, wherein the microorganism having monooxygenase activity is a microorganism belonging to the genus Mycobacterium.