WO2007040267A1

WO2007040267A1 - Method of culturing microorganism capable of producing cobalt-containing nitrile hydratase

Info

Publication number: WO2007040267A1
Application number: PCT/JP2006/319987
Authority: WO
Inventors: Yasushi Kazuno; Tadashi Araki; Kiyoshi Ito
Original assignee: Mitsui Chemicals, Inc.
Priority date: 2005-10-06
Filing date: 2006-10-05
Publication date: 2007-04-12
Also published as: JP2008306928A

Abstract

It is intended to provide a culture method which aims at obtaining a microorganism having an elevated enzyme activity in efficiently producing an amide compound by using cobalt-containing nitrile hydratase. In the multistep culture of a microorganism capable of producing cobalt-containing nitrile hydratase, an appropriate amount of a cobalt ion species is supplied at an appropriate step in the culture to thereby elevate the enzyme activity of the microorganism thus obtained.

Description

Method for culturing cobalt-type nitrile hydratase-producing microorganism

Technical field

[0001] The present invention relates to a method for culturing a microorganism producing a conoleto-tolylhydratase having a high -tolylhydratase activity.

Background art

In recent years, -tolyl hydratase having tolyl hydration activity that hydrates and converts nitrile groups to amide groups has been discovered, and nitriles using microorganisms having nitrile hydratase or -tolyl hydratase, etc. A method for producing a corresponding amide compound from a compound has been studied. This production method is known to have advantages such as higher conversion and selectivity from a nitrile compound to the corresponding amide compound than conventional chemical methods, and is used industrially.

[0003] It is already known that nitrile hydratase has a non-heme iron atom or a non-choline nuclear cobalt atom as a prosthetic molecule at the active center, and iron-tolyl hydratase and And cobalt type nitrile hydratase. Examples of iron-type nitrile hydratase include -tolyl hydratase derived from Rhodococcus sO.N—771, Pseudomonas chlororaphis B23, etc., and cobalt-tolyl hydratase An example of a tase is Rhodococcus rhodochrous-tolylhydratase derived from Pseudonocardia thermopik JCM3095 and the like.

[0004] Here, in order to industrially produce an amide compound from a tolyl compound using nitrile hydratase, it is important to lower the production cost of tolyl hydratase in the production cost of the amide compound It is preferable to increase the hydration activity of the -Torrui compound per unit microbial weight of microorganisms containing nitrile hydratase. At the same time, it is also effective to efficiently produce a microorganism having enhanced hydration activity of such a -Torrui compound.

[0005] For example, a cobalt-tolyl hydratase derived from Syudnocardia 'thermophila is expressed in E. coli by expressing the -tolyl hydratase gene in E. coli. And a method for expressing a protein that activates -tolyl hydratase together with -tolyl hydratase in Escherichia coli (Japanese Patent Laid-Open No. 11-253168), etc. .

[0006] On the other hand, in order to efficiently produce a microorganism having enhanced hydration activity of a nitrile compound, it is generally effective to increase the concentration of the microorganism per unit culture solution. More specifically, a high-density culture method in which the final dry microorganism concentration becomes 20 gZL or more using a stirring type culture tank or the like is applied. In addition, when multi-stage culture of microorganisms producing cobalt-type-tolylhydratase, it is necessary to allow an appropriate amount of cobalt ions to coexist in the final stage of the culture. It is also known that it is necessary for the expression of.

Patent Document 1: Japanese Patent Laid-Open No. 9-275978

Patent Document 2: Japanese Patent Laid-Open No. 11-253168

Disclosure of the invention

Problems to be solved by the invention

[0007] As described above, industrially, (1) the hydration activity of the -Torrois compound per unit microorganism weight is increased, (2) the water of the Nitrile compound by the high-density culture method. It is important to simultaneously achieve the two problems of efficiently producing a microorganism having high sum activity, and the present invention provides a measure for this purpose.

Means for solving the problem

[0008] In general, a high-density culture method of microorganisms includes an inoculum that is lyophilized and stored in an ampule, an inoculum that is stored in a glycerol solution of about 10%, and the like in about 1 to 3 stages. This is a method of increasing the density of microorganisms in the final stage culture after increasing the amount of microorganisms in stages by performing seed step culture. In order to increase the density of microorganisms in this final stage of cultivation, a carbon source that is a raw material for constituents such as proteins, nucleic acids, lipids, and vitamins in microorganisms and that is an energy source necessary for the growth of microorganisms is cultured. It is usual to add to a tank.

[0009] In order to solve the above-mentioned problems, the present inventors have investigated that an appropriate amount of cobalt ion species should be added not only in the final stage of culture but also in other stages of culture. Thus, it is found that a microorganism having a higher activity per unit microorganism weight can be obtained. Further, when supplying the carbon source into the culture solution in the final stage culture, an appropriate amount of cobalt is added to the carbon source solution. By adding, it was found that a microorganism having higher activity per unit microorganism weight was obtained, and the present invention was completed.

That is, the present invention is as described in [1] to [4] below.

[1] In a multi-stage culture of microorganisms producing cobalt-tolylhydratase

A method for culturing a cobalt-type-tolylhydratase-producing microorganism, comprising adding a cobalt ion species to a culture solution in a seed step culture stage other than the final stage.

[2] In a multi-stage culture of a microorganism producing cobalt-tolylhydratase

A method for culturing a cobalt-type-tolylhydratase-producing microorganism, comprising supplying a carbon source solution containing a cobalt ion species into a culture solution at a final stage.

[3] A cobalt-type-tolylhydratase-producing microorganism characterized by combining the culture method described in [1] and the culture method described in [2] in culturing a microorganism that produces cobalt-type-tolylhydratase Culture method.

[4] Nitrile in an aqueous medium in the presence of a microorganism that produces cobalt-tolylhydratase obtained by the culture method according to [1], [2], or [3], or a processed product of the microorganism A method for producing an amide compound by hydrating a compound.

The invention's effect

[0011] Cobalt-tolylhydratase with higher activity per unit microbial weight is produced by supplying cobalt at an appropriate stage of the culture in a multistage culture of microorganisms that produce cobalt-tolylhydratase It is possible to provide microorganisms that can be used. In addition, by supplying a carbon source solution containing cobalt to the culture solution, it is possible to provide a microorganism that produces cobalt-tolylhydratase having higher activity per unit microorganism weight.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The cobalt-type-tolyl hydratase in the present invention has -tolyl hydration activity by coordination of cobalt in the molecule. The cobalt-type tolyl hydratase includes -tolyl hydratase derived from Rhodococcus rhodochrous Jl. And -tolylhydratase derived from Pseudonocardia thermophila TCM3095.

[0013] Cobalt-type in the present invention -. The tolyl hydratase producing microorganism, shea Yu de Nocardioides § Samofuira _{_{(p seu d onoC ardia thermophila)}} TCM3095 or, only strain itself, such as Rhodococcus' Rod Kurousu (Rhodococcus rhodochrous) T1 Also included are transformants in which the tolyl hydratase gene cloned from the strain is expressed in any host. In addition, as an arbitrary host mentioned here, Escherichia coli can be raised as a representative example. Other microbial strains such as fungi are also included. MT-10822 (This transformant was entrusted to the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3-3 Tsukuba-Nakto, Ibaraki Prefecture on February 7, 1996. No. FERM BP — 5785, deposited under the Budapest Convention on the International Approval of Deposits of Microorganisms in Patent Procedures). In addition, transformation using recombinant DNA technology to express mutant-tolylhydratase in which one or more of the constituent amino acids of the enzyme is substituted, deleted, deleted or inserted with other amino acids. The body is also included in the conoleto-tolylhydratase-producing microorganism referred to in the present invention.

[0014] The method for culturing a cobalt-type-tolylhydratase-producing microorganism of the present invention is a culturing method that requires multiple stages, and a cobalt ion species is added to the culture solution in a seed step culturing stage other than the final stage. There is a special feature.

[0015] In the present invention, the seed step culture stage means all stages except the final stage of the main culture in the multistage culture method of microorganisms.

[0016] Cobalt-tolylhydratase is an active enzyme having hydration activity for -tolyl compounds by coordinating cobalt to the active center as a prosthetic metal.

[0017] Therefore, in culturing a microorganism that produces cobalt-tolylhydratase, it is necessary to add a coronate ion species to the culture solution in order to express its function. However, it is generally known that excessive amounts of cobalt ions often inhibit the growth of microorganisms such as E. coli. From these points, in the multi-stage culture of a conoleto-tolyl hydratase-producing microorganism, It was common to add an appropriate amount of coronate ion species without adding excess to the final stage culture without adding the barto ion species.

[0018] However, even in the seed step culture stage of a cobalt-type-tolylhydratase-producing microorganism, by adding a cobalt ion species, the enzyme activity per unit microorganism weight finally recovered is determined as a seed. It is significantly higher than when no cobalt ion species is added at the step culture stage.

[0019] The cobalt ion species include divalent cobalt ions (Co ++) and trivalent cobalt ions (Co +++). In the present invention, divalent cobalt ions are supplied in all seed step culture solutions. There is a need. The supply of cobalt ion species is usually supplied as a salt. Examples of salts that can supply divalent cobalt ions include cobalt salt, cobalt sulfate, and cobalt acetate.

[0020] In the present invention, as described above, it is necessary to supply the divalent coronate ions to the culture solution. However, when the cobalt ions are excessively added to the culture solution, the growth of microorganisms is performed. May cause inhibition. For this reason, the cobalt concentration in the medium during the culture is preferably within a range where the activity expression of conoleto-tolylhydratase is sufficient and does not inhibit the growth of microorganisms. Therefore, the divalent cobalt ion concentration in the medium is preferably maintained in the range of 5 to 200 mgZL, more preferably in the range of 10 to 150 mgZL in terms of cobalt chloride hexahydrate.

[0021] Examples of microorganisms that produce the above cobalt-type-tolylhydratase include Rhodococcus rhodochrous. For example, a transformant obtained by transducing the -tolyl hydratase gene derived from the microorganism of the present invention includes, for example, a -tolyl hydratase gene derived from Pseudonocardia thermophila TCM3095. MT-10822 (deposit number FERM BP-5785) can be mentioned as the introduced transformant, and the cobalt concentration of the medium in the seed step culture stage in the multi-stage culture of these cobalt-type-tolylhydratase-producing microorganisms is as described above. When it is within the range, the effect of improving the enzyme activity per unit microorganism weight is remarkable.

[0022] Other culture conditions for the -tolylhydratase-producing microorganism of the present invention include the -tolylhydratase producing microorganism. A medium containing a carbon source, an organic nitrogen source, an inorganic nitrogen source, an inorganic salt, a buffer solution, etc. may be selected according to the characteristics of the hydratase-producing microorganism and cultured at an appropriate temperature and pH condition.

[0023] For example, in the case of Rhodococcus rhodochrous T1, carbon sources such as fructose, inorganic salts such as magnesium sulfate and ammonium sulfate, pH buffering agents such as phosphate, ethanol, etc. Used as a medium component. In addition, for example, an inducer such as an aqueous solution of 1 to 30 gZL of urea may be used. In the case of Pseudonocardia thermophila TCM3095, yeast extract, polypeptone, inorganic salts such as iron sulfate, pH buffering agents, etc. are used as medium components. A commercially available bouillon medium is also an example of a suitable synthetic medium. Also, for example, an inducer such as an aqueous solution of 1 to 20 gZL of various amide compounds may be used. Both microorganisms are preferably cultured at a pH near neutral. The culture temperature is 30 ° C for Rhodococcus rh odochrous T1, and 50 ° C for Pseudonocar dia thermoDhila TCM3095 because it is a moderately thermophilic bacterium. Local culture is suitable for each. In addition, when using a transformant obtained by transforming a cobalt-tolyl hydratase gene such as the above-mentioned microorganism-derived tolyl hydratase, it is desirable to employ culture conditions suitable for the host cell to be used. For example, when MT-1082 2 is used as a microorganism, the medium is divalent cobalt ion and the host is E. coli, so glucose as the carbon source and polypeptone as the organic nitrogen source. In addition to yeast extract and inorganic salts such as ammonium sulfate, iron sulfate and magnesium sulfate, pH buffering agents and the like are used as medium components. A commercially available LB medium (Luria-Bertani Broth) can also be used as a suitable synthetic medium. The culture temperature is preferably in the range of 25 ° C to 38 ° C, and the pH is preferably in the range of 7.0 to 8.0.

[0024] When culturing microorganisms, it is desirable to supply air in addition to stirring or shaking to maintain a dissolved oxygen concentration sufficient for growth. In the final stage of high-density culture, it is important to maintain the dissolved oxygen concentration because the concentration of microorganisms in the culture solution is increased. Cultivation is performed at a few hundred rpm and with an aeration rate of about lwm. Do.

[0025] During the cultivation of microorganisms, the concentration of microorganisms in the culture medium increases, When culturing is applied, carbon sources such as glucose, which are raw materials for microbial constituents such as proteins, nucleic acids, lipids, and vitamins, and as an energy source necessary for the growth of microorganisms, may be insufficient. In such a case, it is preferable to supply a carbon source to the culture solution.

[0026] Therefore, in a culture that requires multiple stages of a nitrile hydratase-producing microorganism, particularly in a high-density culture in which the concentration of the microorganism is significantly increased and the carbon source is insufficient as the culture proceeds, the growth state of the microorganism A method in which an aqueous solution containing a carbon source (hereinafter also referred to as a carbon source solution) is added in accordance with the above is also a preferred embodiment.

[0027] The second aspect of the microorganism culturing method of the present invention is characterized in that a cobalt ion species is further added to the final stage of culturing, that is, the carbon source solution used in the main culturing.

[0028] As described above, when the cobalt ion species is contained in the carbon source solution, the enzyme activity per unit weight of the microorganisms finally recovered is not contained in the carbon source solution. Significantly higher than the case.

[0029] As the cobalt ion species, there are a divalent cobalt ion (Co ++) and a trivalent cobalt ion (Co ++ +). Need to be. Cobalt ion species are usually added as salts. Examples of salts that can supply divalent cobalt ions include cobalt salt, cobalt sulfate, and cobalt acetate.

[0030] In the second aspect of the culturing method of the present invention, as described above, the force necessary to contain the divalent cobalt ions used in the main culturing in the carbon source solution is excessive. If excessively contained in the carbon source solution, it may cause microbial growth inhibition. For this reason, the concentration of cobalt in the carbon source solution is preferably within a range that is necessary for the expression of the activity of conoleto-tolylhydratase and does not cause microbial growth inhibition. Therefore, the concentration of the divalent cornole ion in the carbon source solution is preferably in the range of 10 to 400 mgZL, more preferably in the range of 100 to 200 mgZL in terms of salt-cobalt hexahydrate.

[0031] The carbon source solution described above may contain an organic nitrogen source, an inorganic nitrogen source, inorganic salts, a buffer solution, and the like as required, in addition to the carbon source and the cobalt ion species. Examples of the carbon source, organic nitrogen source, inorganic nitrogen source, inorganic salts, and buffer solution include the culture medium described above. Examples included in the ground.

[0032] As the culture conditions, appropriate temperature and pH conditions can be selected according to the characteristics of the -tolylhydratase-producing microorganism.

[0033] For example, in the case of Rhodococcus rhodochrous T1, in addition to 100 to 300 g / L of fructose as a carbon source, 40 to: L00 g / L of ethanol, 20 to: LOOg ZL of ammonium sulfate A carbon source solution having a composition such as sulfur is used. The pH of the carbon source solution should be near neutral. In addition, when using a transformant obtained by transforming a conoleto-tolylhydratase gene such as the above-mentioned microorganism, it is desirable to have a carbon source solution composition suitable for the host cell to be used. For example, when MT-10822 is adopted as a microorganism, divalent cobalt ions are preferably contained in the range of 50 to 400 mg / L, more preferably 100 to 200 mg / L in terms of cobalt chloride hexahydrate, Furthermore, it is preferable to carry out the final stage of culture while adding a carbon source solution containing 200 to 600 gZL of dulose as a carbon source. The pH of the carbon source solution is preferably in the vicinity of neutrality as long as it is adjusted as necessary.

[0034] Combining both the addition of the cobalt ion species to the culture medium at the seed step stage described above and the addition of the carbon source solution containing the concerto ion species during microbial culture will further increase the combination.

In addition, the enzyme activity per unit microbial weight of the finally recovered microorganism is improved.

[0035] The method for producing an amide compound in the present invention is a method for synthesizing a corresponding amido compound from a -Torrois compound using a microorganism obtained by the culture method of the present invention or a processed product of the microorganism. If so, there are no particular restrictions on the reaction conditions.

[0036] The treated product of microorganisms in the present invention refers to a microorganism extract or ground product obtained by the culturing method of the present invention, and the extract or ground product strength cobalt-tolylhydratase active fraction is separated and purified. As long as they have -tolyl hydratase activity, they fall under the treated microorganisms of the present invention. In addition, the aqueous medium in the present invention is an aqueous solution in which water or a buffer such as a phosphate, an inorganic salt such as a sulfate or carbonate, an alkali metal hydroxide, an amide compound, or the like is dissolved at an appropriate concentration. Indicates.

[0037] When the corresponding amidy compound is synthesized using the microorganism or the processed microorganism of the present invention, the corresponding amidy compound is used for either a batch reaction or a continuous reaction. Is possible. Moreover, you may use as a suspended bed and may use as a fixed bed.

[0038] The type of the Tolyru complex used in the present invention is a cobalt-type-tolylhydratase-producing microorganism obtained by the culture method of the present invention, or one that is converted into an amide compound by a processed product of the microorganism. If it is, it will not specifically limit. Specifically, it is a -Torroi compound having about 2 to 20 carbon atoms, and includes aliphatic-tolyl, aromatic-tolyl and the like. Among them, acrylo-tolyl and meta-tallow-tolyl are preferable examples.

Example

[0039] The ability to explain the present invention in more detail by the following examples The present invention is not limited in any way by the following examples.

[0040] In this example, the deposited microorganism MT-10822 (Accession No. FERM BP-) in which the -tolylhydratase gene derived from Pseudonocardia thermophila TC M3095 was transduced into the HB101 strain derived from Escherichia coli K-12. 5785) was used as a microorganism having -tolylhydratase activity.

[0041] [Example 1]

Cultivation in which cobalt is added to the medium in the seed step culture stage

Sterilized by high pressure steam 0.08gZL FeSO 7 および 0, and 0.05gZL CoCl 6

4 2 2

培 Medium supplemented with ampicillin to LB medium (ρΗ7.5) containing Ο to reach 0.1 mgZL

2

200 ml of the ground was used as the medium for the seed step culture stage. The above-mentioned microorganisms were inoculated in this medium with the ears of white and the seed step culture started at 33 ° C. The seed step culture stage was terminated when the absorbance at 660 nm, ie, turbidity, of the culture medium in which this culture was performed was in the range of 3.0 to 6.0. The culture solution obtained in the seed step culture stage was inoculated into 5 L of the main culture medium shown in Table 1 which had been autoclaved, and the culture in the main culture stage was started at 33 ° C. As the density of microorganisms increases, the deficient carbon source is intermittently supplied, so the pH of the culture solution is monitored from the start of the main culture, and when the pH exceeds 7.45, 500 gZL of glucose is used as the carbon source solution. 15 ml of aqueous solution was added. At the same time, 1. Ovvm was agitated while venting air into the culture, and cultured for 48 hours. The obtained culture broth was centrifuged to obtain wet cells (microorganism suspension).

[0042] [Table 1] Reagent name Concentration (g / L)

Polypeptone 7.0

F e S 0 ₄ 7 H ₂ 0 0. 09

C o C 1 ₂ 6 H ₂ 0 0. 05

(NH4) ₂ S 0 ₄ 1. 5

Mg S 0 ₄ ■ 7 Η, Ο 2. 0

KH ₂ P0 ₄ 2. 0

K ₂ H P0 ₄ 2. 0

Ade force Nord 0.7

[0043] 16 mg of the obtained microbial suspension was taken out and diluted to 10 g with pure water. The diluted solution lg was diluted to 10 g with 50 mM Tris-HCl buffer (pH 8.1). Next, 3.2 ml of Atari mouth-trill was added and the reaction was carried out for 15 minutes while maintaining at 20 ° C. The reaction was stopped by adding 80 g of 10 mM aqueous phosphoric acid solution, and the acrylamide concentration in the reaction solution was measured by HPLC analysis. As a column in HPLC analysis, YMC-Pack ODS-Α (150Χ6 φ mm) was used, and a 10 mM phosphoric acid aqueous solution containing 3% acetonitrile was used as a moving bed. Atarylamide and acrylonitrile were detected by absorbance at 210 nm and the concentration was measured. Next, the weight concentration of dry microorganisms in the suspension was determined, and the amount of allylamide produced per unit dry microorganism weight was calculated. The results of Example 1 were compared with the amount of allylamide produced per unit dry microorganism weight obtained in Comparative Example 1 as 100%. The results are shown in Table 2.

[0044] [Comparative Example 1]

Culture without adding cobalt to the medium in the seed step culture stage

See Example 1 with the exception of adding CoCl 6Η during the seed step culture stage.

twenty two

Cultivation was performed by the same method. The amount of acrylamide produced per unit dry microorganism weight of the obtained culture broth was calculated in the same manner as in Example 1. The results are shown in Table 2.

[0045] [Example 2]

Cultivation with cobalt added to carbon source solution during main culture

Add 0.lgZL CoCl-6 · to the carbon source solution during the main culture, which is the final stage.

twenty two

The culture was carried out by the same method as in Comparative Example 1 except for. For the obtained culture broth, the amount of acrylamide produced per unit dry microorganism weight was calculated in the same manner as in Example 1. Comparison The results of Example 2 were compared with the amount of acrylamide produced per unit dry microorganism weight obtained in Example 1 as 100%. The results are shown in Table 2.

[Example 3]

Add 0.lgZL of CoCl 6Η to the carbon source solution during the main culture, which is the final stage.

twenty two

The culture was carried out in the same manner as in Example 1 except for. In the obtained culture broth, the amount of acrylamide produced per unit dry microorganism weight was calculated in the same manner as in Example 1. Comparison The results of Example 3 were compared with the amount of acrylamide produced per unit dry microorganism weight obtained in Comparative Example 1 as 100%. The results are shown in Table 2.

[0047] [Table 2]

[0048] Microorganisms used: Syudnocardia Thermophila JCM3095-derived tolylhydratase-containing microorganism

Industrial applicability

[0049] According to the present invention, the enzyme activity per unit microbial weight can be increased by supplying an appropriate amount of conoleto during all the culturing of the cobalt-type-tolylhydratase-producing microorganism. It is useful for industrial implementation because higher microorganisms can be obtained

Claims

The scope of the claims

[1] In a culture that requires multiple stages of a microorganism that produces cobalt-type-tolylhydratase, a cobalt ion species is added to the culture solution of a seed step culture stage other than the final stage. Method for culturing type-tolylhydratase-producing microorganism

[2] Cobalt type-Cobalt type characterized by supplying a carbon source solution containing cobalt ion species to the final stage culture medium for multi-stage cultivation of microorganisms producing tolyl hydratase. A method for culturing a tolyl hydratase-producing microorganism.

[3] In the cultivation of a microorganism that produces cobalt-type tolyl hydratase, the culture method according to claim 1 and the culture method according to claim 2 are combined, A method for culturing a microorganism producing a tase.

[4] In the presence of a microorganism producing the cobalt-tolylhydratase obtained by the culture method according to claim 1, 2 or 3, or a treated product of the microorganism, the nitrile compound is water-treated in an aqueous medium. A method for producing an amide compound by adding together.