JP2020115838A - Methods for preserving nitrile hydratase - Google Patents

Abstract

To provide methods for stably preserving cells or processed cells containing nitrile hydratase without addition of any stabilizer or freezing treatment.SOLUTION: Cells or processed cells containing nitrile hydratase can be preserved with little reduction of enzyme activity by using cells and/or processed cells having activity 100 U per mg dried cells or more and storing them at a temperature of at least the freezing point.SELECTED DRAWING: None

Description

The present invention relates to a method for preserving nitrile hydratase, which is an industrially useful enzyme for production of amide compounds.

As one of the main production methods of amide compounds, a hydration method using a nitrile compound as a raw material is often used, and as an industrial production method of amide compounds such as acrylamide, for example, metal such as Raney copper is used. Is used as a catalyst to hydrate a nitrile compound such as acrylonitrile, or in recent years, a method of hydrating a nitrile compound using a nitrile hydratase-containing bacterium and/or a treated product of the bacterium as a catalyst is known. Has been.

The method of using a bacterium containing nitrile hydratase and/or a treated product of the bacterium as a catalyst has attracted industrial attention because of its high conversion rate and selectivity of a nitrile compound such as acrylonitrile.

Enzymes such as nitrile hydratase must be stored stably before use. In general, for the purpose of suppressing the decrease in the activity, the decrease in the enzyme activity is suppressed by storing in the presence of a stabilizer, a metabolic inhibitor, high-concentration salts and the like. It is known that when the above-mentioned stabilizer is not added, it is preserved while maintaining the enzyme activity by freezing, aeration or stirring.

The following studies have been conducted so far regarding the method for storing nitrile hydratase. For example, Patent Document 1 discloses a method in which at least one compound selected from nitriles, amides, and organic acids or salts thereof is added as a stabilizer to maintain the enzyme activity stable for one day or more. It is disclosed. Further, in Patent Document 2, in a method of stably storing enzyme activity and microbial cells for a long period of time in a state in which a microorganism having an enzyme activity or its immobilized product is suspended in an aqueous medium, the aqueous medium is neutral to weak. Disclosed is a method for storing a suspension of bacterial cells or immobilized bacterial cells, which is an aqueous solution of an inorganic salt that is basic and has a saturation concentration of 100 mM. However, all of these methods involve the addition of stabilizers and other additives, which not only leads to an increase in cost due to the addition of stabilizers, but also of the final products such as acrylamide produced using nitrile hydratase. It goes without saying that the quality is greatly affected, and the production cost is further increased by removing it. Therefore, it is industrially desirable that it can be stored without positively adding a stabilizer.

Further, in Patent Document 3, when storing nitrile hydratase, the storage temperature is −5° C. or lower, and is stored in a frozen state at a nitrogen boiling point (−195.8° C.) or higher. A storage method is disclosed. However, in the storage method by freezing, operations such as freezing and thawing may be complicated, and the enzyme activity may be reduced due to the influence of the operations.

From the above viewpoint, a more efficient method for preserving nitrile hydratase is demanded on an industrial scale.

JP 62-259586A JP-A-8-112089 JP, 2003-219870, A

The problem to be solved by the present invention from the above situation is to provide a method for stably storing a nitrile hydratase-containing bacterium or a treated product of the bacterium, without adding a stabilizer or a freezing step. Is to provide.

The present inventors have conducted intensive research to solve the above problems. As a result, when storing the nitrile hydratase, it was found that the above problems can be solved by using a bacterium containing a nitrile hydratase having an active amount of 100 U or more per 1 mg of dry bacterium or a treated product of the bacterium. The present invention has been reached.

That is, the present invention includes the following items.
[1] a step of preparing a microbial cell containing a nitrile hydratase having an activity amount of 100 U or more per 1 mg of dried microbial cell or a treated product of the microbial cell, the temperature of the microbial cell or the treated product of the microbial cell being above a freezing point The method for storing a nitrile hydratase-containing bacterium or a treated product of the bacterium, which comprises:
[2] The method for storing a nitrile hydratase-containing bacterium or a treated product of the bacterium according to the above [1], wherein the enzyme activity is maintained at 70% or more 90 days after the start of storage.
[3] The method for preserving a nitrile hydratase-containing bacterium or a treated product of the bacterium according to the above [1] or [2], which has a storage temperature of not lower than freezing point and not higher than 40°C.
[4] The method for preserving the nitrile hydratase-containing bacterium according to any one of [1] to [3] or the treated product of the bacterium, wherein the nitrile hydratase is a nitrile hydratase derived from Pseudonocardia.
[5] A method for producing an amide compound, which comprises using the nitrile hydratase-containing bacterium or the treated product of the bacterium that has undergone the storage method according to any one of [1] to [4].

According to the present invention, when the nitrile hydratase is stored, it is possible to suppress a decrease in the activity of the enzyme without adding a stabilizer or the like or performing a freezing step. Further, by producing an amide compound using the nitrile hydratase-containing cells or a treated product of the cells that has undergone such a storage method, there is no concern that the amide compound as the final product will be affected by additives, etc. An amide compound can be obtained at low cost without the need to remove extra components such as a stabilizer by purification.

The details of the method for storing the nitrile hydratase of the present invention will be described below.

The present invention provides a method for preserving cells containing nitrile hydratase or a treated product of the cells.

Nitrile hydratase refers to an enzyme (protein) having the ability to hydrolyze a nitrile compound to produce a corresponding amide compound.

The nitrile hydratase-containing bacterium used in the present invention and/or a treated product of the bacterium is not particularly limited as long as the active amount per 100 mg of dried bacterium is 100 U or more. The bacterium producing nitrile hydratase, Nocardia (Nocardia) genus, Corynebacterium (Corynebacterium) genus, Bacillus (Bacillus) genus, thermophilic Bacillus, Pseudomonas (Pseudomonas) genus, Micrococcus genus , Rhodococcus typified by rhodochrous species, Rhodococcus genus, Acinetobacter genus, Xanthobacter (Xanthobacter) genus, Streptomyces (Streptomyces) genus, Rhizobium (Rhizobium) genus, Klebsiella genus, Representatives of the genus Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium or Thermophila species Examples thereof include Pseudonocardia genus, Bacteridium genus and Brevibacterium genus. These may be used alone or in combination of two or more.
In addition, a transformant in which a nitrile hydratase gene cloned from these cells is highly expressed in an arbitrary host, and one or more constituent amino acids of the nitrile hydratase are replaced with other amino acids by using recombinant DNA technology. Also included are transformants expressing a mutant nitrile hydratase with further improved amide compound resistance, nitrile compound resistance, and temperature resistance by substitution, deletion, deletion, or insertion with. Incidentally, the arbitrary host referred to here includes Escherichia coli as a representative example as in the example described later, but is not particularly limited to E. coli and Bacillus subtilis (Bacillus subtilis) etc. Other strains such as fungi, yeasts and actinomycetes are also included. As an example of such a product, MT-10822 [This strain is the biotechnology industrial research institute of the Ministry of International Trade and Industry, Industrial Technology Institute (currently Kisarazu, Chiba Prefecture) on 1-3 February Higashi 1-3-2, Tsukuba City, Ibaraki Prefecture. 2-5-8 Kazusa City, Kazusa, Japan Under the Budapest Treaty on the international approval of the deposit of bacterial cells for patent procedures, with the deposit number FERMBP-5785 at the Biotechnology Center Biotechnology Center, Patent Biological Deposit Center) Have been deposited. ] Is mentioned.

Among these bacterial cells, in terms of having a highly active and highly stable nitrile hydratase, bacterial cells belonging to the genus Pseudonocardia, and a nitrile hydratase gene cloned from the bacterial cells in any host. A highly expressed transformant and a transformant expressing a mutant nitrile hydratase are preferred. The above transformant is preferable because it further improves the stability of nitrile hydratase and has higher activity per bacterium.

In addition, Rhodococcus rhodochrous J-1, which can highly express nitrile hydratase in the cells, and a transformant in which the nitrile hydratase gene cloned from the cells is highly expressed in any host are also preferable. ..

The bacterium producing the nitrile hydratase can be prepared by a general method known in the fields of molecular biology, biotechnology, and genetic engineering.

By referring to the methods described in the documents described in Examples 1 to 3 described later in the present specification, it is possible to obtain a microbial cell or a treated microbial cell having an active amount of 100 U or more per 1 mg of dried microbial cell. it can.

The recombinant vector according to the present invention contains a gene encoding nitrile hydratase, and can be obtained by linking the gene encoding nitrile hydratase to the vector. The vector is not particularly limited, and for example, a commercially available expression plasmid typified by pET-21a(+), pKK223-3, pUC19, pBluescriptKS(+), pBR322 and the like can be used to encode a gene encoding nitrile hydratase. The expression plasmid of the nitrile hydratase can be constructed by incorporating The host organism used for transformation may be any host organism as long as the recombinant vector is stable and capable of self-propagation and can express the trait of foreign DNA. For example, Escherichia coli can be mentioned as a good example. Not limited to this, a transformant having the ability to produce nitrile hydratase can be obtained by introducing it into Bacillus subtilis, yeast or the like.

The nitrile hydratase-producing bacterial cell as described above may be appropriately cultured and grown by a known method to produce nitrile hydratase. In this case, as the medium used, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of carbon source, nitrogen source, inorganic salts and other nutrients. For example, after inoculating the cells into an ordinary liquid medium such as LB medium and M9 medium, an appropriate culture temperature (generally 20°C to 50°C, but 50°C or higher in the case of a thermophilic bacterium) However, it can be prepared by culturing the cells. The culturing can be carried out in a liquid medium containing the above-mentioned culturing components using a usual culturing method such as shaking culturing, aeration and stirring culturing, continuous culturing, or fed-batch culturing. The culture temperature of the transformant is preferably 15 to 37°C. The culture conditions are not particularly limited, and may be appropriately selected depending on the type of culture and the culture method, as long as the strain grows and nitrile hydratase can be produced.

In the present invention, the nitrile hydratase-producing bacterial cells described above may be subjected to various treatments such as collecting cells by centrifugation or the like, or crushing to prepare a crushed bacterial cell, and a bacterium subjected to any of these treatments. The body is collectively referred to as a treated product of bacterial cells. In the examples of the present invention, the term "collected liquid" means the treated product of bacterial cells.

The form of the cells to be crushed is not particularly limited as long as it contains cells that produce nitrile hydratase, and for example, the culture solution itself containing the cells, the culture solution is separated and recovered by centrifugation. Examples of the harvested cells, and those obtained by washing the harvested cells with physiological saline or the like.

The unit "U" of the active amount in the present invention means that 1 micromol of the corresponding amide compound is produced from the nitrile compound in 1 minute. That is, the active amount per 1 mg of dry cells is the number of micromoles of the amount of the corresponding amide compound produced in 1 minute when the nitrile compound was reacted with 1 mg of dry cells.

The method for measuring the amount of activity is as follows. 10 g of 50 mM tris-hydrochloric acid aqueous solution (pH 8.0) is put in a 100 ml glass container which can be sealed, and nitrile hydratase is suspended therein, and then the temperature is adjusted to 20°C. Then, a nitrile compound is added and the reaction is carried out by stirring for 15 minutes. After 15 minutes, 80 g of a 10 mM phosphoric acid aqueous solution was added to complete the reaction, and then the concentration of the amide compound in the reaction-terminated liquid was measured by HPLC analysis. calculate.

The amounts of the nitrile hydratase and the nitrile compound used are set so that the nitrile compound remains in the reaction solution at the end of the reaction and the amide compound after the reaction is produced to a concentration that can be accurately measured.

Examples of the nitrile compound include an aliphatic nitrile compound having 2 to 20 carbon atoms and an aromatic nitrile compound having 6 to 20 carbon atoms, and may be used alone or in combination of two or more kinds.

Examples of the aliphatic nitrile compound include saturated or unsaturated nitrile having 2 to 6 carbon atoms. Specifically, aliphatic saturated mononitriles such as acetonitrile, propionitrile, butyronitrile, isobutyronitrile, valeronitrile, isovaleronitrile, and capronitrile; aliphatic saturated dinitriles such as malononitrile, succinonitrile, and adiponitrile; acrylonitrile, Aliphatic unsaturated nitriles such as methacrylonitrile and crotonnitrile; and the like.

Examples of the aromatic nitrile compound include benzonitrile, o-, m- or p-chlorobenzonitrile, o-, m- or p-fluorobenzonitrile, o-, m- or p-nitrobenzonitrile, o-. , M- or p-tolunitrile, o-, m- or p-cyanopyridine, benzyl cyanide and the like.

Among the nitrile compounds, acrylonitrile and methacrylonitrile are preferable.

The present invention can obtain the effect of the present invention when using a nitrile hydratase having an active amount of 100 U or more per 1 mg of dry cells, but the active amount is preferably 110 U or more, and the active amount is It is more preferably 120 U or more, still more preferably 150 U or more.

In the present invention, the term "preservation" means that the cells containing the nitrile hydratase or a treated product of the cells are stored in a container such as a tank.

The storage period is not particularly limited and is usually 1 day or more (24 hours or more), but considering industrial use, the storage period is preferably 30 days or more, more preferably 60 days or more, and 90 days or more. Is more preferable.

When the bacterium containing nitrile hydratase or the treated product of the bacterium is preserved by the method of the present invention, the decrease in the enzyme activity is suppressed, and the enzyme activity after the preservation period can be maintained. Regarding the maintenance of the activity after the preservation period with respect to the activity at the beginning of preservation, depending on the preservation period, for example, the enzyme activity at the beginning of preservation is maintained at 50% or more based on the enzyme activity at the beginning of preservation. .. The enzyme activity 90 days after the start of storage is preferably maintained at 70% or more, more preferably 80% or more, still more preferably 90% or more.

In the present invention, the temperature for storing the nitrile hydratase is not particularly limited as long as it is above the freezing point, but in order to suppress the decrease in the activity of the nitrile hydratase, it is preferably above the freezing point and below 40°C, more preferably Is above the freezing point and below 20° C., more preferably above the freezing point and below 10° C. Here, the freezing point means a temperature at which a suspension of a bacterial cell and/or a treated product of the bacterial cell coexists while maintaining a solid state and a liquid state in equilibrium. The suspension of the body and/or processed bacterial cells thereof is maintained in a liquid state.

Of course, as long as the effect of the present invention is not impaired, the composition of the present invention (cells containing nitrile hydratase having an active amount of 100 U or more per 1 mg of dried cells or a treated product thereof is a temperature above freezing point). In addition to the above), an additive treatment as described in the background art may be further performed. Examples of the additive include nitriles, amides and organic acids, or inorganic salts selected from phosphates, nitrates, sulfates, sulfites and hydrochlorides. However, it is preferable that the present invention does not substantially contain additives and the like from the circumstances described in the background art. The phrase "substantially free" means that the present invention is construed narrowly and intentionally, and in addition to the constitution of the present invention, an embodiment in which a small amount of additives and the like are intentionally added is taken. However, such an aspect is a description for avoiding such a controversy as if it were an embodiment outside the scope of the present invention. Although a specific value of “substantially free of additives” and the like cannot be described, it is not preferable for the amide compound which is the final product in the process of the present invention even if additives are included. It is meant to include only amounts that have no effect.

Examples of the present invention will be specifically described below, but the present invention is not limited to these examples.

[Example 1]
[Preparation of cells containing nitrile hydratase]
According to the method described in Example 63 of JP-A-2004-194588, the clone number no. 103 cells were obtained and cultured by the following method to obtain wet cells containing nitrile hydratase.

For the culture of this strain, 45.0 L of medium having the following composition was prepared in a 90 L culture tank, and sterilized by autoclaving at 121° C. for 20 minutes. Ampicillin was added to this medium so that the final concentration was 50 μg/mL, and then No. One platinum loop was inoculated with 103 cloned bacterial cells and cultured at 37° C. and 700 rpm for 40 hours. Only the bacterial cells were separated from the culture solution by centrifugation (15000 G x 15 minutes), and the supernatant was added at the time of centrifugation so that the solid content was about 15% by weight in order to make the separated solution fluid. A harvested liquid of wet cells was obtained.
Medium composition Yeast extract 5.0 g/L
Polypeptone 10.0 g/L
NaCl 5.0 g/L
Cobalt chloride hexahydrate 10.0 mg/L
Ferric sulfate heptahydrate 40.0 mg/L
pH 7.5

Next, the bacterial cell suspension was filled into a 20 L polypropylene container (length 30 cm, width 30 cm, height 45 cm) at a rate of 18 kg and stored in a refrigerator at a temperature of 5°C.

(Measurement of nitrile hydratase activity)
Immediately after starting the storage of the harvested liquid prepared by the above method (0 day), the nitrile hydratase activity was measured by the following method. 10 g of 50 mM tris-hydrochloric acid aqueous solution (pH 8.0) was placed in a 100 ml glass container which was able to be sealed, the bacterial cell suspension was mixed and adjusted to 20° C., and then acrylonitrile was added and stirred for 15 minutes to carry out a reaction. After 15 minutes, 80 g of a 10 mM phosphoric acid aqueous solution was added to complete the reaction, and then the acrylamide concentration in the reaction-terminated liquid was measured by high performance liquid chromatography analysis. The amount of nitrile hydratase used and the amount of acrylamide produced produced the amount of activity. Was calculated, the amount of activity was 120 U per 1 mg of dry cells. Here, the analysis conditions are as follows.
·Analysis conditions:
High-performance liquid chromatograph: LC-10A system (Shimadzu Corporation)
(UV detector wavelength 250nm, column temperature 40°C)
Separation column: SCR-101H (Shimadzu Corporation)
Eluent: 0.005% (volume basis)-phosphoric acid aqueous solution

Further, the harvested liquid prepared by the above method was stored at a temperature of 5° C., and the nitrile hydratase activity after 5, 10, 30, 60, 90 days respectively was measured by the above method. The results of the activity amount are shown in Table 1.
As an index of the nitrile hydratase activity, the residual activity rate (%) was calculated by the following formula. It is an index showing the activity after the storage period as a relative value when the activity at the start of storage (day 0) is 100.
Percentage of remaining activity (%)=(activity at the start of storage/activity after the storage period)×100
The results of the residual activity rate are shown in Table 2.

[Example 2]
[Preparation of cells containing nitrile hydratase]
According to the method described in Example 76 of JP-A-2004-194588, the clone number No. described in Example 76 is used. 116 cells were obtained, cultivated by the method described in Example 1, and then subjected to a centrifugation treatment to obtain a wet cell collection solution containing nitrile hydratase.

Next, the collected liquid was filled into a 20 L polypropylene container (length 30 cm, width 30 cm, height 45 cm) by 18 kg each, and stored in a refrigerator at a temperature of 5°C.

(Measurement of nitrile hydratase activity)
The nitrile hydratase activity was measured in the same manner as in Example 1 immediately after the storage of the harvested liquid prepared by the above method was started (day 0). The active amount per 1 mg of dried bacterial cells was 160 U.

In addition, the harvested liquid prepared by the above method was stored at a temperature of 5° C., and the nitrile hydratase activity after 5, 10, 30, 60 and 90 days respectively was measured by the same method as in Example 1. The activity residual rate (%) was determined.

Table 1 shows the amount of activity of the harvested liquid stored under the above conditions, and Table 2 shows the percentage of residual activity.

[Example 3]
[Preparation of cells containing nitrile hydratase]
According to the method described in Example 51 of WO2010-55666, the transformant No. 92 described in Example 51 was obtained, cultured by the method described in Example 1, and then centrifuged. This was carried out to obtain a liquid collection of wet microbial cells containing nitrile hydratase.

Next, the bacterial cell suspension was filled into a 20 L polypropylene container (length 30 cm, width 30 cm, height 45 cm) at a rate of 18 kg and stored in a refrigerator at a temperature of 5°C.

(Measurement of nitrile hydratase activity)
The nitrile hydratase activity was measured in the same manner as in Example 1 immediately after the storage of the harvested liquid prepared by the above method was started (day 0). The amount of activity was 240 U per 1 mg of dry cells.

In addition, the harvested liquid prepared by the above method was stored at a temperature of 5° C., and the nitrile hydratase activity after 5, 10, 30, 60, 90 days respectively was measured by the method described in Example 1. The activity residual rate (%) was determined.

Table 1 shows the amount of activity of the harvested liquid stored under the above conditions, and Table 2 shows the percentage of residual activity.

[Comparative example]
[Preparation of cells containing nitrile hydratase]
According to the method described in Example 6 described in JP-A-9-275978, the clone number No. described in Example 6 was used. The microbial cell of No. 1 was obtained, cultured by the method described in Example 1, and then subjected to a centrifugation treatment to obtain a liquid collection of wet microbial cell containing nitrile hydratase.

Next, the bacterial cell suspension was filled into a 20 L polypropylene container (length 30 cm, width 30 cm, height 45 cm) at a rate of 18 kg and stored in a refrigerator at a temperature of 5°C.

(Measurement of nitrile hydratase activity)
The nitrile hydratase activity was measured in the same manner as in Example 1 immediately after the storage of the harvested liquid prepared by the above method was started (day 0). The active amount per 1 mg of dried bacterial cells was 60 U. In addition, the harvested liquid prepared by the above method was stored at a temperature of 5° C., and the nitrile hydratase activity after 5, 10, 30, 60 and 90 days respectively was measured by the same method as in Example 1. The activity residual rate (%) was determined.

The amount of activity of the harvested liquid stored under the above conditions is shown in Table 1 (preservation period and amount of activity), and the residual activity rate is shown in Table 2 (preservation period and retention rate of activity).

Claims (5)

A step of preparing a bacterial cell containing a nitrile hydratase having an activity amount of 100 U or more per 1 mg of dried bacterial cell or a processed product of the bacterial cell, and the bacterial cell or the processed product of the bacterial cell at a temperature above a freezing point for 24 hours A method for preserving a nitrile hydratase-containing bacterium or a treated product of the bacterium, which comprises the step of storing over the above. The method for preserving a nitrile hydratase-containing bacterium or a treated product of the bacterium according to claim 1, wherein the enzyme activity is maintained at 70% or more 90 days after the start of preservation. The method for storing a nitrile hydratase-containing bacterium or a treated product of the bacterium according to claim 1 or 2, which has a storage temperature of not less than freezing point and not more than 40°C. The method for preserving a nitrile hydratase-containing bacterium or a treated product of the bacterium according to any one of claims 1 to 3, wherein the nitrile hydratase is a nitrile hydratase derived from the genus Pseudonocardia. A method for producing acrylamide, comprising using a nitrile hydratase-containing cell or a treated product of the cell that has undergone the storage method according to any one of claims 1 to 4.