JP2004305066A - Method for retaining nitrilase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stably retaining extremely unstable nitrilase activity of a solution or dispersion of an immobilized substance of a gram-negative bacterium having nitrilase activity or its enzyme for a long period of time in producing ammonium carboxylate by using the solution or dispersion. <P>SOLUTION: The method for retaining nitrilase activity comprises making at least one kind selected from a nitrile, an amide, a carboxylic acid and their salts as a stabilizer exist in the solution or dispersion of the immobilized substance of the gram-negative bacterium having nitrilase activity or its nitrilase enzyme. <P>COPYRIGHT: (C)2005,JPO&amp;NCIPI

Description

【００３１】
【実施例７〜１１】
［アミド類添加効果］
上記調製例で調製した微生物菌体液を、３０ｍＭリン酸バッファーと表２に示す３０ｍＭアミド類の混合液（ｐＨ＝７．０）で洗浄し、菌体懸濁液（乾燥菌体１０重量％）を得て、実施例１と同様に２時間後及び２日後の反応活性評価を実施した。結果を表２に示す。
【００３２】
【表２】

【００３３】
【実施例１２〜１６】
［ニトリル類添加効果］
上記調製例で調製した微生物菌体液を、３０ｍＭリン酸バッファーと下記表３に示す３０ｍＭニトリル類の混合液（ｐＨ＝７．０）で洗浄し、菌体懸濁液（乾燥菌体１０重量％）を得て、実施例１と同様に２時間後及び２日後の反応活性評価を実施した。結果を下記表３に示す。
【００３４】
【表３】

【００３５】
【発明の効果】
本発明によれば、ニトリラーゼ活性を有するグラム陰性菌又はそのニトリラーゼ酵素の固定化物溶液又は懸濁液を用いてカルボン酸アンモニウムを製造するにあたり、非常に不安定なそれらのニトリラーゼ活性を長期安定的に保持する方法を提供することができる。本発明の方法によれば、８時間を越えるニトリラーゼ活性の保持時間が実現される。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for preventing a time-dependent decrease in nitrilase activity based on nitrilase produced by a specific microorganism and stably maintaining this activity.
[0002]
[Prior art]
A method for synthesizing a target compound using a microorganism having enzymatic activity or its enzyme, or an immobilized product thereof, is capable of simplifying the reaction process because the reaction conditions are mild, or high purity because there are few by-products. In recent years, it has been used for the production of various compounds because of its advantages such as the ability to obtain the reaction product of
[0003]
Among such enzymes, nitrilase is known as an enzyme that hydrates a nitrile compound to produce a corresponding ammonium carboxylate. Conventional techniques relating to this enzyme include, for example, Patent Documents 1 to 4. Among the microorganisms producing the enzyme nitrilase, Acinetobacter and Alcaligenes are known as those of Gram-negative bacteria.
[0004]
By the way, when actually producing ammonium carboxylate using a gram-negative bacterium having such nitrilase activity or an immobilized product of the nitrilase enzyme, it is necessary to stably store the nitrilase activity of the enzyme until use. is there. That is, it is necessary to prevent inactivation or reduction of the catalytic activity of the enzyme.
[0005]
However, examinations by the present inventors have revealed that the activity of the nitrilase is extremely unstable, causing a large decrease in the activity even immediately after the collection of bacteria. In addition, it has been found that this large decrease in activity cannot be prevented even by a general technique such as refrigeration.
[0006]
As another solution to this problem, Patent Document 5 describes long-term storage in a suspension of an aqueous solution of an inorganic salt having a concentration of 100 mM to a saturation concentration. However, in this conventional technique, the use of high-concentration salts increases the cost, and requires a complicated operation such as washing with water before use in the reaction system, or the inorganic salts as impurities in the reaction system. There is a drawback in that the impurities are mixed and the load on a purification system such as ion exchange increases due to the purification.
[0007]
[Patent Document 1]
JP-B-63-2596 [Patent Document 2]
JP-A-63-129988 [Patent Document 3]
JP-A-63-209592 [Patent Document 4]
Japanese Patent Publication No. 2000-501610 [Patent Document 5]
JP-A-8-112089
[Problems to be solved by the invention]
In the present invention, in the production of ammonium carboxylate using a solution or suspension of a gram-negative bacterium having nitrilase activity or a nitrilase enzyme of the gram-negative bacterium, the extremely unstable nitrilase activity of the gram-negative bacteria is stabilized for a long time. It is an object of the present invention to provide a method for retaining the information.
[0009]
[Means for Solving the Problems]
In order to solve such a problem, the present inventors have intensively studied a method for stably storing enzyme activity for a long time in a state in which a gram-negative bacterium having nitrilase activity or an immobilized product of the nitrilase enzyme is suspended in an aqueous medium. did. As a result, it has been surprisingly found that the use of specific substances, that is, nitriles, amides, carboxylic acids and salts thereof is extremely effective for the purpose, and has completed the present invention.
[0010]
That is, the present invention provides the following methods for retaining nitrilase activity of 1 to 7.
1. A method for maintaining nitrilase activity, comprising the steps of: adding nitriles, amides, carboxylic acids and the like as stabilizers to a solution or suspension of a gram-negative bacterium having nitrilase activity or a nitrilase enzyme of the gram-negative bacterium; A method for maintaining nitrilase activity, wherein at least one compound selected from salts is present.
[0011]
2. The nitrilase activity according to 1, wherein the immobilized product solution or suspension contains ammonium carboxylate and the concentration of nitriles, amides, carboxylic acids and salts thereof is 0.1 to 500 mM in total. Retention method.
[0012]
(3) The method for retaining nitrilase activity according to (1) or (2), wherein the pH of the solution or suspension is 6.5 to 11.5.
[0013]
(4) The method for retaining nitrilase activity according to any one of (1) to (3), wherein the retention temperature is a freezing point to 20 ° C.
[0014]
(5) The method for retaining nitrilase activity according to any one of (1) to (4), wherein the Gram-negative bacterium having nitrilase activity is Acinetobacter or Alcaligenes.
[0015]
6. The method for retaining nitrilase activity according to 5, wherein the Gram-negative bacterium having nitrilase activity belongs to the genus Acinetobacter.
[0016]
7. The method for retaining nitrilase activity according to 6, wherein the Gram-negative bacteria having nitrilase activity are Acinetobacter sp. AK226 (FERM BP-2451) and Acinetobacter sp. AK227 (Microtechnical Laboratories No. 8272).
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be specifically described below.
Many Gram-negative bacteria having nitrilase activity referred to in the present invention are known. Typical high activity agents include Acinetobacter and Alcaligenes, but the present invention is not limited thereto. Specifically, Acinetobacter sp. AK226 (FERM BP-2451) and Acinetobacter sp. AK227 (Microtechnical Laboratories No. 8272). These strains are described in JP-B-63-2596. Alternatively, a microorganism into which a natural or artificially improved nitrilase gene has been incorporated by a genetic engineering technique or an enzyme taken out therefrom may be used.
[0018]
As a form for retaining nitrilase activity, the microorganism may be used as it is, but it may be used as it is, or after being subjected to a treatment such as crushing, or from a microorganism or the like, from which the nitrilase enzyme is removed, and further from the removed enzyme. What has been immobilized by any suitable entrapment method, cross-linking method, carrier binding method or the like may be used. Examples of the immobilization carrier for immobilizing those having nitrilase activity include, but are not limited to, glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, and the like. .
[0019]
The stabilizer in the present invention is a compound selected from nitriles, amides, carboxylic acids and salts thereof, and these can be used alone or in combination. Specifically, for example, the following compounds can be exemplified, but the present invention is not limited thereto.
1) Aliphatic nitriles such as acetonitrile, propionitrile, isobutyronitrile and corresponding amides and carboxylic acids or salts thereof. (The term “corresponding” means that an amide group or a carboxyl group is bonded instead of the nitrile group.)
2) Amino nitriles such as glycine nitrile and α-aminopropionitrile, and corresponding amides and carboxylic acids or salts thereof.
3) Hydroxynitrile such as lactonitrile, hydroxyacetonitrile and corresponding amide and carboxylic acid or salts thereof.
4) Unsaturated nitriles such as acrylonitrile and methacrylonitrile and corresponding amides and carboxylic acids or salts thereof.
5) Dinitriles such as malononitrile and succinonitrile, and corresponding amides and carboxylic acids or salts thereof.
6) Monocyanoamides such as cyanoacetamide, cyanoacetate and monocyanoic acid or salts thereof.
7) Aromatic nitriles such as benzonitrile and phenylacetonitrile, and corresponding amides and carboxylic acids or salts thereof.
8) Heterocyclic nitriles such as nicotinonitrile and isonicotinonitrile and corresponding amides and carboxylic acids or salts thereof.
9) Halogenated nitriles such as chloroacetonitrile and β-chloropropionitrile, and corresponding amides and carboxylic acids or salts thereof.
10) A carboxylic acid having an aldehyde group such as glyoxal or a salt thereof.
[0020]
In order to maintain nitrilase activity in the present invention, the above-mentioned compound as a stabilizer and a gram-negative bacterium also having the above-mentioned nitrilase activity or an immobilized solution or suspension of the nitrilase enzyme, together with various buffer solutions , Physiological saline, water or the like. Specifically, the cell culture is first centrifuged to separate the cell pellet and the culture, and then the culture is removed. Next, a liquid containing a stabilizer is added to the cell pellet, mixed and suspended, and then centrifuged again to obtain a cell pellet. Finally, a liquid containing the stabilizer can be added, mixed and suspended. . In the examples described later, this series of operations was performed, which was described as washing of the bacterial cell fluid. By such a method, the nitrilase activity could achieve a retention time exceeding 8 hours, and in Examples described later, the activity was stable even after 2 days.
[0021]
The stabilizer may be added in such an amount that the total concentration of the nitriles, amides, carboxylic acids and salts thereof is in the range of 0.1 to 500 mM. 100 mM. The pH of the solution or suspension is in the range of 6.5 to 11.5, preferably 7 to 9. The temperature may be room temperature for short-term storage, but is preferably a low temperature of freezing point to 20 ° C. for long-term storage, particularly a temperature near the freezing point and not reaching the freezing point.
[0022]
Hereinafter, the present invention will be described with reference to examples. The present invention is not limited to these embodiments, and various changes and modifications can be made without changing the gist of the present invention.
[0023]
The method for measuring the dry cell weight in the unfixed microbial cell liquid was carried out as follows. First, an appropriate amount of a microbial cell suspension having an appropriate concentration was taken, cooled to −80 ° C., and completely dried using a freeze dryer to calculate the cell suspension concentration. The cell suspension having a known concentration was diluted to a plurality of appropriate concentrations, turbidity was measured with a turbidimeter, a calibration curve of the turbidimeter was prepared, and a factor was calculated. The dry cell concentration of an arbitrary microbial cell suspension was calculated from the turbidity indicator value of the turbidimeter.
[0024]
When the biocatalyst is obtained by immobilizing cells, measure the dry cell concentration of the cell suspension before immobilization, and fix the immobilized catalyst in the immobilized catalyst using the mixing ratio of immobilized carrier and cells. The dry weight of the biologically derived component from which the immobilized carrier was subtracted was calculated.
[0025]
Evaluation of nitrilase activity was performed as follows. Using acrylonitrile as a substrate, the activity was evaluated by measuring the amount of ammonium acrylate as a product. Specifically, 100 μL of acrylonitrile was added to a suspension of 5 mL of a Gram-negative bacterium having nitrilase activity or an immobilized product of the nitrilase enzyme in a 30 mM aqueous ammonium acrylate solution (pH = 7), which was previously held at 30 ° C. The reaction was started at the time when was quickly added, and the mixture was reacted at 30 ° C. for 20 minutes in a shaking incubator. The ammonium acrylate concentration in the reaction solution or the supernatant after the reaction was determined by neutralization titration after trapping ammonium ions as hexamethylenetetramine by formalin treatment. At this time, a sample to which acrylonitrile was not added at the same time was also prepared, and treated in the same manner as the sample to which acrylonitrile was added. The titration value was used as a blank value (concentration of ammonium acrylate before the reaction), and the value obtained by subtracting from the titration value was used to generate ammonium acrylate. Concentration. The reaction activity was expressed in terms of the weight of ammonium acrylate produced per 1 g of microbial cells per hour.
[0026]
[Example of preparation of microbial cell fluid]
Acinetobacter sp. Having nitrilase activity. AK226 (FERM BP-2451) was prepared from sodium chloride 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, iron sulfate heptahydrate 0.005%, manganese sulfate An aqueous solution containing 0.005% of pentahydrate, 0.1% of ammonium sulfate, and 0.1% of potassium nitrate (all by weight) is adjusted to pH = 7, and 0.5% by weight of acetonitrile is added as a nutrient source. And aerobically cultured at 30 ° C. At this time, the reaction activity immediately after the completion of the culture was evaluated by the above-mentioned method, and it was 89.5 [g-ammonium acrylate / g-dried cells / hour].
[0027]
Embodiment 1
[Activity retention confirmation experiment]
The microbial cell suspension prepared in the above preparation example was washed with 30 mM ammonium acrylate (pH = 7.0) to obtain a cell suspension (10% by weight of dried cells), and refrigerated at 5 ° C. Two hours later, the evaluation of the reaction activity was carried out by the method described above, and it was 89.0 [g-ammonium acrylate / g-dried cells / hour]. When the reaction activity after further refrigerated storage at 5 ° C. for 2 days was evaluated by the above method, it was 89.2 [g-ammonium acrylate / g-dried cells / hour].
[0028]
[Comparative Example 1]
[Effect of phosphate buffer]
The microbial cell liquid prepared in the above preparation example was washed with a 30 mM phosphate buffer (pH = 7.0) to obtain a cell suspension (10% by weight of dried cells), and refrigerated at 5 ° C. Two hours later, the evaluation of the reaction activity was carried out by the method described above, and it was 71.2 [g-ammonium acrylate / g-dried cells / hour]. Further, the reaction activity after refrigerated storage at 5 ° C. for 2 days was 42.5 [g-ammonium acrylate / g-dried cells / hour] as determined by the above method.
[0029]
[Examples 2 to 6]
[Effect of adding carboxylic acids]
The microbial cell liquid prepared in the above preparation example was washed with a mixed solution (pH = 7.0) of 30 mM phosphate buffer and 30 mM carboxylic acids shown in Table 1 below, and a cell suspension (10% by weight of dried cells) The reaction activity was evaluated after 2 hours and 2 days in the same manner as in Example 1. The results are shown in Table 1 below.
[0030]
[Table 1]

[0031]
Embodiments 7 to 11
[Effects of adding amides]
The microbial cell liquid prepared in the above preparation example was washed with a mixed solution (pH = 7.0) of 30 mM phosphate buffer and 30 mM amides shown in Table 2, and a cell suspension (10% by weight of dry cells) The reaction activity was evaluated after 2 hours and 2 days in the same manner as in Example 1. Table 2 shows the results.
[0032]
[Table 2]

[0033]
Embodiments 12 to 16
[Effects of adding nitriles]
The microbial cell suspension prepared in the above preparation example was washed with a mixed solution (pH = 7.0) of 30 mM phosphate buffer and 30 mM nitriles shown in Table 3 below, and a cell suspension (10% by weight of dried cells) ) Was obtained, and the reaction activity was evaluated after 2 hours and 2 days in the same manner as in Example 1. The results are shown in Table 3 below.
[0034]
[Table 3]

[0035]
【The invention's effect】
According to the present invention, when producing ammonium carboxylate using a solution or suspension of a gram-negative bacterium having nitrilase activity or a nitrilase enzyme thereof, a highly unstable nitrilase activity thereof can be stably maintained for a long time. A method of holding can be provided. According to the method of the present invention, a retention time of nitrilase activity exceeding 8 hours is realized.

Claims (7)

A method for maintaining nitrilase activity, comprising: adding nitriles, amides, carboxylic acids and salts thereof to a gram-negative bacterium having nitrilase activity or a solution or suspension of a nitrilase enzyme of the gram-negative bacteria as a stabilizer. A method for maintaining nitrilase activity, comprising the presence of at least one compound selected from the group consisting of: The nitrilase activity according to claim 1, wherein the immobilized product solution or suspension contains ammonium carboxylate, and the concentration of nitriles, amides, carboxylic acids and salts thereof is 0.1 to 500 mM in total. Holding method. 3. The method for retaining nitrilase activity according to claim 1, wherein the pH of the solution or suspension of the immobilized product is 6.5 to 11.5. The method for retaining nitrilase activity according to any one of claims 1 to 3, wherein the retention temperature is a freezing point to 20 ° C. The method for retaining nitrilase activity according to any one of claims 1 to 4, wherein the Gram-negative bacterium having nitrilase activity is of the genus Acinetobacter or Alcaligenes. The method for retaining nitrilase activity according to claim 5, wherein the Gram-negative bacterium having nitrilase activity is Acinetobacter. The method for retaining nitrilase activity according to claim 6, wherein the Gram-negative bacteria having nitrilase activity are Acinetobacter sp. AK226 (FERM BP-2451) and Acinetobacter sp.