JP2001292762A - Method for producing high enzyme-producing bacterium microbial cell in high-density culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a high enzyme-producing bacterium microbial cell, capable of culturing a bacterium in a high density, not reducing a produced enzyme activity at the latter term of the culture. SOLUTION: A high enzyme-producing bacterium microbial cell controlling reduction in enzyme activity at the latter term of culture is produced by carrying out a high-density culture of successively adding a bacterium to a medium, etc., after completion of the growth of bacterium, changing a culture solution to a hypotonic solution by a diafiltration using a microfiltration membrane or an ultrafiltration membrane and continuing the culture afterwards. A bacterium belonging the genus Arthrobacter, capable of producing nitrilase can be exemplified as the bacterium. Water or an aqueous solution containing an enzyme-inducing substance can be exemplified as the hypotonic solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing microbial cells expressing high enzymes capable of suppressing a decrease in enzyme activity at the latter stage of culture, and more particularly, to a method for producing various nitriles in a high-density culture of microorganisms. The present invention relates to a method for producing a microbial cell which not only highly expresses an enzyme such as nitrilase which is useful as a catalyst for producing a corresponding organic acid by hydrolysis, but also suppresses a decrease in enzyme activity.

[0002]

2. Description of the Related Art Conventionally, microorganisms having nitrilase producing ability include, for example, Caseobacter.
Genus, Pseudomonas genus, Alcaligenes genus, Corynebacterium
terium), Brevibacterium
Genus, Nocardia, Rhodoccus
occus, Arthrobacter, Gordona, Bacillus, Bacteridium, Micrococcus
Microorganisms belonging to the genus Coccus, the genus Acinetobacter, etc. are known (JP-B-63-2596).
JP, JP-A-3-251192, JP-A-4-4
0898, JP-A-8-173152, JP-A-8-173175, International Publication WO96-094
No. 03, WO 97-3230).

[0003] However, microorganisms having nitrilase-producing ability generally have low nitrilase activity.
JP, JP-A-3-251192, JP-A-8-1
As described in 73152 and the like, a method of culturing a microorganism by adding a nitrile compound such as isobutyronitrile and ethylene cyanohydrin or a lactam compound as a nitrilase-inducing substance has been proposed. Although the addition of these nitrilase-inducing substances significantly improved the expression of nitrilase activity, they were still insufficient from a practical standpoint.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have developed a high nitrilase-expressing microbial bacterium in which a microorganism having a nitrilase-producing ability is cultured at a high density, and after a logarithmic growth phase is completed, a carbon source is added and the culture is further matured. A body manufacturing method has been developed (JP-A-11-341979). Although this method is excellent in terms of high expression of nitrilase activity, it has been observed that the nitrilase activity once expressed decreases in the latter stage of culture, and it has been desired to develop a method for suppressing this decrease in activity. . That is, an object of the present invention is to provide a method for producing a high-enzyme-expressing microbial cell in which a microorganism is cultured at high density and the expressed enzyme activity does not decrease even in the latter stage of the culture.

[0005]

Means for Solving the Problems The present inventors cultured the microorganisms at high density, and after the growth of the microorganisms was completed, exchanged the culture solution with a hypotonic solution to reduce the enzyme activity in the latter stage of the culture. The present inventors have found that microbial cells with reduced S. can be obtained, and have completed the present invention.

[0006] That is, the present invention provides an enzyme in which a decrease in enzyme activity in the late stage of culture is suppressed in a high-density culture of microorganisms, wherein the culture solution is replaced with a hypotonic solution after the growth of the microorganism is completed. The method according to claim 1, wherein the culture solution is exchanged for a hypotonic solution by a method for producing high-expressing microbial cells (claim 1) or diafiltration using a microfiltration membrane or an ultrafiltration membrane. The method for producing a microbial cell with high enzyme expression in which the decrease in enzyme activity in the late stage of culturing is suppressed (Claim 2), and the hypotonic solution is water or an aqueous solution containing an enzyme inducer. 3. The method for producing a high-enzyme-expressing microbial cell in which a decrease in enzyme activity at the latter stage of culture according to 1 or 2 is suppressed (claim 3), and the high-enzyme-expressing microbial cell is a nitrilase-high-expressing microbial cell. Any of claims 1 to 3, Enzyme high expression microbial cell manufacturing method decreases the enzyme activity was inhibited in culture late described (claim 4) or, microorganisms, Arthrobacter (Arthrobacter)
5. A microorganism belonging to the genus.
The method for producing an enzyme-overexpressing microbial cell in which a decrease in enzyme activity at the latter stage of culture according to any one of (1) to (5) is described, and a microorganism belonging to the genus Arthrobacter,
Arthrobacter sp. NSSC10
4. The method according to claim 5, wherein the bacterial strain is a high-enzyme-expressing microbial cell in which a decrease in enzyme activity at the latter stage of culture is suppressed.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism that can be used in the present invention is not particularly limited, but a specific example is a microorganism having nitrilase-producing ability. Examples of such a nitrilase-producing microorganism capable of hydrolyzing various nitriles to produce the corresponding organic acid include Caseobacter, Pseudomonas, Alcaligenes, Corynebacterium, Brevibacterium, Nocardia. , Rhodococcus, Arthrobacter, Gordona, Bacillus, Bacterium, Micrococcus, Acinetobacter, and other known nitrilase-producing microorganisms. be able to.

[0008] Among the above microorganisms having nitrilase-producing ability, microorganisms of the genus Arthrobacter, in particular, Arthrobacter sp. NSSC104 strain [FE
RMBP-5829], Arthrobact
er) sp. HR4 strain [FERM P-11302] can be preferably exemplified. Of these, Arthrobacter sp. The NSSC104 strain has been found by the present applicants, and its bacteriological properties are described in detail in WO 97-3230.

In the present invention, high-density culturing of microorganisms means culturing under a condition in which the cell density of microorganisms in a liquid medium is high. High-density culturing of microorganisms is usually achieved by culturing in a batch of medium components. Cell culture at a cell density that cannot be performed and can be achieved by fed-batch culture in which a medium component is added intermittently or continuously sequentially can be suitably exemplified. Other culture conditions other than high-density culture
Conditions suitable for culturing the microorganism can be used.
For example, culture of a microorganism having nitrilase-producing ability is generally carried out under aerobic conditions with aeration and stirring at pH 6 to 10,
The temperature can be controlled in an appropriate range of 25 to 37 ° C.

[0010] As a medium used for culturing microorganisms,
Any of a natural medium and a synthetic medium may be used as long as the microorganism to be used can grow, and it is particularly limited as long as the microorganism contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the microorganism. Although not particularly preferred, those containing an enzyme inducer are preferred. For example, in the case of a microorganism having a nitrilase-producing ability, examples of the enzyme inducer include nitrile compounds such as isobutyronitrile and ethylene cyanohydrin, and ε.
And cyclic amide compounds such as caprolactam.

Examples of carbon sources that can be assimilated by the microorganism include sugars such as glucose, fructose, sucrose, maltose and molasses containing them, carbohydrates such as starch and hydrolyzed starch, and organic acids such as acetic acid and lactic acid. And alcohols such as ethanol, glycerin and propanol, etc. can be used alone or in combination. Nitrogen sources include amines, amino acids, nitrates, ammonia, ammonium salts of various inorganic acids and organic acids, and others. Nitrogen compounds, peptone, tryptone, meat extract,
Yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and their digestions, etc. can be used alone or in combination. Potassium, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used alone or in combination.

In the present invention, terminating the growth of microorganisms refers to a state in which, after the addition of the medium components is completed, the components available for the microorganisms in the culture solution are deficient, and the growth of the microorganisms has been substantially terminated. The termination of the growth of such a microorganism can be known, for example, by measuring the concentration of sugar and disappearing the sugar component from the medium, or by measuring the dissolved oxygen concentration in the medium and detecting an increase in the dissolved oxygen concentration. it can.

[0013] In the present invention, the hypotonic solution means water or an aqueous solution having an osmotic pressure lower than that of a culture solution. Examples of the aqueous solution include an enzyme inducer such as a nitrilase enzyme inducer and sodium chloride and potassium chloride. An aqueous solution such as a salt can be exemplified. The exchange of the culture solution with the hypotonic solution is performed, for example, by microfiltration (M
F) It can be performed by diafiltration using a membrane or an ultrafiltration (UF) membrane. The amount of the exchange solution between the culture solution and the hypotonic solution can vary depending on the concentration of the medium used, but usually, a suitable example is about 0.5 to 2 times the amount of the culture solution. Then, after diafiltration, by culturing the microorganism under the same culture conditions, it is possible to produce the enzyme-overexpressing microbial cells of the present invention in which the decrease in the enzyme activity in the latter stage of the culture is suppressed.

[0014]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

The measurement of nitrilase activity in the examples was performed as follows. The culture was sampled, and the cells obtained by centrifugation were suspended in a 100 mM potassium phosphate buffer (pH 7.5). 500 μl of this suspension
Was placed in an Eppendorf tube and pre-incubated at 35 ° C. for 5 minutes. 10 μl of 2-hydroxy-
Add 4-methylthiobutyronitrile, stir, and add
For 30 minutes. Immediately after the reaction, the cells were removed by centrifugation, and the supernatant was subjected to high performance liquid chromatography to quantify the produced 2-hydroxy-4-methylthiobutanoic acid. 1 μmol of 2-hydroxy-4 per minute
-The amount of cells producing methylthiobutanoic acid was 1 U.

The CSL (corn steep liquor) extract used in the examples was prepared as follows. 400 g of CSL was diluted with water, adjusted to pH 7.0 with 10 N sodium hydroxide, and made up to 1.0 L with water. This was centrifuged to remove insolubles, and a supernatant was obtained. The supernatant was sterilized by filtration and used for culture.

Example 1 In a 10 L jar fermenter, 2.85 L of an initial medium (150 ml of CSL extract, 62 ml of 20% [W /
V] casamino acid, 24 ml of 50% [W / V] glucose,
75 ml of 20% [W / V] ε-caprolactam and 2.54 L of water), and to this, Arthrobacter sp. NSSC104 strain was inoculated and cultured at 33 ° C. with aeration and stirring. Gluco Jr. (Biot Co., Ltd.), a small glucose meter dedicated to industrial measurement of glucose concentration in culture solution
2.19 L of supplemented medium (1.35 L of CS) so that the glucose concentration is 0.1-0.2%.
L extract, 553 ml of 20% [W / V] casamino acid, 2
16 ml of 50% [W / V] glucose and 75 ml of 20% [W / V] ε-caprolactam), followed by 960 ml of 50% [W / V] glucose.

At the time when two days have passed since the start of the culture, the culture solution was pumped by a tube pump while continuing the culture.
niKros hollow fiber module (0.05μ pore size)
m, membrane area of 3,900 cm ² , Toyobo Engineering Co., Ltd.) and 3.0 L (0.5 of culture volume)
The same amount of water was replenished while removing the filtrate of (2). Thereafter, the culture was continued for another 4 days. The nitrilase activity reached a maximum on the 5th day of the culture and was 10.0 U / ml culture. Even on the sixth day of culture, the nitrilase activity was 9.1.
It was a 1 U / ml culture.

COMPARATIVE EXAMPLE 1 2.85 L of an initial medium (same as in Example 1) was prepared in a 10 L jar fermenter. NSSC104 strain was inoculated and cultured at 33 ° C. with aeration and stirring. The glucose concentration in the culture was measured by Gluco Jr.
2.19 L of supplemented medium (as in Example 1) followed by 96
0 ml of 50% [W / V] glucose was fed. As a result of continuing the culture for 6 days, the nitrilase activity reached the maximum on the fourth day of the culture, and was 8.97 U / ml culture solution, which was about 90% of the activity obtained in Example 1. The nitrilase activity was reduced to 5.66 U / ml on the sixth day of culture.

Example 2 In a 10-L jar fermenter, 2.85 L of an initial medium (same as in Example 1) was prepared, and this was added to Arthrobacter sp. NSSC104 strain was inoculated and cultured at 33 ° C. with aeration and stirring. The glucose concentration in the culture was measured by Gluco Jr.
2.19 L of supplemented medium (as in Example 1) followed by 96
0 ml of 50% [W / V] glucose was fed. At the time when two days have passed since the start of the culture, while continuing the culture,
The culture was circulated through a MiniKros hollow fiber module (same as in Example 1) by a tube pump and 6.0
L (1 time of the amount of culture solution) while removing the filtrate.
A 5% [W / V] ε-caprolactam aqueous solution was supplemented. Thereafter, the culture was continued for another 5 days. The nitrilase activity reached a maximum on day 6 of the culture and was 11.5 U / ml culture. Even on the seventh day of the culture, the nitrilase activity was 11.0.
U / ml culture.

Example 3 In a 10 L jar fermenter, 2.85 L of an initial culture medium (same as in Example 1) was prepared, and this was added to an Earthobacter sp. NSSC104 strain was inoculated and cultured at 33 ° C. with aeration and stirring. The glucose concentration in the culture was measured by Gluco Jr.
2.19 L of supplemented medium (as in Example 1) followed by 96
0 ml of 50% [W / V] glucose was fed. At the time when two days have passed since the start of the culture, while continuing the culture,
The culture was circulated through a MiniKros hollow fiber module (same as in Example 1) by a tube pump, and 3.0
The same amount of 0.5% [W / V] ε-caprolactam aqueous solution was replenished while removing the filtrate of L (0.5 times the amount of the culture solution). Thereafter, the culture was continued for another 5 days. The nitrilase activity reached a maximum on the 5th day of culture and reached 10.7 U / ml.
It was a culture solution. On day 7 of the culture, the nitrilase activity was 7.91 U / ml culture.

Example 4 1.86 L of an initial medium (150 ml of CSL extract, 12 ml of 50% [W /
V] glucose, 50 ml of 20% [W / V] ε-caprolactam, and 1.65 L of water). Inoculate NSSC104 strain,
The cells were cultured at 33 ° C. with aeration and stirring. The glucose concentration in the culture was measured by Gluco Jr.
3.18 L of additional medium (2.85 L of C
SL extract, 228 ml of 50% [W / V] glucose,
And 100 ml of 20% [W / V] ε-caprolactam), followed by 960 ml of 50% [W / V] glucose.

At the time when two days have passed since the start of the culture, the culture solution was supplied by a tube pump while continuing the culture.
niKros hollow fiber module (same as Example 1)
And the same amount of 0.5% [W / V] ε-caprolactam aqueous solution was replenished while removing 6.0 L (1 time of the amount of the culture solution) of the filtrate. Thereafter, the culture was continued for another 4 days. Nitrilase activity reached a maximum on day 5 of culture,
It was a 9.42 U / ml culture solution. Even on the sixth day of culture, the nitrilase activity was 8.42 U / ml culture.

Comparative Example 2 1.86 L of an initial medium (same as in Example 4) was prepared in a 10-L jar fermenter, and this was added to Arthrobacter sp. NSSC104 strain was inoculated and cultured at 33 ° C. with aeration and stirring. The glucose concentration in the culture was measured by Gluco Jr.
3.18 L of supplemented medium (as in Example 3), followed by 96
0 ml of 50% [W / V] glucose was fed. As a result of continuing the culture for 6 days, the nitrilase activity reached the maximum on the fourth day of the culture, and was 8.67 U / ml culture solution, which was about 92% of the activity obtained in Example 3. On the sixth day of culture, nitrilase activity decreased to 6.36 U / ml culture.

[0025]

According to the present invention, a decrease in the enzyme activity at the latter stage of the high-density culture can be suppressed, and a microorganism cell overexpressing the enzyme can be obtained.

Claims (6)

[Claims] In a high-density culture of microorganisms, a culture solution is replaced with a hypotonic solution after the growth of the microorganisms is completed. A method for producing cells. 2. The method according to claim 1, wherein the culture solution is exchanged for a hypotonic solution by diafiltration using a microfiltration membrane or an ultrafiltration membrane. A method for producing a suppressed bacterial cell with high expression of an enzyme. 3. The microorganism according to claim 1, wherein the hypotonic solution is water or an aqueous solution containing an enzyme-inducing substance. Manufacturing method. 4. The microorganism cell overexpressing an enzyme is a microorganism cell overexpressing nitrilase.
4. The method for producing a microorganism according to any one of the above, wherein a decrease in enzyme activity in the latter stage of the culture is suppressed. 5. The method according to claim 5, wherein the microorganism is Arthrobacter.
5. The method according to claim 1, wherein the microorganism is a microorganism belonging to the genus cter). 6. The microorganism belonging to the genus Arthrobacter, wherein the microorganism belonging to the genus Arthrobacter is Arthrobacter s.
p. 6. The strain is NSSC104 strain.
A method for producing an enzyme-overexpressing microbial cell in which a decrease in enzyme activity in the latter stage of the culture is suppressed.