JP2002017339A - Method for high-density culturing of bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for obtaining microorganisms by which the growth of a bacterium is improved and nitrile hydratase enzyme activity is highly expressed by a simple means without using an expensive derivative or complicated culture control, when the microorganisms having the ability to produce the nitrile hydratase is cultured. SOLUTION: The culturing is carried out by maintaining the dissolved oxygen concentration during culturing at 1 ppm to the saturation concentration when the bacterium having the ability to produce the nitrile hydratase is cultured. Thereby, the culturing can be performed at a high density and the cultured microbial cell highly expressing the nitrile hydratase activity is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing bacterial cells having high nitrile hydratase enzyme activity in a high yield by culturing.

[0002]

2. Description of the Related Art In recent years, there has been an increasing movement to utilize microorganisms or their enzymes as catalysts. Nitrile hydratase is known as an enzyme that hydrolyzes a nitrile compound to produce a corresponding amide compound, and is particularly useful as a catalyst for producing acrylamide from an acrylonitrile compound. Microorganisms having nitrile hydratase enzymatic activity and methods for culturing the same are described in many publications. For example, Corynebacterium spp. Genus (see JP-A-54-129190), Pseudomonas genus (see JP-A-59-91879), Arthroba (Arthroba)
cter), Microbacterium (see JP-A-61-162193), Rhodococcus (Rho
dococcus) (see JP-A-2-470), Rhizobium (see JP-A-5-236977), Klebsiera (see JP-A-5-30982),
Aeromonas (see JP-A-5-30983), Agrobacterium (see JP-A-8-154691), Bacillus
Bacteria belonging to [See JP-A-8-187092], Pseudonocardia (see JP-A-8-56684) and the like are known.

[0003]

However, in the above publication, an essential expensive inducer and a metal salt contained in the nitrile hydratase enzyme are added as means for highly expressing the nitrile hydratase enzyme. Culture is performed by shaking culture. Inducing agents often include nitrile compounds and amide compounds.A large amount of the nitrile hydratase enzyme causes high expression of the nitrile hydratase enzyme, but the growth rate is remarkably reduced. The enzyme is not overexpressed. The same occurs when urea and urea derivatives are used as inducers. In addition, nitrile compounds and amide compounds can be easily obtained as a reagent, but when industrially cultivated on a large scale, nitrile compounds and amide compounds manufactured as chemical products are limited, so only the inducing agent is used. However, the method has a drawback that the original performance of the bacterium cannot be sufficiently exhibited by culturing, and it has been difficult to obtain a high-yield, highly-active microbial cell by the above method.

[0004] The present invention provides a method for producing bacterial cells having high nitrile hydratase enzyme activity in a high yield by culturing, which solves the above problems.

[0005]

Means for Solving the Problems The present inventors have simplified the culture yield for producing bacterial cells having a high nitrile hydratase activity in a high yield without requiring any special means. As a result of intensive studies on the method of increasing the concentration, surprisingly, the dissolved oxygen concentration in the culture was reduced to 1 ppm.
By maintaining the saturation concentration from, the growth of bacteria is improved,
In addition, they have found that it is possible to obtain bacterial cells in which nitrile hydratase activity is highly expressed, and have completed the present invention.

That is, the present invention is a method for high-density cultivation of bacteria having nitrile hydratase-producing ability to maintain the dissolved oxygen concentration in a culture from 1 ppm to a saturated concentration.

In the present invention, the fact that the expression of nitrile hydratase has an optimum value of the dissolved oxygen concentration in the culture is not described in any literatures or publications, and was not known. is there.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The bacterium which is an object of the present invention is not particularly limited as long as it has nitrile hydratase enzyme activity and can hydrolyze a nitrile compound to produce a corresponding amide compound. Not done. Preferably,
By adjusting the dissolved oxygen concentration in the culture from 1 ppm to a saturated concentration, any bacteria can be used as long as the growth of the bacteria can be improved and the nitrile hydratase enzyme activity can be highly expressed. Specifically, Nocardia sp.N described in JP-B-56-17918
-775, Rhodococcus rhodochrous J-1 described in JP-B-06-55148, JP-A-05-30982
Klebsiella sp.MCI2609 described in
Aeromonas sp.MCI261 described in 05-30983
4, JP 05-30984 described Citrobacter freundii (Citrobacter freundii) MCI2615, JP-A 05-103681 described Agrobacterium rhizogenes (Agrobacterium
rhizogenes) IAM13570 and Agrobacterium tumefaciens, JP-A-05-161
Xanthobacter flavus described in No. 495 (Xanthobacter
flavas) JCM1204, Erwinia Nigrifluence (Er
winia nigrifluens) MAFF03-01435, JP 05-236975 described Enterobacter sp.MCI2707, JP 05-236976 described Streptomyces (Streptomyc
es) sp.MCI2691, Rhizobium (R) described in JP-A-05-236977.
MCI2610, Rhizobium sp.MCI2643, Rhizobium loti IAM13588, Rhizobium legminosarum IAM12609, and Rhizobium merioti IAM12
611, Candida guilliermondii NH-2, Pantoea described in JP-A-05-15384
Agglomerans (Pantoea agglomerans) NH-3 and Klebsiella pneumoniae subsp.pneumoniae NH-
26T2, Agrobacterium radiobacter described in JP-A-06-14786 (Agrobacterium radiobacter) SC-C15-1,
Bacillus Smithy described in JP-A-07-25494 (Bacillus
smithii) SC-J05-1, JP-A 08-56684 Pseudonocardia thermophila (Pseudonocardia thermophil
a) ATCC19285, Pseudonocardia thermophila JC described in JP-A-09-275978
Microorganisms such as M3095 can be mentioned as suitable examples.

[0009] A transformant in which a nitrile hydratase gene cloned from a bacterium is expressed in any host also has the nitrile hydratase of the present invention, and hydrolyzes a nitrile compound to produce a corresponding amide compound. Bacteria that can be included. For example, USP5 using Escherichia coli as a representative example of a host,
The MT-10822 strain described in 807,730 (FERM BP-5785) can also be mentioned as a preferred example.

A general embodiment of the present invention is as follows. That is, as a nitrogen source, such as glucose, fructose, mannitol, sorbitol as a carbon source,
For example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, sodium glutamate and the like, as organic nutrients, for example, yeast extract, meat extract, malt extract, peptone, taste liquid, etc., as inorganic salts,
For example, phosphates, magnesium salts, potassium salts, sodium salts, iron salts, cobalt salts, and other trace metal salts, as enzyme inducers, if necessary, for example, urea, methyl urea, 1,1-dimethyl urea, A medium containing 1,3-dimethyl urea, ethyl urea, 1,1-diethyl urea, 1,3-diethyl urea, methacrylamide, etc. is inoculated with bacteria having nitrile hydratase activity, and the dissolved oxygen concentration is controlled. And culture under aerobic conditions.

In the culturing, the dissolved oxygen concentration may be 1 ppm or more both at the start of the culturing and during the culturing. When the concentration becomes 1 ppm or less during the culture, not only does the growth rate of the bacteria decrease, but also the nitrile hydratase enzyme leaks out of the cells. As a result, not only the specific activity but also the liquid activity decreases, and it becomes difficult to obtain a high-yield, high-activity microbial cell.

In order to maintain aerobic conditions during the culturing, a gas containing oxygen is supplied. Sources are air, pure oxygen,
Any of a mixture of oxygen and nitrogen or another gas at a fixed ratio may be supplied. For example, in the case of a flask with a cotton stopper, oxygen can be dissolved and supplied by shaking using a shaking incubator, and in the case of a jar culture, gas sterilized by a sterilizing filter is dispersed from a sparger or using a membrane. The gas can be diffused and supplied with an increased gas-liquid contact area by stirring.

The pH of the medium is usually 5-10, preferably 7-7.
9. Culture at a temperature of usually 20 to 50C, preferably 25 to 40C, aerobically for 1 to 3 days. By performing the culture under the above conditions, nitrile hydratase activity is highly expressed,
In addition, a culture solution of 10 g / L or more (in terms of dry cell weight) can be obtained.

[0014]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 A 20 ml culture of the Rhodococcus rhodochrous J-1 strain grown under the following pre-culture conditions was cultured under the following main culture conditions, and the nitrile hydratase activity was examined. The dissolved oxygen concentration at the start of the cultivation starts at a saturation value under the culturing conditions. However, since the dissolved oxygen concentration decreases with the growth of the bacterium, the dissolved oxygen concentration during the cultivation after 10 hours when the dissolved oxygen concentration is stabilized at a low level. The stirring number was adjusted and controlled so that the oxygen concentration became 1.5 ppm.

(1) Culture of bacteria Pre-culture conditions: (medium composition) fructose 2% (w / v), taste liquid 2% (w / v)
(Manufactured by Ajinomoto Co., Ltd.), polypeptone 5% (w / v) (manufactured by Nippon Pharmaceutical Co., Ltd.), yeast extract 0.3% (w / v) (manufactured by Oriental Yeast Co., Ltd.), KH ₂ PO ₄ 0.1 % (W /
_{v), K 2 HPO 4 0.1} % (w / v), MgSO 4 · 7H 2 O 0.1% (w /
v) pH7. (Culture method) 100 ml of the medium was dispensed into a 500 ml Erlenmeyer flask, which was stoppered with cotton, and sterilized in an autoclave at 121 ° C for 20 minutes. Rhodococcus rhodochrous J-1 (FERM BP-147
8) The strain was inoculated and cultured with shaking at 30 ° C. for 48 hours.

Main culture conditions: (medium composition) Initial medium: taste liquid 1% (w / v), yeast extract 0.2% (w / v),
KH ₂ PO ₄ 0.1% (w / v), K ₂ HPO ₄ 0.1% (w / v), MgSO
_{4 · 7H 2 O 0.1% (} w / v), CoCl 2 · 6H 2 O 0.002% (w /
v), ammonium sulfate 0.025% (w / v), fructose 2% (w / v),
Urea 2% (w / v), ethanol 0.4% (v / v), Pluronic L61 0.1% (w / v) (Asahi Denka Kogyo Co., Ltd.),
pH 7. Post-supplemented medium: fructose 20% (w / v), ethanol 5% (v / v), ammonium sulfate 6% (w / v), pH 6.5.

(Culture method) Two liters of the initial medium was dispensed into a 3-liter mini jar fermenter (manufactured by Takasugi Seisakusho) and sterilized in an autoclave at 121 ° C for 20 minutes.
However, fructose, ethanol and urea are separately
Filtrate aseptically (Advantech Toyo 45
Micron filter paper was used). Tank pressure 0.0
98MPa, stirring number 50-600rpm, ventilation volume 1vv
The cells were cultured at a temperature of 30 ° C. for 44 hours. From the 20th hour of the culture, a post-addition medium was added at a flow rate of 20 ml / hr until the end of the culture.

(2) Dissolved oxygen measurement method A sterilizable oxygen electrode for fermentation (CSL-1 manufactured by Able) was added to a solution in which copper sulfate hexahydrate was previously added to a sodium sulfite solution to reduce the dissolved oxygen concentration to zero. After immersion and adjustment so that the indication of the dissolved oxygen indicator (MODEL M-1032 manufactured by Able Co., Ltd.) becomes zero, the oxygen electrode was
It was attached to a mini-jar fermenter for liters and sterilized together with the medium. When starting the culture after inoculating the inoculum, the temperature,
The value of the dissolved oxygen indicator was corrected so as to indicate the saturated dissolved oxygen value calculated from the tank pressure and the like. The dissolved oxygen concentration during the culture was controlled from the value of a dissolved oxygen indicator or a hybrid recorder (KL-11-4E, Chino Corporation).

(3) Measurement of nitrile hydratase activity 0.1 ml of culture solution and 1/20 M phosphate buffer (pH 7.
7) Mix 4.90 ml and add another 5.0%
5 ml of a 1/20 M phosphate buffer (pH 7.7) containing (w / v) acrylonitrile was added, reacted at 10 ° C. for 10 minutes, and the cells were filtered off, followed by gas chromatography (GC-14B Shimadzu Corporation). The measurement was performed by quantifying the acrylamide produced in the above. The analysis conditions were 1 m filled with Polapack PS (a column filler manufactured by Waters).
Using glass column, column temperature 230 ° C, detector 2
Performed at 50 ° C. FID.

Hereinafter, the unit of enzyme activity is defined as one unit in which the activity of converting 1 μmol of acrylonitrile to acrylamide per minute in the above activity measurement. The activity was examined for the activity per culture and the activity per cell. The activity per culture solution and the activity per microbial cell were calculated, and the activity value under other culture conditions was defined as the relative activity (%), with the value obtained by culturing in Example 1 as 100.
Indicated by

<Examples 2 to 6> Instead of controlling the dissolved oxygen concentration during culturing to 1.5 ppm in Example 1, culturing the dissolved oxygen concentration during culturing to 2.0 ppm (Example 2) Culture in which the dissolved oxygen concentration during culturing was controlled to 3.0 ppm (Example 3), and the dissolved oxygen concentration during culturing was 4.0 pp.
m (Example 4), culture in which the dissolved oxygen concentration during culture was controlled at 11.0 ppm (Example 5), and culture in which the dissolved oxygen concentration during culture was controlled at 12.0 ppm (Example 6). Was carried out in the same manner as in Example 1 except that the above was carried out. The results are shown in Table 1.

<Comparative Example 1> The procedure of Example 1 was repeated except that the cultivation was carried out at 0.7 ppm, instead of controlling the dissolved oxygen concentration at 1.5 ppm. It carried out by the method similar to Example 1. The results are shown in Table 1.

[Table 1]

Example 7 Rhodococcus rhodochrous
Nocardia sp.N-775 strain (FERM BP-961) instead of J-1 strain
Was carried out in the same manner as in Example 1 except that As for the activity, the activity per culture solution and the activity per bacterial cell were calculated, and the activity value under other culture conditions was shown as a relative activity (%), with the value obtained by culturing in Example 7 as 100.

<Examples 8 and 9> Instead of controlling the dissolved oxygen concentration during culturing to 1.5 ppm in Example 7, culturing while controlling the dissolved oxygen concentration during culturing to 2.0 ppm (Example 8) The procedure was carried out in the same manner as in Example 7 except that the culture (Example 9) in which the dissolved oxygen concentration during the culture was controlled at 12 ppm was performed. The results are shown in Table 2.

<Comparative Example 2> The procedure of Example 7 was repeated, except that the cultivation was carried out at 0.7 ppm, instead of controlling the dissolved oxygen concentration at 1.5 ppm. It carried out by the method similar to Example 7. The results are shown in Table 2.

[Table 2]

Example 10 Pseudomonas chlororafis strain B-23 (FERMBP-1) grown under the following preculture conditions
20 ml of the culture of 87) was cultured under the following main culture conditions, and the nitrile hydratase activity was examined. In addition, the dissolved oxygen concentration during the culture was controlled to be 1.0 ppm. (1) Culture of bacteria Pre-culture conditions: (medium composition) glucose 0.5% (w / v), polypeptone 0.5% (w / v) (manufactured by Nippon Pharmaceutical Co., Ltd.), yeast extract 0.3% ( w / v) (Oriental Yeast Industry Co., Ltd.),
Malt extract 0.3% (w / v) (manufactured by Oriental Yeast Co., Ltd.), pH 7. (Culture method) 100 ml of the medium was dispensed into a 500 ml Erlenmeyer flask, which was stoppered with cotton, and sterilized in an autoclave at 121 ° C for 20 minutes. Pseudomonas chlororafis B-23 strain was inoculated and cultured with shaking at 25 ° C. for 24 hours. Main culture conditions: (medium composition) taste liquid 2% (w / v) (manufactured by Ajinomoto Co.), polypeptone 0.3% (w / v), KH ₂ PO ₄ 0.1% (w / v), K ₂ HP
O ₄ 0.1% (w / v), MgSO ₄ .7H ₂ O 0.1% (w / v), FeSO
_{4 · 7H 2 O 0.005% (} w / v), 0.025% ammonium sulfate (w / v),
_{CuSO 4 · 7H 2 O 0.0005%} (w / v), sucrose 3
% (W / v), Pluronic L61 0.1% (w / v) (Asahi Denka Kogyo Co., Ltd.), pH 7.8. (Culture method) Two liters of the medium was dispensed into a 3-liter mini-jar fermenter and sterilized in an autoclave at 121 ° C. for 20 minutes. Tank pressure 0.049MPa, agitation number 50-600rpm, ventilation volume 1vvm, pH7, temperature 2
The cells were cultured at 5 ° C for 44 hours.

(2) Measurement of nitrile hydratase activity The measurement was carried out in the same manner as in Example 1. The activity was examined for the activity per culture and the activity per cell. Table 3 shows the results. The activity per culture solution and the activity per microbial cell were calculated, and the activity value under other culture conditions was defined as the relative activity (%), with the value obtained by culturing in Example 10 as 100.
Indicated by

<Examples 11 and 12> Instead of controlling the dissolved oxygen concentration during culturing to 1.0 ppm, culturing while controlling the dissolved oxygen concentration during culturing to 3.5 ppm (Example 11)
The procedure was performed in the same manner as in Example 10 except that the culture (Example 12) in which the dissolved oxygen concentration during the culture was controlled at 9.0 ppm was performed. The results are shown in Table 3.

<Comparative Example 3> The dissolved oxygen concentration during culturing was set at 1.
The procedure was carried out in the same manner as in Example 10 except that the culture was performed with the dissolved oxygen concentration being maintained at 0.5 ppm instead of being controlled at 0 ppm. The results are shown in Table 3.

[Table 3]

Example 13 Growed under the following preculture conditions
20 ml of the culture of MT-10822 strain (FERM BP-5785)
Investigate nitrile hydratase activity by culturing under main culture conditions
Was. In addition, the dissolved oxygen concentration during cultivation will be 1.5 ppm.
Managed. (1) Culture of bacteria Pre-culture conditions: (Medium composition) Polypeptone 1% (w / v), yeast extract 0.1%
5% (w / v), NaCl 0.5% (w / v), ampicillin 10
0 (μg / ml), pH 7 (LB medium). (Culture method) Dispense 100ml of medium into 500ml Erlenmeyer flask
Stopper and sterilize by autoclave at 121 ° C for 20 minutes
did. Inoculate MT-10822 strain and shake culture at 37 ℃ for 6 hours
did. Main culture conditions: (Medium composition) Yeast extract 0.5% (w / v), polypeptone
2% (w / v), NaCl 0.06% (w / v), KCl 0.02%
(w / v), MgCl_Two・ 6H_TwoO 0.2% (w / v), CoCl_Two・ 6H_TwoO 0.
002% (w / v), MgSO_Four・ 7H_TwoO 0.243% (w / v), full
Kutose 4% (w / v), Pluronic L61 0.02
% (W / v), pH 7 <Culture method> For 3-liter mini jar fermenter
Dispense 2 liters of the initial medium and heat at 121 ° C for 20 minutes.
Sterilized in a toclave. However, fructose and Mg
SO ₄・ 7H₂O is filtered aseptically separately (Advan
Use 0.45 micron filter paper manufactured by Tech Toyo Co., Ltd.)
Added to medium. Tank pressure 0.025MPa, stirring number 80
0 rpm, aeration 1 vvm, pH 7, 25 at 37 ° C
Cultured for hours.

(2) Measurement of nitrile hydratase activity The measurement was carried out in the same manner as in Example 1. The activity was examined for the activity per culture and the activity per cell. Table 4 shows the results. The activity per culture solution and the activity per microbial cell were calculated, and the activity value under other culture conditions was defined as the relative activity (%), with the value obtained by culturing in Example 13 taken as 100.
Indicated by

<Examples 14 and 15> In Example 13, instead of controlling the concentration of dissolved oxygen in the culture to 1.5 ppm,
Culture in which the concentration of dissolved oxygen during culturing was controlled to 3.5 ppm (Example 14), and the concentration of dissolved oxygen during culturing was 5.8 ppm
The procedure was performed in the same manner as in Example 10 except that the culture (Example 15) was performed. The results are shown in Table 4.

<Comparative Example 4> The procedure of Example 13 was repeated, except that the cultivation was performed with the dissolved oxygen concentration controlled at 0.9 ppm instead of controlling the dissolved oxygen concentration at 1.5 ppm. Performed by a method similar to that in Example 13.
The results are shown in Table 4.

[Table 4]

[0035]

According to the present invention, by increasing the dissolved oxygen concentration during cultivation from 1 ppm to a saturated concentration, it is possible to improve the growth of bacteria and to obtain cells having high expression of nitrile hydratase enzyme activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 1/20 (C12N 1/20 A C12R 1: 365) C12R 1: 365)

Claims (1)

[Claims] 1. A method for high-density cultivation of bacteria having nitrile hydratase-producing ability to maintain the concentration of dissolved oxygen during culturing from 1 ppm to a saturated concentration.