JPH0753104B2 - Bacterial culture method - Google Patents

Description

Description: BACKGROUND OF THE INVENTION The present invention relates to a method for producing a high yield of Rhodococcus spp. Rhodococcus sp. Having high nitrile hydratase activity.

2. Description of the Related Art In recent years, there have been active movements for immobilizing microorganisms, enzymes or the like and using them as catalysts for various unit chemical reactions or complex chemical reactions.

Nitrile hydratase has been found by Yamada et al. Among the present inventors as an enzyme that hydrates nitriles to generate corresponding amides [Agric.Biol.Chem. 46 1165 (1
982)], as a specific application example thereof, a method for producing amides from nitriles using a bacterium having a nitrile hydratase has been proposed [see Japanese Patent Publication No. 59-37951].

Furthermore, the present inventors have previously proposed a method for producing amides, which is particularly suitable for producing amides from aromatic nitriles, using the bacterium of the genus Rhodococcus sp. See Japanese Patent Application No. 63-231744]. In such a case, it would be of great benefit if bacterial cells of Rhodococcus sp. With high nitrile hydratase activity could be obtained in high yield.

[Outline of Invention]

SUMMARY OF THE INVENTION The present invention aims to provide a solution to the above-mentioned problems, and to achieve this object by culturing the bacterium by allowing a specific substance, that is, a specific urea derivative and cobalt ion, to be present in the medium. To do.

Therefore, the method for culturing a bacterium of the genus Rhodococcus of the genus Rhodococcus having a high nitrile hydratase activity according to the present invention is a Rhodococcus rhodochrou sp.
In producing a bacterial cell having nitrile hydratase enzymatic activity by culturing a bacterium belonging to s) and capable of producing nitrile hydratase, it is represented by the following formula I or II in a crotonamide-free medium. The presence of at least one urea derivative and cobalt ion is characterized.

R ₁ R ₂ NCONR ₃ R ₄ [I] (wherein R ₁ , R ₂ , R ₃ and R ₄ are —H and —C, respectively).
It represents of H ₃ or -C ₂ H ₅ group,. However, the case where all are -H is excluded. ) R ₅ R ₆ NCOOC ₂ H ₅ [II] (wherein R ₅ and R ₆ each represent —H, —CH ₃ , or —C ₂ H ₅ group.) Effect In medium containing no crotonamide Incubation of a bacterium of Rhodococcus rhodochrous species in the presence of at least one of the specific urea derivatives represented by the formula I or II and cobalt ion significantly increased the nitrile hydratase activity per unit culture solution. To do.

This increase in nitrile hydratase activity per unit culture is due to an increase in cell concentration (ie, cell yield) and / or cell activity (ie, amount of nitrile hydratase in cells). It is understood as something.

In the present invention, the presence of urea derivative and cobalt ion is particularly effective for the latter.

[Specific Description of the Invention]

1. Rhodococcus rhodochrous bacterium The bacterium used in the present invention is a bacterium of Rhodococcus rhodochrous species having nitrile hydratase activity and capable of hydrating a nitrile, particularly an aromatic nitrile, to produce a corresponding amide. is there. Specifically, for example, the Rhodococcus rhodochrous J- strain described in Japanese Patent Application No. 63-231744 is used.
1) The Micro Works Research Institute No. 1478 (FERM BP-1478) can be mentioned. The details of this bacterium are described in the above-mentioned Japanese Patent Application No. 63-231744, specifically as follows.

(1) Origin and Deposit The J-1 strain was collected by the present inventors from soil in Sakyo Ward, Kyoto City and deposited on September 18, 1987 at the Institute for Microbial Technology, Institute of Industrial Science and Technology. Obtained the accession number of FERM BP-1478.

(2) Bacteriological properties (a) Morphology (1) Shape and size of cells 0.9 to 1.0 μ × 3 to 10 μ (2) Presence or absence of polymorphism in cells
It shows a club-shaped growth without snapping and with snapping, and then ruptures into short rod-shaped fungi (3) No motility (4) No spores (5) Gram stain positive (6) Acid-negative (7) Metachromatic bodies are observed (b) Growth condition in each medium (30 ℃) (1) Meat broth agar plate culture 1 mm in diameter (48 hours) round, irregular, smooth and dry on the surface, flat, opaque, pale orange pink (2) Meat broth agar slant culture Filamentous, smooth surface, slightly raised cross section, slightly dry, pale orange pink (3) Liquid broth broth Membrane formation and vigorous growth.
Medium turbidity and precipitation occur as it grows.

(4) Meat broth gelatin stab culture It grows well on the surface and grows like a funnel along the stab, but hardly grows in the lower layer. Liquefaction of gelatin is not observed.

(5) Litmus milk unchanged (c) Physiological properties (1) Sulfate reduction positive (2) Derooming reaction negative (3) MR test negative (4) VP test negative (5) Indole formation positive (6) Generation of hydrogen sulfide Positive (7) Hydrolysis of starch Negative (8) Utilization of citric acid Coser medium: Negative Christensen medium: Positive (9) Inorganic nitrogen source utilization Nitrate: Positive Ammonium salt: Positive (10) Dye Negative (11) Urease positive (12) Oxidase negative (13) Catalase positive (14) Cellulose hydrolysis negative (15) Growth range pH: 5-10 Temperature: 10-41 ° C (16) Attitude toward oxygen Aerobic (17) Tyrosine degradation positive (18) Adenine degradation positive (19) Phosphatase positive (20) Tween80 hydrolysis positive (21) OF test 0 (weak) (22) Heat resistance (10% skimmed (72) in acid, 15 minutes) None (23) Production of acid and gas from sugar Production of acid Production of gas L-arabinose-D-xylose-D-glucose + -D-mannose-D-fructose +- Maltose ＋ － Sucrose ＋ － Lactose －－Trehalose －－D-Sorbit ＋ － D-Mannitol ＋ －－ Glycerin ＋ －－ (24) Grow as a single carbon source Inositol-maltose ＋ D-Mannitol ＋ Rhamnose －D-Sorbit ＋ m-Hydroxybenzoic acid ＋ Sodium adipate ＋ Sodium benzoate ＋ Sodium citrate ＋ Sodium lactate ＋ Testtetron ＋ L-tyrosine ＋ Glycerol (1%) (W / V) (＋) Trehalose (＋) p-Hydroxybenzoate Acid (1%) (W / V) + (+) Weak but positive.

(25) Fatty acid and cell wall analysis Contains unsaturated, saturated straight chain fatty acid, and tuberculostearic acid. TLC of mycolic acid
Gives a single spot.

Based on the above bacterial characteristics, Bergy's Bacterial Classification Document (Bergy's M
When classified based on the anual of Systematic Bacteriology (1986), the J-1 strain is an aerobic, gram-positive, weak acid-fast, catalase-positive, endospore-free bacillus that does not set flagella. In addition, in the early stage of development, it appears to be a Nocardia-type bacterium by exhibiting long-rod-like fungal shape, showing growth accompanied by branching (Branching), and later rupturing into short-rod fungi. .

Analysis of fatty acid composition includes unsaturated, saturated straight chain fatty acids including tuberculostearic acid. Mycolic acid TLC is distinguished from Mycobacterium because it gives a single spot showing the same Rf as the standard strain Rodococcus rhodochrous (IFO 3338). Also, from the composition (number of carbon atoms) of mycolic acid
Distinguished from the genus Nocardia. From the examination of other biochemical properties, this bacterium is recognized as Rhodococcus rhodochous.

2. Urea Derivative In the present invention, the urea derivative acts as an enzyme inducer, but the fact that the urea derivative is effective for the induction of nitrile hydratase as in the present invention is completely unexpected from the conventional knowledge.

Examples of the compound represented by the formula I include methylurea, ethylurea, 1,1-dimethylurea, 1,3-dimethylurea and the like.

Examples of the compound represented by the formula II include urethane and methylurethane.

To allow the urea derivative to be present in the medium, it is added to the medium at one time or sequentially. “Sequential” means either continuous or intermittent.

3. Cobalt ion Since nitrile hydratase cannot be obtained even when the above-mentioned enzyme derivative, urea derivative, is present in the medium, it is essential in the present invention that cobalt ion is present in the medium (of the bacteria of the present invention). The fact that the presence of cobalt ion is essential for the production of nitrile hydratase is described in Japanese Patent Application No. 63-23
144 as described in the specification).

Cobalt ions are usually produced by adding a water-soluble cobalt compound to the medium, rather than the medium being aqueous. Water-soluble cobalt compounds are clarified in chemical dictionaries, and it will be easy for those skilled in the art to select and use an appropriate cobalt compound. Which gives a typical cobalt compounds are for example Co ⁺⁺ or Co ^+++, especially those providing a CO ^++, a a, specifically cobalt chloride, cobalt sulfate, cobalt acetate, cobalt bromide, boric acid Cobalt can be exemplified.

In addition, vitamin B ₁₂ and metallic cobalt can also be used as a cobalt source in the present invention. Vitamin B ₁₂
Cobalt is contained in the complex as a complex and is ionized during culture. In addition, metallic cobalt is ionized by the oxidizing power of microorganisms in the culture.

4. Culture / Production of Nitrile Hydratase The culture of the Rhodococcus spp.
There is no limitation with respect to other conditions as long as it is purposeful, except that the urea derivative and cobalt ion are present in the medium.

For example, at a temperature of about 15 to 50 ° C., preferably about 20 to 45 ° C., particularly preferably about 30 ° C., with a predetermined amount of urea derivative and cobalt ion present in the following basic medium,
The culture may be performed at pH 7 to 9 for about 30 hours or longer, preferably 40 hours or longer (upper limit is 120 hours, for example).

The concentration of the urea derivative in the medium is 1 to 30 g / liter, preferably 2 to 20 g / liter, more preferably 5 to 15 g / liter, and the degree and the concentration of cobalt ion are 5 in terms of CoCl _2.
It is about 15 mg / liter.

Basal medium: Medium A Ingredient Quantity (medium in 1 _{liter) K 2 HPO 4 13.4g KH 2} PO 4 6.5g NaCl 1.0g MgSO 4 · 7H 2 O 0.2g vitamin mixture ^{* 1} 0.1 ml distilled water Balance (pH 7.0 ) * 1 Composition (in 1 liter of solution) Biotin 2.0 μg Calcium pantothenate 0.4 mg Inositol 2.0 mg Nicotinic acid 0.4 mg Thiamine hydrochloride 0.4 mg Pyridoxine hydrochloride 0.4 mg p-Aminobenzoic acid 0.2 ng Riboflavin 0.2 mg Folic acid 0.01 ng Distilled water Remaining medium _{B K 2 HPO 4 0.5g KH 2} PO 4 0.5g MgSO 4 · 7H 2 O 0.5g yeast extract 3.0g distilled water balance (pH 7.2) medium C glucose _{10g K 2 HPO 4 0.5g KH 2} PO 4 0.5g MgSO ₄ · 7H ₂ O 0.5g yeast extract 1.0g peptone 7.5g distilled water balance (pH 7.2) 5. experiment for measuring the activity and definitions (1) nitrile hydratase assays nitrile hydratase activity of the active is 3 as substrate -Cyanopyridine (1M), 1.0 ml potassium phosphate buffer (0.1
M, pH 7.0) 0.5 ml and 2 ml of a reaction mixture containing a predetermined amount of cells (separated from the culture solution) were reacted at 20 ° C. for a predetermined time, and then 0.2 ml of 1N HC1 was added. It was measured by stopping the reaction.

(2) Definition of activity Regarding activity, specific activity (SA) and total activity (TA) were examined. These are defined as follows.

SA: μmol each amide / mg-bacterial body / minute TA: μmol each amide / ml-medium / minute Example 1 A predetermined amount of urea was added to the basal medium C containing 10 mg / liter of CoCl ₂ and each of them was added. 4 ml of a preculture liquid (using the above-mentioned basic medium C) of Rhodococcus rhodochrous J-1 strain (FERM BP-1478) was added to 60 ml of the medium, and shake culture was carried out at 28 ° C. for 96 hours.

As a urea derivative, methylurea or
Not only with CoCl ₂ alone but also with thiourea and acetamide as urea derivatives other than the formulas [I] and [II], respectively,
The same culture was performed for the one to which only CoCl ₂ was added.

The results are shown in Table-1. Table 1 shows the numerical values when the highest total activity (TA) was obtained in the measurement of the activity during that period.

From this, it can be seen that the addition of both the specific urea derivative and CoCl ₂ is essential for increasing the production of nitrile hydratase.

Claims (4)

[Claims] 1. A Rhodococcus spp.
When a bacterium belonging to cus rhodochrous) having the ability to produce nitrile hydratase is cultured to produce a bacterial cell having nitrile hydratase enzyme activity, it is shown in the following I or II in a crotonamide-free medium. A method for culturing a bacterium of the genus Rhodococcus sp. Rhodococcus, which comprises the presence of at least one urea derivative and cobalt ions. R ₁ R ₂ NCONR ₃ R ₄ [I] (wherein R ₁ , R ₂ , R ₃ and R ₄ are —H and —C, respectively).
It represents of H ₃ or -C ₂ H ₅ group,. However, the case where all are -H is excluded. _{) R 5 R 6 NCOOC 2 H} 5 [II] ( wherein R ₅ and R ₆ each represent -H, -CH ₃ or -C ₂ H ₅ group,.) 2. A bacterium belonging to the genus Rhodococcus, which is capable of producing nitrile hydratase, is a Rhodococcus strain J-1 strain (FERM BP-1478).
The method for culturing bacteria according to claim 1, which is 3. The method for culturing bacteria according to claim 1, wherein the concentration of the urea derivative in the medium is 1 to 30 g / liter. 4. The method for culturing bacteria according to claim 1, wherein the cobalt ion concentration in the medium is ₅ to 15 mg / liter in terms of CoCl ₂ .