JPH0753103B2 - 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.
(1982)], 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. 63-72766]. 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]

The purpose of the present invention is 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, urea and cobalt ion, to be present in the medium. Is.

Accordingly, the method for culturing a bacterium of the genus Rhodococcus having a high nitrile hydratase activity according to the present invention is a method for culturing a bacterium of the genus Rhodococcus rhodochrou.
When culturing a bacterium belonging to s) that has the ability to produce nitrile hydratase to produce bacterial cells having nitrile hydratase enzymatic activity, the presence of urea and cobalt ions in a crotonamide-free medium , Are characterized.

EFFECTS When urea and cobalt ions are present in a crotonamide-free medium to cultivate a bacterium of the genus Rhodococcus sp. Rhodococcus, the nitrile hydratase activity per unit culture medium is significantly increased.

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 and cobalt ions is particularly effective for the latter.

[Specific Description of the Invention]

1. Rhodococcus rhodochrous bacterium The bacterium used in the present invention has a nitrile hydratase activity and is of a Rhodococcus rhodochrous species capable of hydrating a nitrile (particularly an aromatic nitrile) to produce a corresponding amide. It is a bacterium. Specifically, for example, the Rhodococcus rhodochrous J-1 strain (Rhodococcus rhodochrous) described in the above-mentioned Japanese Patent Application No. 63-72766 is used.
J-1) Micro Works Research Article No. 1478 (FERM BP-1478) can be mentioned. The details of this bacterium are described in the above-mentioned Japanese Patent Application No. 62-72766.
The description is 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) Growth as a single carbon source Inositol-maltose ＋ D-mannite ＋ Rhamnose-D -Sorbit + m-Hydroxybenzoic acid + Sodium adipate + Sodium benzoate + Sodium citrate + Sodium lactate + Testtetron + L-Tyrosine + Glycerin (1%) (W / V) (+) Trehalose (+) p- Hydroxybenzoic acid (1%) (W / V) + (+) Weak but positive.

(25) Fatty acid and cell wall analysis Unsaturated, saturated straight chain fatty acid,
And tuberculostearic acid. TL of mycolic acid
C gives a single spot.

Based on the above bacterial characteristics, Bergy's Bacterial Classification Document (Bergy's Ma
According to the classification based on the nual 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 distinguishes it from the genus Mycobacterium by giving a single spot showing the same Rf as the standard strain Rodoccocus 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. Urine Urea acts as an enzyme inducer in the present invention, but the fact that urea is effective in inducing nitrile hydratase as in the present invention is completely unexpected from the conventional knowledge. Moreover, it is surprising that the effect is far higher than that of conventional enzyme inducers, especially when urea is used alone without being used in combination with other enzyme inducers. Further, since urea is cheaper than other enzyme inducers, it is economically advantageous in industrializing the present invention.

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

3. Cobalt ion Since nitrile hydratase cannot be obtained even when urea, which is the above-mentioned enzyme inducer, is present in the medium, it is essential in the present invention that cobalt ion is present in the medium (the nitrile of the bacterium of the present invention). It is as described in the above-mentioned Japanese Patent Application No. 63-72766 that the presence of cobalt ion is essential for the production of hydratase).

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.
Other than the presence of urea and cobalt ions in the medium, there is no limitation with respect to other conditions as long as it is purposeful.

For example, urea and cobalt ions are present in a predetermined amount in a basal medium as described below at about 15 to 50 ° C, preferably 20 to 50 ° C.
At a temperature of about 45 ° C, particularly preferably about 30 ° C, a pH of 7 to
9, the culture may be performed for about 30 hours or longer, preferably 40 hours or longer (upper limit is 120 hours, for example).

The concentration of urea in the medium is 1 to 30 g / liter, preferably 2
~ 20 g / liter, more preferably 5 to 15 g / liter, the degree and the concentration of cobalt ions are ₅ to 15 mg / CoCl ₂ equivalent.
It is about a 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 balance 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. measurement and definition of experiment activity (1) nitrile hydratase assays nitrile hydratase activity activity, as substrate Benzonitrile (20 mM) 1.0 ml, 3-cyanopyridine (1M) 1.0 ml or Acrylonitrile (1M) 1.0 ml, potassium phosphate buffer (0.1 M, pH 7.0) cells in 0.5ml and a predetermined amount (as isolated from the culture broth) for the reaction mixture 2ml containing 2
The measurement was performed by allowing the reaction to proceed at 0 ° C. for a predetermined time and then stopping the reaction by adding 0.2 ml of 1N HC1.

(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.

Incidentally, for comparison, the culture was similarly performed for the one to which only urea or CoCl ₂ was added.

The results are shown in Table-1.

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

Example 2 For the media A, B and C, as shown in Table-2,
The J-1 strain was cultured at 28 ° C. for 48 to 120 hours in the same manner as in Example 1 with or without addition of predetermined amounts of CoCl ₂ and the enzyme inducer (urea, acetonitrile, crotonamide).

Table 2 shows the numerical values when the maximum total activity (TA) was obtained in the measurement of the activity during that period.

From this, it can be seen that the sole use of urea as the enzyme inducer is extremely effective in increasing the production of nitrile hydratase.

Claims (4)

[Claims] 1. A Rhodococcus spp.
of bacterium belonging to cus rhodochrous) and capable of producing nitrile hydratase by culturing a bacterium having nitrile hydratase enzymatic activity, in which urea and cobalt ions are present in a crotonamide-free medium A method for culturing a bacterium of the genus Rhodococcus, which is characterized in that 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 urea 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 .