Method for cultivation of bacteria.

BACKGROUND OF THE INVENTION

0001The present invention relates to a method of producing in a high yield cells of a microorganism of the species <u style="single">Rhodococcus</u><u style="single">rhodochrous</u> having a high nitrile hydratase activity.

0002In recent years, there have been increasing attempts to utilize microorganisms and enzymes as they are or in immobilized state as catalysts for various single or complex chemical reactions.

0003Nitrile hydratase has been found by Hideaki Yamada, one of the present inventors, et al. as an enzyme capable of hydrating nitriles to produce the corresponding amides. (Reference: Agric. Biol. Chem. <u style="single">46</u> 1165 (1982)) As one example of the utilization of this enzyme, a method for preparation of amides from nitriles in the presence of bacteria having nitrile hydratase has been proposed. (References: Japanese Patent Pub. No. 37951/1984 and U.S. Patent No. 4,637,982)

0004Further, we have proposed a method for preparation of amides, especially suitable for preparation of amides from aromatic nitriles. (References: Japanese Patent Appln. No. 231744/1988 and U.S. Patent Appln. Ser. No. 243,986) Under this situation, a method that can ensure the production of cells of <u style="single">Rhodococcus</u><u style="single">rhodochrous</u> bacteria having a high nitrile hydratase activity in a high yield would be remarkably beneficial.

SUMMARY OF THE INVENTION

0005An object of the present invention is to solve the above problem by adding specific substances, i.e., urea or its specific derivative and cobalt ion, to a culture medium in the cultivation of the bacteria.

0006Thus, the method for cultivation of bacteria of the species <u style="single">Rhodococcus</u><u style="single">rhodochrous</u> having a high nitrile hydratase activity according to this invention comprises adding to a culture medium at least one of urea and urea derivatives of the following formulae [I] to [III]:         R₁R₂NCONR₃R₄   [I] wherein R₁, R₂, R₃ and R₄ each are -H, -CH₃ or -C₂H₅, all the substituents not being -H;         R₅R₆NCOOC₂H₅   [II] wherein R₅ and R₆ each are -H, -CH₃ or -C₂H₅; and         NH₂CSNH₂   [III] and cobalt ion in the preparation of cells of bacteria having nitrile hydratase activity by cultivating <u style="single">Rhodococcus</u><u style="single">rhodochrous</u> bacteria capable of producing nitrile hydratase.

0007The addition of at least one of urea and specific urea derivatives of formulae [I] to [III] and cobalt ion to the culture medium during the cultivation of <u style="single">Rhodococcus</u><u style="single">rhodochrous</u> bacteria remarkably increases the nitrile hydratase activity per unit culture fluid.

0008This increase in nitrile hydratase activity per unit culture fluid is presumably traceable to the increase in cell concentration (i.e., yield) and/or cell activity (i.e., quantity of the nitrile hydratase in the cells).

0009In the present invention, the addition of urea or a derivative thereof and cobalt ion is especially effective in increasing the cell activity.

DETAILED DESCRIPTION OF THE INVENTION

I.

Rhodococcus

rhodochrous

bacteria

0010The bacteria used in the present invention are <u style="single">Rhodococcus</u><u style="single">rhodochrous</u> bacteria having nitrile hydratase activity and the capability of hydrating nitriles, particularly, even aromatic nitriles, to produce the corresponding amides. A specific example of such bacteria is <u style="single">Rhodococcus</u><u style="single">rhodochrous</u>, strain J-1 (FERM BP-1478), disclosed in Japanese Patent Appln. No. 231744/1988 and U.S. Patent Appln. Ser. No. 243,986 mentioned earlier. The details of the strain J-1 are given in these patent applications as follows.

1. Origin and Deposition

0011The strain J-1 was isolated by us from the soil in Sakyo-ku, Kyoto, Japan, and deposited on September 18, 1987 with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry of Japan, where it was assigned the accession number FERM BP-1478 under the Budapest Treaty.

2. Bacteriological Characteristics

(a) Morphological Characteristics

0012<ul id="ul0001" list-style="none"><li>(1) Shape and size of cell: 0.9-1.0 » × 3-10 »</li><li>(2) Polymorphism:An elongated rod-shaped cell in the initial stage of cultivation grows to form a straight stick with snapping, and then is divided into short bacillus form.</li><li>(3) Motility: Immotile</li><li>(4) Formation of spores: None</li><li>(5) Grain staining: Positive</li><li>(6) Acid-fast property: Negative</li><li>(7) Heterophile granulocyte: Observed</li></ul>

(b) Cultural Characteristics on Various Culture Media (30°C)

0013<ul id="ul0002" list-style="none"><li>(1) Bouillon-agar plate culture: Circle with 1-mm diameter (48 hours), irregular, smooth, rather dry on the surface, flat, opaque, and pale orange-pink.</li><li>(2) Bouillon-agar slant culture: Filament with a smooth surface and a slightly convex, rather dry cross section, and pale orange-pink.</li><li>(3) Bouillon liquid culture: Abundant growth with formation of membrane. The culture fluid becomes fairly turbid and a precipitate is formed as the cell grows.</li><li>(4) Bouillon-gelation stab culture: Good growth on the surface in the shape of a funnel along the stabbed area, but scant growth in the undersurface. Gelatin is not liquefied.</li><li>(5) Litmus-milk: No change.</li></ul>

(c) Physiological properties

0014<tables id="tabl0001" num="0001"><img file="EP0362829B1_D0001.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0362829B1_D0002.tif" /></tables>

0015According to the characterization of the above listed bacteriological properties in the light of Bergy's Manual of Systematic Bacteriology (1986), the strain J-1 is an aerobic, Gram-positive, weakly acid-fast, catalase-positive and non-endospore forming bacillus with no flagellum. This strain is in the shape of an elongated bacillus and a mycelium in the initial stage of growth, grows with branching and then is divided into short bacillus form. In view of these features, the strain J-1 is considered to fall under <u style="single">Nocardia</u> type bacteria.

0016The analysis of the fatty acid composition has revealed that the bacterium contains unsaturated and saturated straight-chain fatty acids including tuberculostearic acid. Since the TLC of mycolic acid gives a single spot having the same Rf value as the standard bacterium <u style="single">Rhodococcus</u><u style="single">rhodochrous</u> (IFO 3338), the bacterium is distinguished from those of the genus <u style="single">Mycobacterium</u>. This bacterium is also distinguished from <u style="single">Nocardia</u> bacteria in view of the composition (number of carbon atoms) of the mycolic acid.

0017As a result of investigation of other biochemical properties, this bacterium has been identified as <u style="single">Rhodococcus</u><u style="single">rhodochrous</u>.

II. Urea and its derivatives

0018In the present invention, urea and urea derivatives of the formulae [I] to [III] shown hereinbefore function as enzyme inducers, but, from what we have heretofore known, i.e., typical enzyme inducers are nitriles or amides, <u style="single">inter</u><u style="single">alia</u>, crotonamide, it is wholly unexpected that urea and its derivatives can effectively induce nitrile hydratase. Surprisingly, urea and its derivatives, when used singly and not in combination with other enzyme inducers, exhibit far higher efficacy than conventional enzyme inducers. Furthermore, since urea is less expensive than other enzyme inducers, the method of the present invention can be advantageously applied to industry from economy viewpoint.

0019Examples of the compounds of the formula [I] among the urea derivatives used in the present invention are methylurea, ethylurea, 1,1-dimethylurea, and 1,3-dimethylurea.

0020Exemplary compounds of the formula [II] are urethane and methylurethane.

0021The compound of the formula [III] is thiourea.

0022Urea or its derivatives are added to the culture medium in one batch at one time or sequentially. The term "sequentially" as used herein is intended to mean both "continuously" and "incrementally".

III. Cobalt ion

0023Nitrile hydratase cannot be obtained simply by adding urea or its derivatives to the culture medium, and it is essential in the present invention that cobalt ion be added to the culture medium. (The presence of cobalt ion is essential for the production of nitrile hydratase by the bacterium of the present invention as has been set forth in Japanese Patent Appln. No. 231744/1988 and U.S. Patent No. 243,986 mentioned earlier.)

0024Ordinarily, cobalt ion is formed by adding a water-soluble cobalt compound to the culture medium which is aqueous. The water-soluble cobalt compounds are as defined in chemical encyclopedias, so that it may be easy for those skilled in the art to suitably select and use one of such compounds.

0025Typical examples of the cobalt compounds are those which afford Co⁺⁺ or Co⁺⁺⁺, particularly Co⁺⁺, such as cobalt chloride, cobalt sulfate, cobalt acetate, cobalt bromide and cobalt borate.

0026Additionally, vitamin B₁₂ and metallic cobalt can also be used as cobalt sources. Vitamin B₁₂ contains cobalt in the form of a complex which is ionized by autoclave treatment, while metallic cobalt is ionized by the oxidizing function of microorganisms during cultivation.

IV. Cultivation/Production of nitrile hydratase

0027The <u style="single">Rhodococcus</u><u style="single">rhodochrous</u> bacteria of the present invention can be cultivated under any conditions suitable for the purpose except that urea or its derivatives and cobalt ion are added to the culture medium.

0028For example, predetermined amounts of urea or its derivatives and cobalt ion are added to basal media listed below, and cultivation may be carried out at a temperature of about 15 to 50°C, preferably about 20 to 45°C, and more preferably about 30°C at a pH of 7 to 9 for about 30 hours or longer, preferably for 40 hours or longer (up to, for example, 120 hours).

0029The overall concentration of the urea or its derivatives in the culture medium is about 1 to 30 g/ℓ, preferably about 2 to 20 g/ℓ, and more preferably about 5 to 15 g/ℓ, while the concentration of the cobalt ion is about 5 to 15 mg/ℓ as calculated in terms of CoCl₂.

Basal medium:

0030<tables id="tabl0003" num="0003"><img file="EP0362829B1_D0003.tif" /></tables>

0031<tables id="tabl0004" num="0004"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="left">Culture medium B</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">K₂HPO₄</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5 g</entry></row><row><entry namest="col1" nameend="col1" align="left">KH₂PO₄</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5 g</entry></row><row><entry namest="col1" nameend="col1" align="left">MgSO₄·7H₂O</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5 g</entry></row><row><entry namest="col1" nameend="col1" align="left">Yeast extract</entry><entry namest="col2" nameend="col2" align="char" char=".">3.0 g</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Distilled water</entry><entry namest="col2" nameend="col2" align="left">Balance (pH 7.2)</entry></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="left">Culture medium C</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Glucose</entry><entry namest="col2" nameend="col2" align="char" char=".">10 g</entry></row><row><entry namest="col1" nameend="col1" align="left">K₂HPO₄</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5 g</entry></row><row><entry namest="col1" nameend="col1" align="left">KH₂PO₄</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5 g</entry></row><row><entry namest="col1" nameend="col1" align="left">MgSO₄·7H₂O</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5 g</entry></row><row><entry namest="col1" nameend="col1" align="left">Yeast extract</entry><entry namest="col2" nameend="col2" align="char" char=".">1.0 g</entry></row><row><entry namest="col1" nameend="col1" align="left">Peptone</entry><entry namest="col2" nameend="col2" align="char" char=".">7.5 g</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Distilled water</entry><entry namest="col2" nameend="col2" align="left">Balance (pH 7.2)</entry></row></tbody></tgroup></table></tables>

V. Experimental Examples

Measurement and Definition of Enzyme Activity

(1) Method for Measuring Nitrile Hydratase Activity

0032The nitrile hydratase activity was determined as follows.

00332 ml of a reaction solution comprising 1.0 ml of benzonitrile (20 mM), 1.0 ml of 3-cyanopyridine (1 M) or 1.0 ml of acrylonitrile (1 M) as a substrate; 0.5 ml of potassium phosphate buffer (0.1 M, pH 7.0); and a predetermined amount of bacterium cells (isolated from a culture fluid) was caused to react at 20°C for a predetermined time period, and the reaction was then terminated with the addition of 0.2 ml of 1N HCl.

(2) Definition of Nitrile Hydratase Activity

0034The activity was determined for the specific activity (S.A.) and the total activity (T.A.) as defined below. <dl id="dl0001"><dt>S.A.:</dt><dd>»mole product amide/mg-cells/min.</dd><dt>T.A.:</dt><dd>»mole product amide/ml-culture medium/min.</dd></dl>

Example 1

0035Predetermined amounts of urea were each added to the above basal medium C containing 10 mg/ℓ of CoCl₂. To 60 ml each of the resulting culture medium was added 4 ml of a preculture fluid of <u style="single">Rhodococcus</u><u style="single">rhodochrous</u>, strain J-1 (FERM BP-1478) (obtained using the basal medium C), and shake culture was carried out at 28°C for 96 hours.

0036For comparison purposes, culture was conducted similarly in a medium containing either urea or CoCl₂ alone.

0037The results obtained are summarized in TABLE 1.

0038From the TABLE, it will be noted that the addition of both urea and CoCl₂ is essential for increased production of nitrile hydratase.

Example 2

0039Strain J-1 was subjected to culture similarly as in Example 1 at 28°C for 48 to 120 hours in the basal medium C mentioned above in the presence of 10 mg/ℓ of CoCl₂, while adding or not adding predetermined amounts of enzyme inducers (urea and crotonamide) as set forth in TABLE 2.

0040Presented in TABLE 2 are T.A. and S.A. values obtained when the maximum T.A. values were marked during the measurements of the activity.

0041As is apparent from the TABLE, the use of urea alone as an enzyme inducer contributes effectively toward increasing production of nitrile hydratase. <tables id="tabl0006" num="0006"><img file="EP0362829B1_D0004.tif" /></tables><tables id="tabl0007" num="0007"><table frame="all"><title>TABLE 2</title><tgroup cols="6" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="26.25mm" /><colspec colnum="2" colname="col2" colwidth="26.25mm" /><colspec colnum="3" colname="col3" colwidth="26.25mm" /><colspec colnum="4" colname="col4" colwidth="26.25mm" /><colspec colnum="5" colname="col5" colwidth="26.25mm" /><colspec colnum="6" colname="col6" colwidth="26.25mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" rowsep="0" align="center">Culture Medium</entry><entry namest="col2" nameend="col2" rowsep="0" align="center">CoCl₂ (mg/ℓ)</entry><entry namest="col3" nameend="col3" rowsep="0" align="center">Urea (g/ℓ)</entry><entry namest="col4" nameend="col4" rowsep="0" align="center">Crotonamide (g/ℓ)</entry><entry namest="col5" nameend="col6" align="center">Benzonitrile</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" /><entry namest="col4" nameend="col4" /><entry namest="col5" nameend="col5" align="center">T.A.</entry><entry namest="col6" nameend="col6" align="center">S.A.</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">C</entry><entry namest="col2" nameend="col2" align="right">10</entry><entry namest="col3" nameend="col3" align="char" char=".">-</entry><entry namest="col4" nameend="col4" align="char" char=".">2.0</entry><entry namest="col5" nameend="col5" align="char" char=".">23.2</entry><entry namest="col6" nameend="col6" align="char" char=".">6.0</entry></row><row><entry namest="col1" nameend="col1" align="left">C</entry><entry namest="col2" nameend="col2" align="right">10</entry><entry namest="col3" nameend="col3" align="char" char=".">-</entry><entry namest="col4" nameend="col4" align="char" char=".">4.0</entry><entry namest="col5" nameend="col5" align="char" char=".">24.6</entry><entry namest="col6" nameend="col6" align="char" char=".">6.1</entry></row><row><entry namest="col1" nameend="col1" align="left">C</entry><entry namest="col2" nameend="col2" align="right">10</entry><entry namest="col3" nameend="col3" align="char" char=".">-</entry><entry namest="col4" nameend="col4" align="char" char=".">7.5</entry><entry namest="col5" nameend="col5" align="char" char=".">16.6</entry><entry namest="col6" nameend="col6" align="char" char=".">5.8</entry></row><row><entry namest="col1" nameend="col1" align="left">C</entry><entry namest="col2" nameend="col2" align="right">10</entry><entry namest="col3" nameend="col3" align="char" char=".">5.0</entry><entry namest="col4" nameend="col4" align="char" char=".">2.0</entry><entry namest="col5" nameend="col5" align="char" char=".">32.6</entry><entry namest="col6" nameend="col6" align="char" char=".">6.5</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">C</entry><entry namest="col2" nameend="col2" align="right">10</entry><entry namest="col3" nameend="col3" align="char" char=".">7.5</entry><entry namest="col4" nameend="col4" align="char" char=".">-</entry><entry namest="col5" nameend="col5" align="char" char=".">213</entry><entry namest="col6" nameend="col6" align="char" char=".">42.2</entry></row></tbody></tgroup></table></tables>

Example 3

0042Predetermined amounts of urea derivatives were each added to the above basal medium C containing 10 mg/ℓ of CoCl₂. To 60 ml each of the resulting culture medium was added 4 ml of a preculture fluid of <u style="single">Rhodococcus</u><u style="single">rhodochrous</u>, strain J-1 (FERM BP-1478) (obtained using the basal medium C), and shake culture was carried out at 28°C for 96 hours.

0043For comparison purposes, culture was conducted similarly in a medium containing methylurea alone or CoCl₂ alone.

0044The results are shown in TABLE 3 in which are presented T.A. and S.A. values obtained when the maximum T.A. values were marked during the measurements of the activity. In this TABLE are added for reference the results obtained for 7.5 g/ℓ of urea.

0045As is apparent from the TABLE, the use of both a urea derivative and CoCl₂ is essential for increased production of nitrile hydratase. <tables id="tabl0008" num="0008"><img file="EP0362829B1_D0005.tif" /></tables>