Method for cultivation of pseudomonas bacteria

Background of the invention

0001The present invention relates to a method of producing in a high yield cells of Pseudomonas bacteria having a high nitrile hydratase activity.

0002In recent years, there have been increasing attempts to utilize microorganisms and enzymes as they are or in an immobilized state as catalyst 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. 461165 (1982)). As one example of the utilization of this enzyme, a method for preparation of acrylamide from acrylonitrile in the presence of bacteria having nitrile hydratase has been proposed. (References: Japanese Patent Laid-Open Pub. No. 86093/1983 (Japanese Patent Appln. No. 184688/1981) and Agric. Biol. Chem. 46 1183 (1982)).

0004Under these circumstances, a method that can ensure the production of cells of Pseudomonas 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 to a culture medium a specific substance, i.e., at least one enzyme inducing agent selected from propionitrile, isobutyronitrile, propionamide, and isobutyramide in a specific manner, i.e., "sequentially" (as defined hereinafter), in the process of cultivation of the bacteria.

0006Thus, a distinguishing feature of the method for cultivation of Pseudomonas bacteria having a high nitrile hydratase activity according to this invention is the sequential addition of at least one compound selected from the group consisting of propionitrile, isobutyronitrile, propionamide, and isobutyramide to a culture medium in the preparation of cells of bacteria having nitrile hydratase activity by cultivating Pseudomonas bacteria capable of producing nitrile hydratase.

0007We have found that, by adding one or more of the enzyme inducing agents designated above to the culture medium sequentially during the cultivation of bacteria of the genus Pseudomonas, the nitrile hydratase activity per unit culture fluid increases remarkably. For example, the sequential addition of the enzyme inducing agent can increase the nitrile hydratase activity per unit culture fluid to a value nearly twice that obtained when the same compound is added at one time.

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

0009In the present invention, the propionitrile and other compounds named hereinbefore are sometimes called enzyme inducing agents in view especially of the latter factor although these compounds are effective not only in increasing the cell activity as has been set forth above.

Detailed description of the invention

Pseudomonas bacteria

0010The bacteria used in the present invention are Pseudomonas bacteria having nitrile hydratase activity and the capability of hydrating nitriles, particularly acrylonitrile, to produce the corresponding amides, particularly acrylamide. Specific examples of such bacteria are Pseudomonas chlororaphis, strain B 23 (Ferm BP-187), and Pseudomonas sp., strain PS 1 (Ferm BP-188), disclosed in Japanese Patent Laid-Open Pub. No. 86093/1983. The principal mycological properties of these bacteria are as follows. <tables id="tabl0001" num="0001"><img file="EP0109083B1_D0001.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0109083B1_D0002.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP0109083B1_D0003.tif" /></tables>

Enzyme inducing agent

0011In the present invention, propionitrile, isobutyronitrile, propionamide, and isobutyramide are used as enzyme inducing agents. These compounds can be used singly or in the form of a mixture of two or more members.

0012In accordance with the present invention, these compounds are added to the culture medium sequentially. The term "sequentially" as used herein is intended to mean both "continuously" and . "intermittently" and not "at one time in a single batch".

Cultivation-Practice of the present invention

A preferred embodiment of this invention will be described below.

0013Pseudomonas bacteria having nitrile hydratase activity are inoculated into a culture medium, especially an aqueous culture medium, containing carbon sources such as glucose, fructose, sucrose, dextrins, glycerol, ethanol, and succinic acid; nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea; organic nutriment sources such as yeast extract, meat extract, malt extract, casein hydrolyzate, and peptone; inorganic salts such as phosphates; magnesium, potassium, and iron and other metals in trace amounts; and other substances. Cultivation is carried out under aerobic conditions while at least one of propionitrile, isobutyronitrile, propionamide, and isobutyramide is added sequentially as an enzyme inducing agent.

0014The sequential addition of enzyme inducing agents is essential in order to obtain bacterium cells having a high nitrile hydratase activity in a high yield as will be apparent from the experimental examples set forth hereinlater. Ordinarily, the concentration of the enzyme inducing agent in the culture medium is adjusted preferably to lower than 15 g/I (in total when two or more compounds are used as such), and more preferably to 10 g/I or lower although this may vary depending on the cultivation time, temperature and other conditions. If the concentration becomes 15 g/I or higher, the nitrile hydratase activity of the bacteria will be lowered. The pH of the culture medium is of the order of 6 to 9, preferably of the order of 7 to 8, while the cultivation temperature is of the order of 20 to 37°C, preferably of the order of 25 to 30°C, and the cultivation time is about 1 to 3 days.

0015After the cultivation has been completed, the cells or nitrile hydratase can be collected or utilized in accordance with a procedure which will be described hereinlater in the experimental examples.

0016In the disclosure, the letter "I" indicates "liter".

Experimental Examples

0017In the following experimental examples, 1 ml of a culture fluid was added to 9 ml of a phosphate buffer solution (pH 7.5) containing 2.8% by weight of acrylonitrile, and the resulting solution was caused to react at 10°C for 10 to 60 minutes. The quantity of acrylamide obtained was measured by means of gas chromatography, and the hydratase activity of the bacteria exhibited in the hydration of acrylonitrile was determined on the basis of the data thus obtained, the capability of producing 1 µmole of acrylamide per ml of a culture fluid per minute being designated as 1 unit.

Example 1

0018100 ml of a precultivation culture medium (pH 7.2) comprising 10 g/l of glucose, 5 g/l of peptone, 3 g/I of yeast extract, and 3 g/I of malt extract was sterilized in a 500-ml Erlenmeyer flask. Pseudomonas chlororaphis, strain B 23 (Ferm BP-187), was inoculated into the sterilized culture medium, and subjected to shaking cultivation at 25°C for 24 hours.

0019Separately, 100 ml of a culture medium (pH 7.2) comprising 10 g/I of sucrose, 0.5 g/I of KH₂PO₄, 0.5 g/I of K₂HPO₄, 0.5 g/I of MgSO₄- 7H₂O, and 10 mg/I of FeSO₄- 7H₂O was sterilized in a 500-ml Erlenmeyer flask.

0020Into this culture medium was inoculated 1 ml of the seed culture fluid obtained by the precultivation, and shaking cultivation was carried out aerobically at 25°C with sequential addition of isobutyronitrile and isobutyramide as an enzyme inducing agent respectively in two instances.

0021For comparison purposes, cultivation was carried out similarly with the addition of the enzyme inducing agent at one time in a singla batch.

0022The culture fluids obtained by the foregoing cultivation procedures carried out for specific time periods were sampled respectively, and the nitrile hydratase activity of each of the culture fluid samples exhibited in the hydration of acrylonitrile was measured.

0023The relationship between the conditions under which the enzyme inducing agents were added and the nitrile hydratase activities of the culture fluids is shown in Table 1. <tables id="tabl0004" num="0004"><img file="EP0109083B1_D0004.tif" /></tables>

0024As is apparent from Table 1, the enzymatic activity of the culture fluid obtained when isobutyronitrile or isobutyramide was added sequentially increased to a Value nearly twice that obtained when substantially the same quantity of the enzyme inducing agent was added at one time. In the case where the concentration of the enzyme inducing agent exceeds 15 g/I during cultivation (30 hours after the initiation of cultivation), however, the enzymatic activity decreased afterward even when the enzyme inducing agent was added sequentially.

0025When the enzyme inducing agent was added at one time at the initiation of the cultivation at a concentration of 15 g/I or higher, almost no enzymatic activity was exhibited.

Example 2

0026100 ml of a precultivation culture medium (pH 7.2) comprising 10 g/l of glucose, 5 g/l of peptone, 3 g/I of yeast extract, and 3 g/I of malt extract was sterilized in a 500-ml Erlenmeyer flask. Pseudomonas sp., strain PS-1 (Ferm BP-188), was inoculated into the sterilized culture medium, and subjected to shaking cultivation- at 25°C for 24 hours.

0027Separately, 100 ml of a culture medium (pH 7.2) comprising 10 g/I of glycerol, 0.5 g/I of KH₂P0₄, 0.5 g/I of K₂HPO₄, 0.5 g/I of MgSO₄. 7H₂0, and 10 mg/I of FeSO₄ .7H₂O was sterilized in a 500-ml Erlenmeyer flask.

0028This culture medium was inoculated with 1 ml of the seed culture fluid obtained by the precultivation, and shaking cultivation was carried out aerobically at 25°C with sequential addition of propionitrile and propionamide as an enzyme inducing agent respectively in two instances.

0029For comparison purposes, cultivation was carried out similarly with addition of the enzyme inducing agent at one time in a single batch.

0030The nitrile hydratase activity of each of the culture fluids after the elapse of specific time periods exhibited in the hydration of acrylonitrile was measured.

0031The relationship between the conditions under which the enzyme inducing agents were added and the nitrile hydratase activities of the culture fluids is summarized in Table 2. <tables id="tabl0005" num="0005"><img file="EP0109083B1_D0005.tif" /></tables>

0032As is apparent from Table 2, the enzymatic activity of the culture fluid obtained when propionitrile or propionamide was added sequentially increased to a value nearly twice that obtained when substantially the same quantity of the enzyme inducing agent was added at one time.

Example 3

00331.3 of a culture medium (pH 7.2) comprising 20 g/I of sucrose, 1 g/I of KH₂P0₄, I g/I of K₂HP0₄, 1 g/I of MgSO₄- 7H₂0, and 20 mg/l of FeS0₄- 7H₂0 dissolved in tap water was sterilized in a small-sized 2-l jar fermenter.

0034This culture medium was inoculated with 50 ml of a seed culture fluid obtained similarly as in Example 1 by the precultivation of Pseudomonas chlororaphis, strain B 23 (Ferm BP-187), and cultivation was carried out at 25°C for 45 hours at an aeration rate of 2 I/min. and an agitation speed of 500 rpm. Isobutyronitrile was added intermittently in a quantity amounting to a total of 20.8 g/1.3 I by the completion of the cultivation while the concentration thereof in the culture fluid was controlled so as not to exceed 10 g/I. The pH of the culture fluid was adjusted to 7 to 8 with an aqueous solution of sulfuric acid or caustic soda.

0035For comparison purposes, cultivation was carried out similarly except that 5.2 g/1.3 I of isobutyronitrile was added at one time at the initiation of the cultivation.

0036In the case where isobutyronitrile was added sequentially, the nitrile hydratase activity reached 166 units (the cell concentration being 7.8 g/I) after 45 hours' cultivation while, in the case where this enzyme inducing agent was added at one time, the nitrile hydratase activity reached the maximum (14 units) 12 hours after the initiation of the cultivation and then started to decrease to 12 units (the cell concentration being 4.2 g/I) as measured after 45 hours' cultivation.

Example 4

0037The procedure of Example 2 was followed except that a total of 24.7 g/1.3 I of isobutyronitrile was added through a pump continuously over a period of 40 hours while the concentration thereof in the culture fluid was controlled so as not to exceed 10 g/I.

0038As a result, 171 units (the cell concentration being 7.4 g/I) of nitrile hydratase activity was obtained after 44 hours' cultivation.

Example 5

00391.3 I of a culture medium (pH 7.2) comprising 25 g/I of glucose, 2 g/I of ammonium sulfate, 1 g/I of KH₂PO₄, 1 g/I of K₂HPO₄, 1 g/I of MgS0₄- 7H₂0, and 20 mg/I of FeS0₄. 7H₂0 dissolved in tap water was sterilized in a small-sized 2-1 jar fermenter.

0040This culture medium was inoculated with 50 ml of a seed culture fluid obtained similarly as in Example 1 by the precultivation of Pseudomonas chlororaphis, strain B 23 (Ferm BP-187), and cultivation was carried out at 25°C for 42 hours at an aeration rate of 2 I/min. and an agitation speed of 500 rpm. The pH of the culture fluid was adjusted to 7 to 8 with an aqueous solution of sulfuric acid or caustic soda. A total of 20.8 g/1.3 I of isobutyronitrile was added intermittently from 8 hours after the initiation of the cultivation until the completion thereof, while the concentration of the isobutyronitrile in the culture fluid was controlled so as not to exceed 10 g/I.

0041For comparison purposes, cultivation was conducted similarly except that 10.4 g/1.3 I of isobutyronitrile was added at one time 8 hours after the initiation of the cultivation.

0042In the case where isobutyronitrile was added sequentially, 170 units (the cell concentration being 9.0 g/I) of nitrile hydratase activity was obtained after 42 hours' cultivation, while, in the case where this enzyme inducing agent was added at one time, the nitrile hydratase activity reached 42 units 27 hours after the initiation of the cultivation and then started to decrease to 30 units (the cell concentration being 6.7 g/1) as measured after 42 hours' cultivation.