Method for cultivation of Pseudomonas bacteria

Cells of Pseudomonas bacteria having a high nitrile hydratase activity can be obtained in a high yield by adding to a culture medium at least one amide compound selected from the group consisting of acrylamide, methacrylamide, crotonamide, and n-butyramide in the preparation of cells of bacteria having nitrile hydratase activity by cultivating Pseudomonas bacteria capable of producing nitrile hydratase.

BACKGROUND OF THE INVENTION 
The present invention relates to a method of producing in a high yield 
cells of Pseudomonas bacteria having a high nitrile hydratase activity. 
In recent years, there have been increasing attempts to utilize 
microorganisms and enzymes as they are or in an immobilized state as 
catalysts for various single or complex chemical reactions. 
Nitrile 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. 46 1165 (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)) 
Under 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. 
From the foregoing point of view, some of us have proposed a method in the 
concurrently filed U.S. patent application. The method for cultivation of 
Pseudomonas bacteria disclosed in this U.S. patent application comprises 
adding sequentially 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. 
SUMMARY OF THE INVENTION 
An object of the present invention is to solve the above problem by 
substantially the same means as is disclosed in the concurrently filed 
U.S. patent application mentioned previously on the basis of the discovery 
that specific amide compounds other than those named hereinbefore have 
similar advantages. 
Thus, a distinguishing feature of the method for cultivation of Pseudomonas 
bacteria having a high nitrile hydratase activity according to this 
invention is the addition of at least one amide compound selected from the 
group consisting of acrylamide, methacrylamide, crotonamide, and 
n-butyramide to a culture medium in the preparation of cells of bacteria 
having nitrile hydratase activity by cultivating Pseudomonas bacteria 
capable of producing nitrile hydratase. 
We have found that, by adding amide compounds to the culture medium during 
the cultivation of Pseudomonas bacteria, the nitrile hydratase activity 
per unit culture fluid increases remarkably. 
This increase in 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). 
In the present invention, amide compounds 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 
The 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. 
______________________________________ 
B 23 PS 1 
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(a) Morphology 
1 Shape and size 
bacillus bacillus 
of cell 0.8-1.1 .times. 1.6- 
0.8-1.1 .times. 1.3- 
2.7 .mu.m 1.9 .mu.m 
2 Polymorphism none none 
3 Motility motile motile 
one to three with polar fla- 
polar flagella 
gella 
4 Formation of none none 
spores 
5 Gram staining 
-- -- 
6 Acid-fast -- -- 
property 
(b) Growth on vari- 
ous culture 
media 
1 Bouillon-agar 
spherical, smooth, homoge- 
plate culture 
convex, glos- 
neous, glossy, 
sy, translucent 
and mucoidal 
and yellow 
2 Bouillon-agar 
small colony smooth, glossy, 
slant culture 
formed translucent, 
and yellow 
3 Bouillon li- precipitated 
quid culture 
4 Bouillon-gela- 
liquified (+) 
-- 
tin stab 
culture 
5 Litmus-milk acidic: pepto- 
alkaline: pepto- 
nized, not nized, not 
coagulated coagulated 
(c) Physiological 
properties 
1 Reduction of + - 
nitrate 
2 Denitrifica- + - 
tion 
3 MR test - - 
4 VP test - - 
5 Formation of - - 
indole 
6 Formation of - - 
hydrogen 
sulfide 
7 Hydrolysis of 
- - 
starch 
8 Utilization of 
Simon's cul- Simon's cul- 
citric acid ture: + ture: + 
9 Utilization of 
ammonium ammonium 
inorganic salt: + salt: + 
nitrogen source 
10 Formation of King-A cul- King-A cul- 
pigments ture: - ture: - 
King-B cul- King-B cul- 
ture: + ture: + 
green (water- 
green (water- 
soluble) soluble) 
11 Urease - - 
12 Oxidase + + 
13 Catalase + + 
14 Growth range pH: 6.0-9.9 
temperature: 
5-36.5.degree. C. 
15 Behavior toward 
aerobic aerobic 
oxygen 
16 O-F Test oxidized oxidized 
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17 Formation of 
Forma- Forma- 
Forma- Forma- 
acid & gas tion of tion of 
tion of tion of 
from saccharide 
acid gas acid gas 
D-glucose + - + - 
D-mannose + - + - 
D-fructose - - - - 
D-galactose + - + - 
maltose - - - - 
sucrose - - - - 
lactose - - - - 
trehalose - - 
D-mannitol - - - - 
glycerol - - - - 
starch - - - - 
18 Nutritive re- 
none none 
quirements 
19 Other proper- 
See remarks 
ties 
______________________________________ 
Remarks: Aminopeptidase + 
Formation of levan 
+ 
from saccharose 
Formation of poly-.beta.- 
- 
hydroxybutyrate 
GC content 64.6% 
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ENZYME INDUCING AGENT 
In the present invention, acrylamide, methacrylamide, crotonamide, or 
n-butyramide is used as an enzyme inducing agent. These compounds can be 
used singly or in the form of a mixture of two or more members. 
In accordance with the present invention, these compounds are added to the 
culture medium at one time or sequentially. The term "sequentially" as 
used herein is intended to mean both "continuously" and "intermittently".

CULTIVATION - PRACTICE OF THE PRESENT INVENTION 
A preferred embodiment of this invention will be described below. 
Pseudomonas 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, a 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 species of the 
aforementioned amide compounds is added at one time or sequentially. 
Ordinarily, the concentration of the enzyme inducing agent in the culture 
medium is adjusted preferably to lower than 50 g/l (in total when two or 
more compounds are used as such), and more preferably to 10 g/l or lower 
although this may vary depending on the cultivation time, temperature and 
other conditions. If the concentration becomes 50 g/l 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.degree. to 
37.degree. C., preferably of the order of 25.degree. to 30.degree. C., and 
the cultivation time is about 1 to 3 days. 
After 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 example. 
In the disclosure, the letter "l" indicates "liter". 
EXPERIMENTAL EXAMPLE 
1. Cultivation of Bacteria 
1.4 ml of a seed culture fluid obtained from Pseudomonas chlororaphis, 
strain B 23 (FERM BP-187), grown under the following precultivation 
conditions was cultivated under the subsequent cultivation conditions to 
determine the acrylamide-producing activity of the bacteria. 
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(1) Precultivation Conditions 
MY culture medium (pH 7.6): 
peptone 5 g/l 
yeast extract 3 g/l 
malt extract 3 g/l 
glucose 5 g/l 
Cultivation temperature: 28.degree. C. 
Cultivation time: 18 hours 
a 500-ml (net capacity: 50 ml) Sakaguchiflask 
was used. 
(2) Cultivation Conditions 
Culture medium (pH 7.6): 
sucrose 10 g/l 
KH.sub.2 PO.sub.4 0.5 g/l 
K.sub.2 HPO.sub.4 0.5 g/l 
MgSO.sub.4 .7H.sub.2 O 0.5 g/l 
FeSO.sub.4 .7H.sub.2 O 20 mg/l 
L-cystin 2 g/l 
L-glutamic acid 2 g/l 
L-proline 2 g/l 
amide compound(s) 5 g/l 
Cultivation temperature: 25.degree. C. 
a 500-ml (net capacity 70 ml) Sakaguchiflask 
was used. 
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2. Measurement of Nitrile Hydratase Activity 
1 ml of a culture fluid was admixed with 4 ml of a 1/10M phosphate buffer 
solution (pH 7.0), and 5 ml of a 1/10M phosphate buffer solution (pH 7.0) 
containing 5.0% by weight of acrylonitrile was added thereto. The 
resulting solution was caused to react at 10.degree. C. for 10 minutes, 
and the bacterium cells therein were separated by filtration. The nitrile 
hydratase activity of the cells exhibited in the hydration of 
acrylonitrile to produce acrylamide was determined by measuring the 
quantity of the acrylamide (AA) thus obtained by means of gas 
chromatography. 
The activity was determined for the specific activity (SA) and the total 
activity (TA) as defined below. 
SA: .mu.mole AA/mg-cells/min. 
TA: .mu.mole AA/ml-culture medium/min. 
The results obtained are summarized in Table 1. 
TABLE 1 
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Amide Culti- Cell concen- 
Enzymatic 
com- vation tration activity 
pound* time(hr) pH (g/l) SA TA 
______________________________________ 
AA 30 7.00 2.15 5.14 11.05 
37 7.10 2.56 6.77 17.33 
44 7.55 2.38 2.21 5.26 
53 8.00 2.50 0 0 
CA 19 6.90 4.05 28.88 117.0 
24 7.15 4.65 32.41 150.7 
30 7.68 3.93 29.92 117.6 
37 8.00 3.64 27.09 98.6 
MA 15 7.35 3.10 34.50 107.0 
19 7.30 4.47 55.93 250.0 
23 7.45 5.36 62.65 335.8 
27 7.60 5.96 50.69 302.1 
BA 15 7.10 5.30 7.64 40.5 
19 7.00 7.45 5.81 43.3 
23 7.18 8.46 4.93 41.7 
27 7.25 8.40 4.71 39.6 
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*AA: acrylamide 
CA: crotonamide 
MA: methacrylamide 
BA: nbutyramide