Strain of Rhodococcus rhodochrous as a producer of nitrile hydratase

This invention provides a new strain Rhodococcus rhodochrous having a high nitrile hydratase activity and capable of hydrating aliphatic and aromatic nitriles to corresponding amides. An isolated culture of Rhodococcus rhodochrous VKM Ac-1515D is also disclosed for use in the production of nitrile hydratase. An enzymatic inducer is not required in the growth medium, however, the growth medium does include a salt, a carbon source, and a nitrogen source.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to biotechnology and to preparing a new 
strain of bacteria having a high nitrile hydratase activity, which strain 
is intended for use in processes for producing amides from nitriles. 
2. Description of Related Art 
An enzyme of a nitrile hydratase capable to catalyze the conversion of 
nitriles into amides has been detected in a number of genera of bacteria. 
In order to produce nitrile hydratase in the process of growing bacteria, 
it is necessary to add an inducer to the nutrient medium. Nitriles and 
amides of organic acids (Ref. U.S. Pat. No. 4,555,487; European Patent No. 
0 109 083; European Patent No. 0 204 555), urea or its derivatives 
(European Patent Application No. 0 362 829) may be used as inducers. Known 
in the prior art are strains of Corynebacterium N 774 (U.S. Pat. No. 
4,248,968) and Rhodococcus sp. S-6 (U.S. Pat. No. 5,179,014) for which no 
inducer is required. Disadvantages of the strain N774 reside in its low 
nitrile hydratase activity (its specific activity is 50-60 units/mg, here 
and further on measured in mcM of acrylamide/min/mg of cells, based on dry 
matter weight), narrow range of substrate nitriles (only aliphatic 
nitriles), a low thermal stability of the enzymes (the optimum effect 
temperature for N774 is 35.degree. C.). A disadvantage of the strain S-6 
resides in its capacity to hydrate the produced amides to acids, whereby 
the quality of the amides is sharply affected. Furthermore, the strain S-6 
has a low thermal stability of nitrile hydratase (not higher than 30 C.) 
and a low productivity (it is capable of accumulating not more than 20% of 
acrylamide in the solution). Expensive components of nutrient media (such 
as peptone, yeast extract, meat extract) are used to grow both strains, 
N774 and S-6. 
In terms of its technical essence and the result achieved, the strain 
Rhodoccus rhodochrous J1 (European Patent Application No. 0 362 829) 
possessing a nitrile hydratase activity with respect to aliphatic and 
aromatic nitriles comes nearest to the present invention. However, the 
above-cited strain is disadvantageous by the fact that it is necessary to 
use a nutrient medium containing vitamins, yeast extract and peptone to 
cultivate it. Another disadvantage lies in the fact that the strain J1 
necessitates the use of urea as inducer in high concentrations (7.5-12 
g/l). Thus, when the strain is cultivated in a medium without urea, the 
specific nitrile hydratase activity is as low as 3.35 units/mg (total 
activity is 17.7 units/ml, here and further when measured in micro-moles 
of acrylamide/min/ml of a cultural liquid). With the presence of 7.5 g/l 
of urea in the medium, the specific activity reaches 497 units/mg, the 
total activity being 2480 units/ml. It should be noted that the urea plays 
a dual role, namely: it is used as a source of nitrogen, and as an inducer 
for the nitrile hydratase. An amount of urea which does not exceed 2 g/l 
is sufficient for growth of the strain (whereby the specific nitrile 
hydratase activity of the strain J1 reaches 36.5 units/mg, the total 
activity being 189 units/ml), while high urea concentrations are necessary 
only for induction. For this reason, a substantial amount of the urea 
remains in the medium after cultivating therein the strain J1. 
SUMMARY DESCRIPTION 
It is the object of the present invention to produce a strain featuring a 
high nitrile hydratase activity, while being grown in simple synthetic 
media which does not contain any vitamins, amino acids, or other compounds 
serving as an inducer for synthesis of nitrile hydratase (nitriles, amides 
or urea). 
The above-formulated object is accomplished by developing a strain 
Rhodocosccus rhodochrous M33 which would be capable in the absence of 
inducers to constitutively produce nitrile hydratase for catalyzing the 
hydrolysis of aliphatic nitriles, such as, e.g. acrylonitrile, and 
aromatic nitriles, such as, e.g. 3-cyano pyridine, into corresponding 
amides, such as, e.g. acrylamide and nicotinamide. The strain of the 
present invention is capable, in the absence of urea, to produce nitrile 
hydratase having a specific activity of 200-457 units/mg, and a total 
activity of 360-350 units/ml (depending on the carbon source used), and 
with 2 g/l of urea in the medium, of 180 units/mg and 1,368 units/ml, 
respectively. Another advantageous feature of the strain M33 resides in 
the practically complete absence of amidase activity. The point is that 
the amidase is an enzyme catalyzing hydrolysis of the produced amides into 
corresponding acids, e.g. acrylamide into acrylic acid, whereby the 
quality of the produced amides is sharply deteriorated. 
The new strain grows in simple synthetic media comprising a carbon source, 
such as pyruvate, acetate or glucose, in concentrations of from 0.I to 4%, 
and a nitrogen source, such as ammonium, nitrate salts or urea, in 
concentrations of from 0.4 to 2%, no vitamins, amino acids, yeast extract 
being required for cultivation of the strain M33. 
The Rhodococcus rhodochrous strain M33 is derived from the strain 
Rhodococcus rhodochrous M8 (RF Patent No. 1 731 814) by direct selection 
in two steps using selective media containing, chloracetamide, in the 
first stage, and acetamide, in the second stage. 
The new strain in accordance with the present invention is capable of 
producing nitrile hydratase constitutively, in the absence of inducers, 
such as nitriles, amides or urea in the medium (Tab.1). 
TABLE 1 
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Specific activity 
Nitrile hydratase** 
Amidanse*** 
Cultivation conditions* 
M8 M33 M8 M33 
______________________________________ 
Inducer-free medium 
8 457 0.0I 0.00I 
Medium with an inducer 
315 425 0.3 0.015 
(urea, 10 g/l) 
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*The strains were grown in Erlenmeyer flasks (750 ml volume), each 
containing 150 ml of a nutrient medium with pyruvate (5 g/l) and NH.sub.4 
Cl (2 g/l) during 24 hours at 30.degree. C., under intensive aeration 
conditions. Upon expiration of 24 hours, and the cultures thus prepared 
were divided into two parts and urea, as an inducer (in an amount of I0 
g/l) was added to one of them, whereupon the cultivation procedure was 
continued for 48 hours more. Cells were isolated by centrifuging and used 
to determine the activities of the nitrile hydratase and amidase. The 
nitrile hydratase activity of each of the prepared cultures was determine 
using acrylonitrile as a substrate under standard conditions, whereas the 
amidase activity was determined by the formation of ammonium from 
acrylamide used as a substrate. The ammonium concentration was determined 
by the Nessler method. 
**mcM of acrylamide/min/mg of cells, dry weight. 
***mcM of ammonium/min/mg of cells, dry weight. 
It is obvious from the above table, that the specific activity of the 
nitrile hydratase reaches 457 units/mg, when the strain M33 has been grown 
in a nutrient medium in the absence of urea. Under the same conditions, 
the nitrile hydratase activity of the original strain M8 is 8 units/mg. It 
is known that the specific activity of the nitrile hydratase strain J1, 
when grown in urea-free media, is 3.4 units/mg (Ref. European Patent 
Application No. 0 362 829; U.S. Pat. No. 5,089,411). 
Thus, the strain M33, as distinct from the original strain M8 and strain 
J1, is capable of constitutively producing nitrile hydratase even in the 
absence of an inducer in the medium and has an amidase activity that is 20 
times weaker. 
The strain M33 has been deposited on Dec. 6, 1993 at the Institute of 
Biochemistry and Physiology of Microorganisms of The Russian Academy of 
Science (IBFM-VKM), Pushchino-na-Oke, 142292 Moscow Region, Russian 
Federation under accession number VKM Ac-1515 D in the All-Russian 
National Collection of Microorganisms and is characterized by the 
following morphologico-cultural and physiologico-biochemical 
characteristics: 
Morphological properties. Cells of the strain M33 are nonmotile and 
gram-positive. No spores are produced, the cells are not acid resistant. 
At an age of 18-20 hours, the cells form long (up to 20 mcM), slightly 
ramified filaments which, after some 48-72 hours, undergo fragmentation 
into short rod- and cocci-shaped elements. 
Cultural properties. After a 48-hour growth on glucose yeast extract agar 
belonging to dense nutrient media (such as MPA, Hottinger), the strain M33 
produces round smooth colonies having a diameter of 1 mm and coloured from 
pale pink to pink-orange. When growth is carried out in a meat-peptone 
broth, a film and a sediment are formed, no changes in Litmus milk being 
observed. 
Physiological properties. The strain represents an obligate aerobe, it 
reduces nitrates. MR and VP tests are negative. The strain generates 
hydrogen sulphide, it is oxidase negative, and catalase and phosphatase 
positive. The strain does not hydrolyze starch and cellulose, but it does 
hydrolyze Tween 60 and 80. No adenine is utilized. Cells are not resistant 
to heating in skim milk at 72.degree. C. for 15 minutes. The strain M33 
grows at pH 6-9 and at a temperature of 5-45 C. Acid is generated from the 
following sugars and alcohols: glucose, fructose, maltose, saccharose, 
sorbitol, mannitol, and glycerol. No gas generation is observed from a 
single sugar. It uses ammonium compounds, nitrates and urea as a single 
nitrogen source, while as a single carbon source, use is made of maltose, 
mannitol, sorbitol, glucose, glycerol, lactate, pyruvate, benzoate, p- and 
m-hydroxybenzoate, tyrosin. It does not use rhamnose, galactose, inositol, 
a-ketoglutarate. 
Biochemical analysis has shown that the cell wall of the strain M33 
contains meso-diaminopimelic acid, arabinose and galactose, which feature 
is characteristic of Coryneform bacteria having a Type IV cell wall. The 
cells also contain lipide A (LCH), which is characteristic of Rhodococci. 
Thus, the strain M33 possesses characteristics which are typical of 
Coryneform bacteria. In view of the above-listed properties and in 
accordance with Bergy's Manual of Systematic Bacteriology and with 
Nesterenko's Classification Manual, the strain M33 can be related to the 
genus Rhodococcus, species rhodochrous. 
To obtain M33 cells featuring a high nitrile hydratase activity, an M33 
culture is placed into nutrient media, followed by incubation at 
25.degree.-30.degree. C. for 24-72 hours. 
Cell suspensions obtained by centrifuging the cultures, followed by 
resuspending the cells in a I0-mM phosphate buffer are used to transform 
the nitriles into amides. 
The cells obtained by this procedure have a high nitrile hydratase activity 
and are capable of catalyzing hydrolysis of aliphatic and aromatic 
nitriles into corresponding amides over a wide range of temperatures 
extending from 4 to 50 C. and at pH values ranging from 3 to 10. 
The standard test for measuring the nitrile hydratase activity is conducted 
in the following manner: 1 ml of a 2%-solution of nitrile in I0 mM of a 
phosphate buffer having a pH of 7.6 is mixed with 1 ml of a cell 
suspension containing 0.04 mg of cells based on the dry matter content. 
The reaction is conducted at 20.degree. C. for 5 minutes, whereupon the 
reaction is stopped by adding 20 ml of concentrated HCl. The concentration 
of the amides thus-formed is determined by gas chromatography. 
The nitrile hydratase activity is expressed in the following units: 
One unit is defined as the amount of an enzyme required to produce amide 
from nitrile under the above-described conditions at a rate of 1 
mcM/minute. 
Specific activity is expressed in mcM amide/min/mg of cells, based on the 
dry matter content, or in units/mg; 
Total activity is expressed in mcM amide/min/ml of the culture, or in 
units/ml.

DETAILED DESCRIPTION--EXAMPLE 
Example 1 
cells of strain M33 were cultivated in the absence of urea in a nutrient 
medium containing sodium nitrate as a nitrogen source. Five ml of a strain 
M33 culture, preliminarily grown in a medium of the following composition 
for 48 hours at 30.degree. C., were placed into an Erlenmeyer flask (750 
ml volume) filled with 150 ml of a nutrient medium having the same 
composition (g/l): 
______________________________________ 
K.sub.2 HPO.sub.4 
-- 0.5 KH.sub.2 PO.sub.4 
-- 0.6 
MgSO.sub.4 -- 0.5 FeSO.sub.4 
-- 0.005 
CoCl.sub.2 -- 0.01 glucose 
-- 5 
NaNO.sub.3 -- 1 
______________________________________ 
The flask was incubated on a shaker for 48 hours at 30.degree. C. The 
nitrile hydratase activity of the cells was determined using acrylonitrile 
as a substrate. The specific activity of the nitrile hydratase in the 
strain M33 reached 200 units/mg, its total activity being 360 units/ml. 
Thus, the specific activity of the strain M33 cultivated in a urea-free 
medium is more than 60 times greater than the activity of the strain J1. 
What is more, commercially readily available components of the medium, 
namely, glucose and sodium nitrate, are used to grow the strain M33. 
Example 2 
Five ml of a culture of the strain M33 preliminarily grown for 48 hours at 
30.degree. C. in a medium of the same composition as in Example 1, with 
the exception that glucose was taken in an amount equal to 20 g/l and urea 
in an amount of 2 g/l was used instead of sodium nitrate, was introduced 
into an Erlemmeyer flask (750 ml volume) containing 150 ml of the same 
nutrient medium. The flask was incubated on a shaker at 30.degree. C. for 
48 hours. The nitrile hydratase activity was determined using 
acrylonitrile as a substrate. The cell yield was 7.6 g/l, specific 
activity--180 units/mg, total activity--1,386 units/ml. 
Example 3 
The strain M33 was grown using the same procedure as described. The cells 
were separated by centrifuging, washed with I0 mM of a phosphate buffer, 
and resuspended in a I0 mM of a phosphate buffer. Reaction was conducted 
under standard conditions using various nitriles as substrates. The 
reaction was discontinued by addition of concentrated HCl, the 
concentration of the amides thus-formed being determined by gas 
chromatography. 
TABLE 
______________________________________ 
Substrate Specific activity, %* 
______________________________________ 
Acetonitrile 140 
Acrylonitrile 100 
3-cyanopyridine 
54 
4-cyanopyridine 
70 
2-cyanopyridine 
22 
______________________________________ 
*Specific activity with acrylonitrile used as a substrate (200 mcM/min/mg 
taken as I00%. 
It transpires from the table above that the strain M33 is capable of 
catalyzing hydrolysis of both aliphatic and aromatic nitriles. 
Example 4 
The strain M33 was grown as in Example 1. A biomass containing M33 cells up 
to a concentration of 0.5 mg/ml and 3-cyanopyridine up to a concentration 
of 8% was added into a I00-ml flask containing 40 ml of distilled water 
(pH 7.6). The flask was incubated at 30.degree. C. under stirring 
conditions. After 60 minutes, a I0%-nicotinamide solution was obtained. 
Nicotinic acid and 3-cyanopyridine were not present in the reaction 
mixture. 
Example 5 
Into a steel reactor having a volume of 1.5 l and provided with a 
mechanical stirrer and thermostattable within the temperature range of 
from 12.degree. to 20.degree. C., 400 ml of distilled water were poured 
(pH 7.6), followed by resuspending in it 272 mg of a biomass (based on the 
dry matter content) of the strain of Rhodococcus rhodochrous M33 grown as 
described in Example 1. Then a pure acrylonitrile was added to the 
reaction mixture at such a rate that its concentration in the solution 
would not exceed 2%. The qualitative and quantitative compositions of the 
solution were determined by gas-liquid chromatography. All in all, 173 g 
of acrylonitrile were placed into the reactor. A 46% (weight/volume) 
solution of acrylamide was obtained after 8 hours. The yield of acrylamide 
was close to 99%. No acrylonitrile and acrylic acid were detected as 
by-products. 
Thus, the claimed strain M33 has a high nitrile hydratase activity and is 
capable of hydrating both aliphatic and aromatic nitriles into 
corresponding amides. As distinct from its prior-art analogues, the mutant 
strain M33 is capable of constitutively producing nitrile hydratase when 
cultivated in media containing no inducers for synthesis of nitrile 
hydratase. This advantageous feature makes it possible to use media 
without vitamins, but containing glucose as a carbon source, and sodium 
(or potassium) nitrite as a nitrogen source, for cultivation of the cells. 
These media contain no special compounds as inducers of nitrile hydratase 
(such as amides, nitriles or urea taken in high concentrations). Another 
advantageous feature of the strain M33 resides in the virtually complete 
absence of amidase activity which blocks the formation of acids from the 
amides in the process of catalytically assisted conversion of the 
nitriles. 
The strain Rhodococcus rhodochrous M33 can be recommended as a producer of 
a nitrile hydratase enzyme and it can be used in processes to prepare 
aliphatic and aromatic amides from nitriles. 
Although certain presently preferred embodiments of the invention have been 
described herein, it will be apparent to those skilled in the art to which 
the invention pertains that variations and modifications of the described 
embodiment may be made without departing from the spirit and scope of the 
invention. Accordingly, it is intended that the invention be limited only 
to the extent required by the appended claims and the applicable rules of 
law.