WO2002081659A1

WO2002081659A1 - Bacterial strain alcaligenes denitrificans producing nitrilase

Info

Publication number: WO2002081659A1
Application number: PCT/RU2002/000141
Authority: WO
Inventors: Svetlana Viktorovna Poltavskaya; Tatiana Nikolaevna Kozulina; Igor Singirtsev; Sergei Vladimirovich Kozulin; Sergei Petrovich Voronin
Original assignee: Zakrytoe Aktsionernoe Obschestvo 'bioamid'
Priority date: 2001-04-03
Filing date: 2002-04-01
Publication date: 2002-10-17
Also published as: RU2177034C1

Abstract

The invention relates to biotechnology and can be used for preparation of acrylic and other organic acids. The strain was isolated by the method of step-by-step adaptation from samples of soil taken from chemical plant with the use of acrylonitrile as selection agent. The strain requires neither reach nutrient media nor additional inducers for its cultivation; it can grow on simple hemi-synthetic medium containing acetate as a carbon source and corn extract as a source of additional growth factors. Strain C-32 was relates to species denitrificans of genera Alcaligenes on the base of its morphological, culture, and biochemical properties. Strain Alcaligenes denitrificans C-32 VKM B-2243 D possesses high nitrilase activity on acrylonitrile [555.6 mmole/(h*g) at 30 °C]. Maximum nitrilase activity of cells of the strain [1606.0 mmole/h*g)] occurs at 65 °C. Enzymatic activity of the strain appears in a wide pH range (4 to 12). 3 Tables are enclosed.

Description

BACTERIAL STRAIN ALCALIGENES DENITRIFICANS PRODUCING NITRILASE

The invention relates to biotechnology and concerns the isolation of new bacterial strain Alcaligenes denitrificans C-32 NKM B-2243 D, which produces nitrilase enzyme catalyzing the direct hydrolysis of nitriles to corresponding organic acids.

Various microorganisms possessing nitrilase enzyme are described in literature. Among them, there are strains related to fungi, such as Fusarium [1], actinomycetes, such as Rhodococcus [2] and Nocardia [3], Gram-positive bacteria, such as Corynebacterium [4] and Arthrobacter [5], and Gram-negative bacteria, such as Alcaligenes [6] and Klebsiella [7]. However, most of the described nitrilases can hydrolyze only aromatic and heterocyclic nitriles.

An ability of direct hydrolysis of aliphatic nitriles to give their corresponding organic acids is reported for strains Rhodococcus rhodochrous J-l [8], Rhodococcus rhodochrous ΝCrMB 40757 and ΝCIMB 40833 [9], Rhodococcus rhodocrhous K22 [10], Alcaligenes sp. AK866Ν [11], Alcaligenes sp. NKM B-6706 [12].

Literature data and our experience are show that main characteristics of the strains proposed for use as biocatalysts are their high enzymatic activity and ability of giving large cell harvest on simple and cheap media.

A need of toxic, highly fugitive nitriles as inducers, amino acids and vitamins as additional growth factors, or expensive peptone, which are to be added to medium during cultivation, is a general disadvantage of strains Rhodococcus rhodochrous J-l, Rhodococcus rhodochrous ΝCIMB 40757, Rhodococcus rhodochrous ΝCIMB 40833, Rhodococcus rhodochrous K22, and Alcaligenes sp. B-6706. On the other hand, these strains are characterised by low specific nitrilase activity. Thus, strain Rhodococcus rhodochrous J-l requires peptone for its growth, and isovaleronitrile or caprolactame for appearance of nitrilase activity. The maximum specific activity of the strain on acrylonitrile is 212.4 mmole/(h*g) at the reaction temperature of 20°C. The maximum specific activity of strains Rhodococcus rhodochrous ΝCJV1B 40757 and Rhodococcus rhodochrous ΝCIMB 40833, even if grown on media containing vitamins and also acrylonitrile as inducer, not exceeds 82.98 mmole/(h*g) at the reaction temperature of 30°C. Strain Alcaligenes sp. B-6706 requires an introducing casamino acid to the cultivation medium, and its specific nitrilase activity is not above 234 mmole/(h*g) at 30°C. Strain Alcaligenes sp. AK866N, whose cells show high nitrilase activity in a temperature range 0 to 30°C upon cultivation on a medium containing peptone, propionitrile, or acetonitrile, is the mostly close to the proposed invention by its properties. Thus, the maximum specific activity of the strain on acrylonitrile is 506.0 mmole/(h*g) at the reaction temperature of 30°C. The disadvantage of this strain is, primarily, the necessity of introduction of expensive peptone and additional inducers, such as propionitrile or acetonitrile, to the cultivation medium. Furthermore, the enzyme thermolability does not allow of performance of nitrile hydrolysis at the temperature above 30°C.

The goal of the present invention was isolation of strain possessing high nitrilase activity at wide temperature range, and requiring neither inducers nor expensive medium components for its cultivation.

The undertaken aim is reached by isolation of strain Alcaligenes denitrificans C-32, which does not require a use of expensive nutrient components, such as peptone, casamino acid, yeast extract, or vitamins, and highly toxic, fugitive nitriles as special inducers during the cultivation process. Strain Alcaligenes denitrificans C-32 possesses high nitrilase activity [555.6 mmole/(h*g) at 30°C] and characterized by the enzyme thermostability.

The claimed strain Alcaligenes denitrificans C-32 was deposited in the Russian collection of microorganisms under collection number B-2243 D. It has the following morphological, cultural, and biochemical properties.

Cultural and morphological characteristics. Cells of strain Alcaligenes denitrificans C- 32 having age of 20 to 22 hours are rod-shaped and ovoid. They dispose lonely, sometimes in pairs. Sizes of cells are 0.7 to 0.8 by 0.9 to 2.0 μ. Cells are Gram-negative. Cells form neither spores nor capsules. They are motile.

Bouillon-agar culture: colonies are circle, of 2 to 3 mm diameter, convex, spread on agar with an age. Colonies are brilliant, translucent in passed light. They have uneven edge. Colonies are pasty. Uniform turbidifϊcation of the whole medium and also formation of ring on the surface close to walls take place during the growth on peptone bouillon.

Physiological and biochemical properties. The strain is aerobe with obligatory respiratory metabolism. It is able of nitrate respiration; reduces nitrate to nitrite. The optimum growth temperature is 30 to 32°C. It does not grow at 42°C. The optimum growth pH value is 7.5± 0.5. It shows positive tests on catalase and oxidase. The strain is chemoorganotroph; uses various organic acids and amino acids as carbon sources. It can grow on synthetic media containing acetonitrile, propionitrile, or acrylonitrile as single carbon and/or nitrogen source. It is able of hydrolysis of nitriles without formation of amides as intermediates. The strain causes alkalization if grown on media containing salts of organic acids, nitriles, or amides. It does not form acid in O/F tests on medium with sugars and polyols. It hydrolyzes neither starch nor cellulose. The strain has no gelatinase. It does not form pigments on the media <<King A» and «King B». It shows weak growth on the medium with 6.5 % NaCl. The strain forms neither indole nor hydrogen sulphide.

The strain is non-toxic and non-pathogenic for human organism.

Basing on its above-mentioned properties and according to the Bergey's manual of determinative bacteriology, strain C-32 was related to species denitrificans of genera Alcaligenes.

Strain C-32 was cultivated on semi-synthetic nutrient media for 72 hours at the temperature of 32°C to obtain cells having high nitrilase activity.

Enzymatic activity of cells was determined using nitrile of acrylic acid as substrate. A unit of specific nitrilase activity was defined as quantity of the enzyme, which catalyzed formation of 1 mmole of acid during a time unit (1 hour) and which contained in 1 gram of cells (by the dry weight).

The essence of the invention is explained by the following examples.

Example 1. Isolation of strain Alcaligenes denitrificans C-32 NKM B-2243 D.

The claimed strain was isolated by the method of step-by-step adaptation from samples of soil, which was taken from a plant producing acrylonitrile.

Sample of soil (1.0 g) was suspended in 10 ml of sterile saline and allowed to stand for a hour. Supernatant (5 ml) was then transferred to 300-ml Erlenmeyer flasks filled each by 1/6 with a medium of the following composition (g): glucose, 0.5; peptone, 1.0; K₂HPO₄, 0.1; MgSO₄, 0.1; acrylonitrile, 0.01; distilled water, 1000 (pH 7.2+0.2).

Cultivation was carried out at 28°C under round shaking (160 r.p.m.). A half of the culture fluid from each the flask was removed daily, and residue in flask was diluted to the total volume of 50 ml with the medium described above. Acrylonitrile concentration was increased step-by-step from 0.01 to 3.0 g 1. Glucose and peptone were gradually removed from the medium during the 30 days' adaptation. Thus obtained isolations were seeded onto agarized medium having above-mentioned composition. The grown colonies were re-seeded twice onto agarized nutrient medium to ensure their purity and then analysed on ability of transformation of acrylonitrile to acrylic acid. h order to do that, the microorganism was subgrown on bouillon for a day. The obtained cell suspension was centrifuged at 6,000 r.p.m. for 20 min. Cells were washed with 0.01 M phosphate buffer pH 7.5+0.2 and then resuspended up to optical density of 1.0 (wavelength of 540 nm, 5.07-mm cuvette) in 1 ml of the same buffer containing 10.0 g/1 acrylonitrile. Transformation was performed at the temperature of 30°C. After a hour, reaction was terminated by addition of 6 N HCl (0.05 ml) to the reaction solution. A quantitative determination of acrylic acid formed was carried out by gas chromatography.

Example 2. Growth of strain Alcaligenes denitrificans C-32 NKM B-2243 D in a laboratory fermenter.

Cells of the strain Alcaligenes denitrificans C-32 were subgrown in 300-ml Erlenmeyer flasks filled by 1/6 with the Hottinger bouillon for a day under round shaking (160 r.p.m.) at the temperature of 30°C. Thus obtained culture fluid was used for seeding the laboratory fermenter.

Fermentation was performed in 3-1 laboratory fermenter filled by 1/2 of its volume with the medium having the following composition:

Νa₂HPO₄ *12 H₂O, 6.0 g/1;

KH₂PO₄, 2.0 g/1;

MgSO₄*7 H₂0, 0.7 g/1;

CH₃COONH₄, 10.0 g/1;

corn extract, 5.0 g/1; distilled water, up to 1 1; pH 7.3.

Cultivation was carried out at the temperature of 32°C under aeration of 1.5 min^'1 and continuous stirring (560 r.p.m.).

During the fermentation process, upon the growth substrate exhaustion in the medium, a solution of acetic acid was introduced into the fermenter using a principle of reverse connection.

Periodically, samples of the culture fluid were taken from the fermenter to determine cell harvest and nitrilase activity. Cell concentration was estimated by photocolorimetry at the wavelength of 540 nm in 5.07-mm cuvette. A unit of optical density corresponded to cell concentration of 0.58 g/1 (by the dry weight). Cell nitrilase activity was determined in the following manner. The tested cell suspension was centrifuged at 6,000 r.p.m. for 5 min. A deposit was washed with 0.01 M phosphate buffer pH 7.5. Cells were resuspended in the same buffer to the optical density of 1.0. The cell suspension was transferred to the plastic test-tubes (by 1.0 ml into each one), which were then sealed and incubated in a water bath at the temperature of 20°C. Acrylonitrile was added to the samples up to the final concentration of 20.0 g/1. Transformation was performed for 30 min under continuous stirring. Reaction was stopped by addition of 6 N HCl (0.05 ml) to the reaction mixture. Concentration of acrylic acid in a supernatant was estimated by spectrophotometry at the wavelength of 255 nm.

By 72 hours, cell harvest was 9.3 g/1 (by the dry weight), specific nitrilase activity of cells on acrylonitrile reached 342 mmole/(h*g).

Example 3. Effect of the reaction temperature on nitrilase activity of cells of strain Alcaligenes denitrificans C-32.

Dependence of cell nitrilase activity of strain Alcaligenes denitrificans C-32 on temperature was studied in a process of hydrolysis of acrylonitrile to acrylic acid. Cells were grown and prepared to the transformation process as it was described in Example 2 above. Conditions of transformation were analogous to that of described for the prototype strain.

Samples of reaction mixture (1.0 ml) containing 0.3 mg cells (by the dry weight) in of 0.01 M phosphate buffer pH 7.5 were incubated for 5 min at the temperatures from 0 to 70°C. Then acrylonitrile was added to the reaction mixture up to the final concentration of 20.0 g/1. Transformation was carried out in a water bath at the above-mentioned temperatures under shaking. Reaction was stopped by addition of 6 N HCl (0.05 ml). Cells were precipitated by centrifugation at 14,000 r.p.m. for 2 min. Concentration of acrylic acid in a supernatant was determined. Dependence of cell specific nitrilase activity on temperature is shown in Table 1.

The data presented in the Table 1 show that cells of strain Alcaligenes denitrificans C-32 possess higher nitrilase activity and thermostability of the enzyme [specific nitrilase activity at 30°C is 555.6 mmole/(h*g), temperature range, in which enzymatic activity exists, is 0 to 70°C] than that of cells of the prototype strain Alcaligenes sp. AK866N [specific nitrilase activity at 30°C is 505.0 mmole/(h*g), temperature range of the enzymatic activity existence is 0 to 30°C]. Maximum nitrilase activity of cells of strain C-32 is detected at the temperature of 65°C [1606 mmole/(h*g)].

Example 4. Effect of reaction medium pH on cell nitrilase activity of strain Alcaligenes denitrificans C-32. Cells were grown and prepared to the transformation process as it was described in Example 2 above. Samples of the reaction mixtures containing 0.58 mg/ml cells (by the dry weight) in 0.01 M phosphate buffer having pH 4.0 to 8.0 or in 0.01 M NaOH/glicine buffer having pH 9.0 to 12.0 were incubated for 5 min at 20°C. Then acrylonitrile was added to the reaction mixtures (each of 1.0 ml volume) up to the final concentration of 20.0 g/1. Transformation was carried out in a water bath at 20°C under stirring for 20 min. Reaction was stopped by addition of 6 N HCl (0.05 ml). Cells were precipitated by centrifugation at 14,000 r.p.m. for 2 min. Concentration of acrylic acid in a supernatant was estimated by spectrophotometry at the wavelength of 255 nm. Dependence of cell nitrilase activity on reaction mixture pH is presented in Table 2.

As it is seen from the data presented in the Table, cells of strain Alcaligenes denitrificans C-32 exhibit nitrilase activity in a more wide pH range (4 to 12) as compared with cells of the prototype strain (pH range 6 to 10).

Example 5. Effect of acrylonitrile concentration on cell nitrilase activity of strain Alcaligenes denitrificans C-32.

Nitrilase activity of cells was estimated as in Example 3 above with the exception that cell concentration in the reaction samples was 0.23 mg/ml, and initial acrylonitrile concentrations corresponded to ones presented in Table 3. The reaction temperature was 30°C.

As it is seen from the data shown in the Table 3, cell nitrilase activity of strain Alcaligenes denitrificans C-32 is not essentially depended on initial acrylonitrile concentration in the reaction mixture up to the substrate solubility limit.

Thus, the claimed bacterial strain Alcaligenes denitrificans C-32 VKM B-2243 D possesses constitutive nitrilase, which performs direct hydrolysis of acrylonitrile to acrylic acid in wide ranges of temperature (0 to 70°C) and medium pH (4 to 12). High nitrilase activity of cells is reached upon the strain growth in the medium, which does not contain expensive components or additional inducers. Specific nitrilase activity of the strain on acrylonitrile is 555.6 mmole/(h*g). Maximum nitrilase activity of strain C-32 occurs at 65°C.

Strain Alcaligenes denitrificans C-32 NKM B-2243 D can be recommended as a producer of nitrilase enzyme and used in biotechnological processes of preparation of organic acids. Sources of information

1. Harper D.B. Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fuzarium solani II Biochem. J. - 1977.- Vol. 167, No. 3.- P. 685-692.

2. Mathew CD., Nagasawa T., Kobayashi M., Yamada H. Nitrilase - catalized prodaction of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous Jl // Appl. Environ. Microbiol.- 1988.- Vol. 54, No. 4.- P. 1030-1032.

3. Collins PA, Knowles C.J. The utilization of nitriles and amides by Nocardia rhodochrous II J. Gen. Microbiol.- 1983.- Vol. 129, No. 3.- P. 711-718.

4. Fukuda Y.,Fukui M., Harada T., Izumi Y. Formation of α-aminoacid from - aminonitrile by cell suspensions of a strain of Corinebacterium II J. Ferment. Technol.- 1971.- Vol. 49, No. 12.- P. 1011-1016.

5. Bandyopadhyay A.K., Nagasawa T., Asano Y., Fujishiro K., Tani Y., Yamada H. Purification and characterization of benzonitrilases from Arthrobacter sp. strain J-l // Appl. and Environ. Microbiol.- 1986.- Vol. 51, No.2.- P. 302-306.

6. Nagasava T., Mauger J., Yamada H. A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3. Purification and characterization // Eur. J. Biochem.- 1990.- Vol. 194, No. 3.- P.

765-772.

7. Stalker D.M., Malyj L.D., Mc.Bride K.E. Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the boon gene // J. Biol.Chem.- 1988.- Vol. 263, No. 13.- P. 6310-6314.

8. Nagasawa T., Nakamura T., Yamada H. ε-Caprolactam, a new powerful inducer for the formation of Rhodococcus rhodochrous Jl nitrilase // Arch.Microbiol.- 1990.- Vol.155.- P. 13- 17.

9. Pat. 5,998,180 USA, Int.Cl.⁶ C12 P007/60, C12 N 009/78, C12 N001/20. Nitrilase from Rhodococcus rhodochrous for converting acrylonitrile directly to acrylic acid / Armitage Y.Ch., Hughes J., Webster N.A. - 18 pp.

10. Kobayashi M., Yanaka N., Nagasawa T., Yamada H. Hyperinduction of an aliphatic nitrilase by Rhodococcus rhodochrous K22 // FEMS Microbiol. Lett.- 1991.- Vol.77.- P. 121- 124.

11. Appl. 1-132392 JP, Int.Cl.⁴ C12 P 7/40, C12 Rl:05.Microbiologocal production of monocarboxylic acid / Kawakami K., Tanabe T., frioue Sh.- 16 pp. 12. Russian pat. 2081169 C 1 RU, Int.Cl.⁶ C12 N 9/78, C12 P7/40, 13/02, C12 R 1:05. Bacterial strain Alcaligenes species producing nitrilase / Zabaznaya EN., Kozulin SN., Kulikova L.K., Voronin S.P. - 10 pp.

Table 1

Table 3

Claims

Claim

Strain Alcaligenes denitrificans C-32 VKM B-2243 D producing nitrilase.