RU2160778C1 - Bacterial strain bacillus cereus producing nitrilehydratase - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: bacterial strain produces nitrilehydratase on simple organomineral medium. Activity of the latter with regard to acetonitrile is 220 un. and to acrylonitrile 200 un. Nitrilehydratase is thermally stable and may be used for biotechnological synthesis of acrylamide from acrylonitrile in aqueous solutions. Strain is characterized by elevated activity, satisfactory heat resistance and homogeneity of culture. EFFECT: intensified biotechnological production of acrylamide. 5 tbl, 5 ex

Description

 The invention relates to biotechnology and the microbiological industry and relates to the production of a new strain of bacteria having high nitrile hydratase activity, which can be used to produce important products, in particular acrylamide from acrylonitrile. Nitrile hydratase is an enzyme that catalyzes the process of hydrolysis of nitriles of carboxylic acids to the corresponding amides.

 Microorganisms are known that produce the nitrile hydratase enzyme and belong to the genera Pseudomonas, Rhodococcus, Brevibacterium, Agrobacterium. In particular, strains capable of transforming acrylonitrile into acrylamide are known: Rhodococcus rhodochrous J-1 [1], Rhodococcus sp. N771 [2], Rhodococcus sp. N774 [3], Pseudomonas chlororophis B23 [4], Brevibacterium R312 [5], Agrobacterium radiobacter SC-C15-1 [7].

 The closest analogue to the present invention is a strain of Rhodococcus rhodochrous M8 [6], which is used for the synthesis of concentrated aqueous solutions of acrylamide from acrylonitrile [8,9].

 The disadvantage of Rhodococcus bacteria is relatively slow growth and the associated difficulties in culturing and maintaining the homogeneity of the culture during the industrial production of the strain, as well as low activity.

 The objective of the invention is a fast-growing, easily cultivated strain with high activity and satisfactory thermal stability of nitrile hydratase. To solve this problem, a bacterial strain Bacillus cereus B5, a nitrile hydratase producer, is proposed.

The inventive bacterial strain on the basis of a taxonomic study is assigned to the species Bacillus cereus. This is the first known strain of the species Bacillus cereus with the ability to convert nitriles to amides. This strain has an inducible nitrile hydratase that hydrolyzes aliphatic nitriles to amides. Induction of nitrile hydratase is achieved by growing Bacillus cereus B 5 cells on a mineral medium with cobalt ions containing nitriles and amides of organic acids, for example, acetonitrile or acetamide, as an inducer, and glucose as a carbon source. All components of the medium are commercially available compounds, which is especially important in the case of industrial use of the strain. An important technological advantage of using the strain is the high growth rate and temperature stability of the enzyme. The maximum activity of the enzyme is observed at 55 ^o C.

 This microorganism is characterized by rapid growth on a simple organomineral medium with high nitrile hydratase activity and the simplicity of maintaining culture homogeneity.

 The strain Bacillus cereus B5 can be used in the biotechnological process of producing amides, in particular acrylamide.

 The strain is characterized by the following cultural, morphological and physiological and biochemical characteristics.

 Cultural properties: when grown on meat and peptone agar, the strain exhibits mushroom growth, forms colorless flat colonies with a rhizoid edge, and the surface is dull. With growth on meat and peptone broth forms a film and sediment.

 Morphological signs. Gram-positive cells, motile, optionally anaerobic, rod-shaped, sizes 0.9-1.2 x 2.0-5.0 microns, form heat-resistant endospores.

Physiological properties: the strain reduces nitrates. Test with methyl red, the Voges-Proskauer reaction is positive. The oxidase-catalase-positive strain, urease-negative. It grows at pH 6-9, the optimum pH is 7.2, at a temperature of 0-45 ^o C, the optimum temperature is 34 ^o C. The strain gives an acid reaction when grown on glucose, fructose, does not form acid from arabinose and mannitol. Hydrolyzes starch and casein. As sources of nitrogen, it uses ammonium compounds and nitrates.

 Sensitivity to antibacterial drugs: the strain is sensitive to chlorafenicol, benzylpenicillin, ampicillin, kanamycin, monomycin, tetracycline.

 Pathogenicity: the strain is not pathogenic.

 The invention describes this microorganism for use in the hydration process of various nitrile compounds and to obtain the corresponding amido compounds, including any mutant thereof.

 The culture of the bacterial strain used in this invention can be obtained on various types of media, including various sources of carbon and nitrogen, organic and inorganic salts, which are widely used to obtain a culture of ordinary bacteria.

 Enzymatic activity was determined using nitriles of carboxylic acids as substrates. The unit of specific nitrile hydratase activity was taken as the amount of nitrile hydratase contained in 1 mg of dry cell weight, catalyzing the formation of 1 μmol amide per minute (μM amide / mg dry wt. • min). The invention is illustrated by the following examples.

 Example 1. Isolation of a strain of Bacillus cereus B5.

 The inventive strain was isolated from the soil. 1.0 g of soil was introduced into 100 ml of medium of the following composition (table. 1).

Cultivated for 3 days at a temperature of 30 ^o C and aeration. 2 ml of the resulting suspension was added to 100 ml of the same medium.

After two days, 1 ml of culture fluid was taken, serially diluted in physiological saline and plated on agar medium (1.5% agar-agar) with the same components. After incubation for 24-48 hours at 28-30 ^° C, individual colonies were selected and their ability to transform acrylonitrile into acrylamide was studied. As a result, a strain of Bacillus cereus B5 with nitrile hydratase activity was isolated.

 Example 2. The use of a strain of Bacillus cereus b 5 for the transformation of acetonitrile into acetamide.

 Cells were cultured in the following medium, pH 7.2 (Table 2).

 Samples were periodically taken from the reaction medium to determine the concentration of cells and their nitrile hydratase activity. The cell concentration was determined photometrically at a wavelength of 540 nm.

 Nitrile hydratase activity was evaluated as follows.

1 ml of culture fluid was centrifuged, the cells were washed with 0.01 M phosphate buffer, pH 7.2. Cells were resuspended in the same buffer to an optical density of 0.2-0.5 (A 540 nm). Acetonitrile was added to 2 ml of the cell suspension in an amount of 25 μl. The reaction was carried out at 20 ^° C. for 10 minutes and then stopped by the addition of 0.1 ml of 1 n HCl. Bacterial cells were separated by centrifugation. The concentration of acrylamide in the supernatant was analyzed by gas-liquid chromatography.

 The activity of nitrile hydratase in relation to acetonitrile reached 220 units.

 Example 3. The use of a strain of Bacillus cereus B5 for the transformation of acrylonitrile into acrylamide.

 The strain Bacillus cereus B5 was prepared for the transformation process according to example 2. The activity of nitrile hydratase was evaluated analogously to example 2. Acrylonitrile was added as a substrate in an amount of 25 μl.

 The activity of nitrile hydratase in relation to acrylonitrile reached 200 units.

 The measurement of activity depending on the time of cell growth is presented in table 3. It is obvious that the strain of Bacillus cereus b 5 has a higher growth rate and activity than the prototype.

 Example 4. Purification of a culture of Bacillus cereus B5 from contamination.

 The resulting strain of Bacillus cereus B 5 was cultured in Erlenmeyer flasks with a volume of 500 ml, containing 150 ml of the medium of the composition shown in table. 4.

At OD _{540 of} suspension 0.1, the sterility of the medium was deliberately violated by the addition of 0.5 g of soil. They were cultured for 24 hours on a rotary shaker (120 rpm) at 30 ^° C. Then, the culture flask was heated at 80 ^° C for 15 minutes. After this, cultivation was continued under the same conditions for 24 hours.

 Samples of the culture thus obtained were dispersed to separate colonies on an agar medium of a similar composition and on meat peptone agar. In all cases, the culture was homogeneous.

 Example 5. The effect of temperature on the activity of nitrile hydratase strain Bacillus cereus B5.

 Bacillus cereus B5 bacteria were grown for 24 hours and prepared for transformation according to Example 2. Samples with a reaction mixture containing 25 μl of acrylonitrile and 0.04 mg of cells by dry weight in 2 ml of 0.025 M phosphate buffer, pH 7.6, were incubated in 5 minutes at various temperatures. The concentration of acrylamide formed in the reaction mixture was determined using gas-liquid chromatography. The change in the activity of nitrile hydratase depending on the temperature of the transformation reaction for the inventive strain is presented in table. 5.

 Thus, the nitrile hydratase from the inventive strain has satisfactory thermal stability.

 The inventive strain can be used for biotechnological synthesis of acrylamide by hydration of acrylonitrile in aqueous solutions.

List of references
1. Brennan, BA, Alms, G., Nelson, MJ, Dumey, LT and Scarrow, RC (1996b) Nitrile hydratase from Rhodococcus rhodochrous J1 contains a non-corrin cobalt ion with two sulfur ligands. J. Am. Chem. Soc. 118, 9194-9195.

 2. Nagamune, T., Honda, J., Cho, WD, Kamiya, N., Teratani, Y., Hirata, A., Sasabe, H. and Endo, I. (1991) Cristallization of a photosensitive nitrile hydratase from Rhodococcus sp. N-771. J. Mol. Biol. 220, 221-222.

 3. Korf, M.-A.., Honda, J., Teratani, Y., Kobayashi. Y., Nagamune, T., Sasabe, H., Hirata, A., Ambe, F. and Endo, I. (1992) Light-induced oxidation of iron atoms in a photosensitive nitrile hydratase. FEBS Lett. 301, 177-180.

4. Nagasava, T., Nanba, H., Ryuno, K. and Yamada, H. (1987) Nitrile hydratase of Pseudomonas chlororaphis B23. Purification and characterization. Eur. J. Biochem. 162, 691-698
5. Bonnet, D., Artaud, I., Petre, D. and Mansuy, D. (1996) Key role of alkanoic acids on the spectral properties, activity, and active-site stability of iron-containing nitrile hydratase from Brevibacterium R312 . Eur. J. Biochem. 240, 239-244.

 6. Patent N 1731814 SU.

 7. Patent N 05395758 US.

 8. Patent N 1811698 SU.

 9. Patent N 2112804 RU.

Claims (1)

 The bacterial strain Bacillus cereus is a producer of nitrile hydratase.

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