BG65589B1 - Method for the decomposition of nitrile compounds - Google Patents

Abstract

The method for the decomposition of nitrile compounds of thermostable bacterial nitrilase is used for biological detoxication of nitrile-containing wastes and in the chemical synthesis. The advantages are found in the decomposition of a large number of nitrile compounds and in the high degree of the enzyme half-life after treating the cells at high temperatures. By the method, hydrolysis of the nitrile compounds takes pace in the presence of intact bacterial cells or degraded bacterial cells, or immobilized bacterial cells, containing the nitrilase enzyme. The pure culture cells of thermophilic bacterial strain Basillus sp. UG-5B. registered in NBIMCC under No. 8021. The hydrolysis occurs at temperatures from 40 to 70 degrees C for from 5 to 15 min.

Description

Technical field

The invention relates to a method for the degradation of nitrile compounds with thermostable bacterial nitrilase for use in biological detoxification of nitrile-containing wastes and in chemical synthesis.

BACKGROUND OF THE INVENTION

Methods for the degradation of nitrile compounds using enzymes are known. Nitrilase activity is more prevalent in mesophilic bacteria.

Bacterial nanitrylase producers are isolated from soil samples, from industrially contaminated soils and waters, coastal marine sediments, activated sludge.

Bacterial nitrile hydrolysis is known to occur via two enzymatic pathways. One involves hydrolysis of the nitrile molecule to carboxylic acid and ammonia via an intermediate amide with the participation of two enzymes - nitrile hydratase and amidase (a) and the other by direct hydrolysis to carboxylic acid and ammonia (b), (1).

R-CN + H ₂ O nitrile hydratase R-CONH ₂ amidase + H ₂ O

R-CN + 2H ₂ O nitrilase R -COOH + NH ₃ / b /

RCOOH + NH ₃ / a /

The disadvantages of the known methods for the degradation of nitrile compounds using mesophilic bacteria are that they are highly thermolabile.

Also known are thermophilic nigril degrading enzyme systems in temperate thermophiles: Bacillus palidus Dac 521/2 /, Bacillus RAP c8 / 3 /, and a new thermophilic actinomycetes Pseudonocardia thermophila, as well as Bacillus smithii strain SC-JO51 / 4 /. The disadvantages of these thermophilic enzyme systems are their insufficient thermal stability and the longer time to achieve optimal hydrolysis activity.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for the degradation of nitrile compounds by cell suspension, cell-free extract or immobilized cells of a thermophilic bacterial strain producing thermostable nitrilase capable of degrading a wide range of nitrile compounds.

The problem is solved by using in the process according to the invention a newly isolated thermophilic bacterial wastewater strain obtained by cultivation of various selective nutrient media containing nitrile compounds. The strain is Basillus sp. UG-5B, registered with NBPMCC under No. 8021.

The strain is a gram-positive spore-forming rod with a centrally located spore. The morphological, cultural, physiological and biochemical properties of the strain are presented in Tables 1 and 2.

Obtaining a pure culture from the strain is carried out by culturing it on a Bioflo fermenter with a working volume of 1.01 at 50 ° C. 400 rpm and aeration - 0.6 v / v / m. Under these cultivation conditions, the maximum nitrilase activity is reached at the 7th hour of cultivation.

Cultivation was performed using a culture medium containing in g / 1: peptone (Oxoid) 5.0; yeast extract (Difko) 5.0; NaCl 1.0; K ₂ NRA ₄ 5.0; MgSO ₄ 0.1; FeSO 4 O 0.03 and benzonitrile 20 mM, pH 7.5.

According to the method of the present invention, a pure culture of intact bacterial cells, degradation bacterial cells containing the enzyme or immobilized enzyme preparations containing cells of the thermophilic bacterial strain are capable of hydrolyzing the following compounds: propionitrile, valeronitrile, 4-chlorobutyronitrile, cyclohexyltril, cyclohectyl

65589 B1 isocyanide, adiponitrile, acrylonitrile, allyl cyanide, crotonitrile, fumarodinitrile, benzonitrile, 4-chlorobenzonitrile, 4-nitrobenzonitrile, tolunitrile, cyanopyridine and 4-cyanopyridine to the corresponding acid.

The advantages of the process according to the invention are the degradation of a large number of nitrile compounds over a period of 5 to 15 minutes at temperatures of 40 to 70 ° C.

This fact is further emphasized by the long half-life of the enzyme (5 min) after treatment of the cells at high temperature (70 ° C).

Examples of carrying out the invention

Example 1.

Pure culture was obtained from the strain by culturing it on a Bioflo fermenter with a working volume of 1.01 at 50 ° C, 400 rpm and aeration 0.6 v / v / m. The culture fluid was centrifuged at 4000 g for 10 min. The precipitated bacterial cells were washed with buffer (pH 7.2) and resuspended in the same buffer to the appropriate protein concentration (2 mg / ml). To 600 µl of the resulting bacterial suspension was added 10 µm (1.2 M benzonitrile (final concentration 20mM) and the reaction was carried out for 15 min at 45 ° C. As a result of hydrolysis, 12 mM ammonia is obtained as a final product, respectively 12 mM benzoic acid.

Example 2.

Pre-treat the cell suspension for 5 min at 70 ° C. The suspension thus obtained is then hydrolyzed according to Example 1. 6 mM ammonia is obtained as a final product, which means a 5 min 5 half-life of nitrilase from the bacterial cells tested.

Claims (1)

Claims A method for the degradation of nitrile compounds by hydrolysis in the presence of intact bacterial cells or degraded bacterial cells, or immobilized bacterial cells containing the nitrilase enzyme, characterized in that the cells are from the pure culture of the thermophilic bacterial strain Bacillus sp. UG-5B, registered in NBPMCC under No. 802 of 9/11/2001, the hydrolysis being carried out at a temperature of 40 to 70 ° C for a time of 5 to 15 min.