RU2352635C2 - Method of producing immobilised biocatalyst and method of producing aqueous amide solutions with using this biocatalyst - Google Patents

Abstract

FIELD: chemistry; biotechnologies.

SUBSTANCE: method of producing immobilised biocatalyst includes trapping of Rhodococcus type microorganism cells of nitrile-hydrase activity on carbon carriers by delivering cellular suspension through the column, filled with the carrier, or by joint centrifugation of cells and carriers. Herewith carbon carriers are active carbons made of vegetative and polymer raw materials with porous structure parameters, including total pore space 0.4-1.8 cm³/g and micropore volume concentration 45-92%. Produced biocatalyst is used in method to produce aqueous amide solutions by carboxylic acid nitrile hydration.

EFFECT: high amide yield and increased time for amide synthesis biocatalyst application.

4 cl, 2 tbl, 5 ex

Description

The invention relates to biotechnology and relates to a method for producing an immobilized biocatalyst based on cells of microorganisms with nitrile hydratase activity, as well as a method for producing concentrated aqueous solutions of amides, in particular acrylamide and nicotinamide, by hydration of nitriles of carboxylic acids using an immobilized biocatalyst.

Amide compounds are currently used in all industries. In particular, acrylamide is widely used as a monomer for the synthesis of polymers and copolymers for various purposes. The most promising for obtaining aqueous solutions of amides, in particular acrylamide, are biotechnological methods for hydration of nitriles of carboxylic acids using microorganisms having nitrile hydratase activity as a biocatalyst. Biotechnological processes for producing amides, in contrast to the chemical method, have several important advantages: nitrile hydration takes place at normal temperatures and pressures, is characterized by a high yield of the target product (99.9%), low energy consumption and low waste production.

Known strains of microorganisms producing the nitrile hydratase enzyme that convert nitriles to the corresponding amides: Rhodococcus erythropolis E84 (patent RU No. 2196822, priority from 07.25.2001), Rhodococcus ruber GT (patent RU No. 2223316, priority from 06.12.2001), etc.

Known methods for producing aqueous solutions of acrylamide and other amides in capacitive type reactors using a suspension of microorganisms (bacteria) with nitrile hydratase activity [1-3]. The disadvantages of these methods is that the biocatalyst is introduced in the form of a suspension, used once, after the synthesis, laborious procedures for separating the biocatalyst sludge are required. There is also a method for producing aqueous solutions of amides using bacterial cells with nitrile hydratase activity, immobilized by incorporation of polyacrylamide into the gel [4, 5]. The disadvantages of these methods is the reduction of the catalytic activity of bacteria included in the gel and the rate of amide synthesis, due to:

- the inevitable damage (covalent modification) of the nitrile hydratase enzyme molecules and cells during the polymerization of polyacrylamide;

- a significant decrease in the mass transfer rate of the substrate (nitrile) to bacterial cells containing nitrile hydratase and the outflow of the product (amide) from them due to diffusion restrictions in the granules of the polyacrylamide gel;

- the possibility of swelling and erosion of the polyacrylamide gel.

The closest in technical essence to the proposed solution analogue is a method of industrial production of concentrated solutions of acrylamide [3], adopted as a prototype. The method is carried out by hydration of acrylonitrile in an aqueous solution using a strain of microorganisms Rhodococcus rhodochrous M33, used in the form of a suspension at a concentration of 0.64-4.1 g / l of tap or distilled water at a temperature of 13-22 ° C and a pH of 6.8-7 , 8 for 4-6 hours with the introduction of acrylonitrile to a concentration not exceeding 0.1%. The disadvantages of this method are the single use and high consumption of the biocatalyst, the presence in the resulting solution of a suspension of slurry of the biocatalyst after synthesis and the need for purification from it, limiting the concentration of the substrate - acrylonitrile (up to 0.1%).

The technical task of the present invention is to increase the yield of the final product in the process of amide synthesis, carried out by hydration of nitriles, and the ability to carry out synthesis both in batch and in flow mode in reactors of known designs. The task is carried out by immobilizing the catalytically active biomass of bacteria, the ease of separation of the bulk of the biocatalyst from a solution of synthesized amide, as well as the multi-cycle operation of the biocatalyst or running in flow mode.

The solution to this problem is achieved by the fact that bacteria with nitrile hydratase activity are immobilized by adsorption from a cell suspension on a porous carbon carrier. Active carbons with parameters of the porous structure — with a total pore volume of 0.4-1.8 cm ³ / g — are used as a carrier.

To obtain a biocatalyst, microorganism strains having nitrile hydratase activity are used, for example, Rhodococcus ruber GT (Patent RU No. 2223316), Rhodococcus erythropolis E84 (Patent RU No. 2196822).

The immobilization of bacteria - producers of nitrile hydratase is carried out by adsorption on carbon carriers (sorbents), in particular active carbons from plant and polymer raw materials, which allows to obtain a biocatalyst for the synthesis of amide compounds in batch reactors by multi-cycle use, and in continuous mode in column reactors or capacitive type. After each synthesis cycle in capacitive reactors, the biocatalyst is easily separated from the liquid phase - the amide solution by draining the liquid phase, filtering or low-speed centrifugal separation (centrifugation or separation at an angular speed of up to 3000 rpm). The production of amides by the proposed method can be carried out in flow reactors of a column, membrane, hollow fiber type or any other known construction, without the need for a filtration or separation step. The resulting amide solution at the outlet of the reactor does not contain an immobilized biocatalyst.

Active carbons quite firmly bind Rhodococcus cells, which prevents their desorption during synthesis and exit into the solution containing the reaction product. These sorbents have a protective and stabilizing effect on the biocatalyst, expressed in a decrease in nitrile suppression of the nitrile hydratase activity of Rhodococcus cells and an increase in the thermal stability of the biocatalyst, which allows you to add a substrate (acrylonitrile or other nitrile) to a concentration of 20% and carry out up to 7-10 repeated synthesis cycles. All these advantages allow us to solve the problem, namely, to increase the yield of the final product in the synthesis of amides.

An advantage is also the fact that the carbon carriers used are manufactured on an industrial scale and have a low cost.

Rhodococcus cells (R.ruber GT or R.erythropolis E84) are grown on a medium of the following composition (g / l): glucose - 1-5; NH ₄ Cl (or urea) - 0.3-1; KH ₂ PO ₄ - 1.0; K ₂ HPO ₄ × 3H ₂ O - 1.6; NaCl - 0.5; MgSO ₄ × 7H ₂ O - 0.5; FeSO ₄ × 7H ₂ O — 0.002; CoCl ₂ × 6H ₂ O — 0.01; pH 7.4 for 30-45 hours until a suspension density of 4-10 g / 100 ml is reached. The biomass is separated from the medium by centrifugation, centrifugal separation, filtration or any other known method and suspended in distilled or tap water or in a buffer solution to a concentration of 1-30 g / L.

Bacterial immobilization is carried out by incubation of carriers (active carbons) with suspended Rhodococcus cells for 15-35 min, or by passing (pumping) the suspension at a linear velocity of up to 0.03 m / s through the volume of the reactor filled with sorbent (active carbon), or by co-centrifuging the cell suspension and sorbent. After that, the biocatalyst is washed with water or a buffer solution. As carriers for immobilization, for example, active carbons are used: raw birch coal; BAU; SKT-3; FTD (coal properties are given in table 1).

The amide production process is carried out in an aqueous solution at a pH of 6.5-8.3 in a batch mode - with a ratio of the amount of biomass immobilized on a carrier to the total volume of the reaction medium 0.1-10 g / l (dry weight) or in flow mode. Acrylonitrile (or other aliphatic nitrile) is introduced into the medium to a concentration not exceeding 20%. The reaction time is 1-5 hours. At the end of the synthesis, a solution of acrylamide or another aliphatic amide with a concentration of 7-55% is obtained.

The enzymatic activity of bacterial biomass is determined by the change in the concentration of substrates - nitriles of carboxylic acids and reaction products - amides. The unit of specific nitrile hydratase activity is the amount of nitrile hydratase contained in 1 mg of dry cell weight, catalyzing the formation of 1 μmol of amide per minute (μmol of amide / mg dry weight / min). The concentration of acrylamide is measured spectrophotometrically by the value of optical density (OD ₂₄₀ ) or by gas-liquid chromatography.

The method is illustrated by the following examples:

Example 1. Obtaining bacterial cells immobilized on carbon carriers.

Rhodococcus cells (for example, R.ruber GT or R.erythropolis E84) are grown on a synthetic medium of the composition (g / l): glucose - 1-5; NH ₄ Cl (or urea) - 0.3-1; KH ₂ PO ₄ - 1.0; K ₂ HPO ₄ × 3H ₂ O - 1.6; NaCl - 0.5; MgSO ₄ × 7H ₂ O - 0.5; FeSO ₄ × 7H ₂ O — 0.002; CoCl ₂ × 6H ₂ O — 0.01; pH 7.4 for 30-45 hours until a suspension density of 4-10 g / 100 ml and nitrile hydratase activity of at least 100 units are reached.

The grown cells are separated from the culture medium by centrifugation at 8000 rpm for 10 minutes. The resulting biomass is diluted in 10 mm sodium phosphate buffer, pH 6.5-8.3, distilled or tap water to a concentration of 1-30 g / L.

The immobilization of bacteria is carried out in one of 2 ways:

(1) A flow reactor with a ratio of height and diameter of 4: 1-10: 1, the outlet opening of which is closed with filtering material with a filtering depth of 10-200 μm, is filled with a carbon sorbent in a bulk state for 4/5 of the volume, and a suspension is passed through it Rhodococcus cells in a uniform flow with a linear velocity inside the reactor up to 0.03 m / s for 10-25 minutes After that, the liquid phase is drained and the biocatalyst is washed with distilled, softened or tap water with a linear velocity inside the reactor up to 0.03 m / s for 10-25 minutes. The biocatalyst obtained is discharged from the reactor, or the flow reactor filled with it can be directly used for the synthesis of amides.

(2) A suspension of Rhodococcus cells is centrifuged 5-7 times with sorbents at 3000-8500 rpm for 10-20 minutes, sequentially separating the supernatant and adding a suspension of cells. The biomass with sorbents is resuspended in distilled or tap water or in 10 mM sodium phosphate buffer, pH 6.5-8.3, with a sediment to liquid ratio of 1: 2, incubated for 1-35 min on a rotating platform (rocking chair) with mixing speed up to 100 rpm The carrier with immobilized cells is separated from the aqueous medium by filtration through a paper, nylon, asbestos or ceramic filter or through a nylon or metal mesh and washed with an aqueous phase.

The number of sorbed cells is determined by the difference in optical density (OD ₅₄₀ ) of the culture medium before and after sorption. Sorption capacity in relation to cells is expressed in mg of dry cell weight per g of sorbent.

Under these conditions, from 10 to 16 mg of cells (by dry weight) / g of carrier are firmly adsorbed (Table 1). When immobilized by co-centrifugation, up to 78 mg dry are firmly adsorbed. weight C. / g carrier. The sorbent with immobilized cells is separated from the culture medium by filtration through a paper, nylon, asbestos or ceramic filter or through a nylon or metal mesh.

To determine the thermal stability of the biocatalyst, immobilized cells and cells in suspension are incubated at temperatures of 50, 60, and 70 ° C for 10, 30, 60, and 180 minutes, after which nitrile hydratase activity is measured. The nitrile hydratase activity of the immobilized cells of the R.ruber GT strain in comparison with the activity of cells in suspension is presented in Table 2.

Example 2. Transformation of acrylonitrile into acrylamide by immobilized Rhodococcus cells.

The transformation of acrylonitrile using immobilized Rhodococcus biomass is carried out in an aqueous medium (distilled or tap water) or potassium phosphate buffer, pH 6.5-8.3. Acrylonitrile contribute to a concentration of 0.5-20%. With complete transformation into the amide, a new portion of acrylonitrile is added.

The activity of cell nitrile hydratase after immobilization is evaluated as follows: cells in an amount of 0.5-4.5 mg by dry biomass immobilized on a carbon support in Example 1 are incubated in 10 ml of 10 mM sodium phosphate buffer or water, pH 6.5- 8.3, with acrylonitrile in a concentration of up to 20% for 1-10 minutes, and then the reaction is stopped by adding 0.5 ml of 6 N. HCl. The carrier with cells is separated from the reaction medium by filtration through a paper filter, the filtered solution is centrifuged for 5 minutes at 12,000 rpm.

The concentration of acrylamide in the supernatant is analyzed by gas-liquid chromatography. The preservation of nitrile hydratase activity in immobilized cells is evaluated by comparison with the nitrile hydratase activity of cells in suspension.

The result is a solution of acrylamide with a concentration of 40-55%.

Example 3. The use of immobilized Rhodococcus cells for the transformation of 3-cyanopyridine into nicotinamide or other nitriles into the corresponding amides.

Rhodococcus bacteria with high nitrile hydratase activity are immobilized and prepared for transformation according to Example 1. Transformation of nicotinamide using immobilized Rhodococcus biomass is carried out in an aqueous medium (distilled or softened water) with a ratio of the volume of immobilized biocatalyst and the aqueous phase 1: 7-1: 20. The mixing or flow rate is set so that the linear velocity of the fluid does not exceed 0.03 m / s. As a substrate, 3-cyanopyridine or another nitrile is added to a concentration of 10 g / L. The concentrations of residual 3-cyanopyridine and nicotinamide are determined by high pressure liquid chromatography (HPLC). As 3-cyanopyridine is completely converted to nicotinamide, new portions of the substrate (or other nitrile to the corresponding amide) are added.

The result is a solution of nicotinamide in a concentration of 20-23% or a solution of another amide in the copepods of at least 20%.

Example 4. The multi-cycle use of immobilized cells of the strain Rhodococcus in the conversion of acrylonitrile to acrylamide.

Each cycle of the conversion of acrylonitrile to acrylamide is carried out in reactors with a volume of from 0.001 to 10 m ³ . The reaction is catalyzed by cells immobilized on carbon carriers with an activity of 100-460 μmol / mg / min, taken in an amount of 0.1-2.5 g / l, calculated on dry weight. The preservation of nitrile hydratase activity during sequential complete conversion of acrylonitrile introduced in each cycle to a concentration of 10% in distilled or tap water or in a potassium phosphate buffer pH 6.5-8.3 is evaluated. Samples for determining the activity are taken after 10 minutes from the start of the reaction. A complete conversion cycle is carried out for 20 minutes, after which the sorbents with immobilized cells are washed with water. The operational stability of immobilized cells is compared with the operational stability of cells in suspension, ceteris paribus.

Under these conditions, immobilized cells do not lose nitrile hydratase activity after 6-7 cycles of synthesis, while bacteria in suspension by the second cycle retain only half the activity. The results are presented in table.3. The shown stabilization of activity during immobilization on a carbon carrier allows the biocatalyst to be used repeatedly.

Under similar conditions, maintaining the concentration of acrylonitrile in the reactor of 0.5-10%, concentrated solutions of acrylamide (40-55%) are obtained. The yield of acrylamide is 1600-5400 g of acrylamide per 1 g of cells (by dry weight). At the same time, in the synthesis of acrylamide catalyzed by a suspension of Rhodococcus cells, the yield of acrylamide is 180-1640 g of acrylamide per 1 g of cells (by dry weight).

Example 5. The synthesis of amide in flow mode.

Flow reactor, which is a hermetically sealed collapsible stainless steel cylinder with a diameter to height ratio of 1 / 4-1 / 12 and a height of at least 0.5 m, equipped with nozzles for solution inlet and outlet and filters in the form of a nylon or metal mesh located at the ends, or a reactor of another known design is filled with an immobilized biocatalyst prepared as in example 1. Acrylonitrile solution with a concentration of not more than 20% and a linear flow rate inside the reactor is pumped through the reactor 0 , 002-0.005 m / s. Under such conditions, the output is a solution of acrylamide with a concentration of up to 20%. The following approaches are used to synthesize concentrated solutions of acrylamide: (1) the use of reactors longer than 1.2 m; (2) cascading 3-6 reactors with the addition of acrylonitrile to the reaction medium before each subsequent reactor; (3) cyclic multiple passing of a solution of acrylonitrile through the reactor with the addition of new portions of acrylonitrile as it is transformed, or a combination of these approaches. With cyclic repeated pumping of the solution, the flow velocity can be increased, but not more than up to 0.04 m / s. Under these conditions, for 0.3-5 hours of transformation, a solution of acrylamide with a concentration of 38-55% is obtained. The yield of acrylamide in the preparation of concentrated solutions is more than 2200 g of acrylamide per 1 g of cells (by dry weight).

Thus, the claimed method provides the following advantages over the known methods for producing amides: the yield of the target product is increased due to the stabilization of the catalytic activity of cells immobilized on carbon carriers, the synthesis of amide compounds can be carried out both in a batch mode and in a flow mode in all known types of reactors, the biocatalyst is easily separated from the synthesis product and used repeatedly, which solves the problem of separation of sludge and increases the purity of the product.

Table 1
Characterization of carbon carriers, the number of Rhodococcus cells immobilized on them, and acrylamide adsorption Property Carbon carrier Raw Birch Coal BAU SKT-3 FTD Feedstock birch wood birch charcoal peat phenol formaldehyde resin Bulk density, g / dm ³ 280 ± 5 220 ± 20 530 ± 29 480 ± 20 The total volume of macropores, cm ³ / g 0.95-1.15 1.35-1.8 0.38-0.43 0.08-0.1 Volume fraction of macropores,% 91-92 85-86 47-51 (45-80) 7-12 Sorption capacity in relation to cells, mg / g 13.8 ± 2.3 14.6 ± 1.4 13.2 ± 1.7 12.4 ± 2.3 Sorption capacity in relation to acrylonitrile, mg / g 187.3 ± 17.3 350.0 ± 6.5 273.5 ± 1.5 217.4 ± 1.1 Sorption capacity of the sorbent with cells in relation to acrylamide, mg / g 0.1 ± 0.1 6.6 ± 2.9 7.5 ± 0.8 9.5 ± 0.9

table 2
Preservation of nitrile hydratase activity of cells of the strain R.ruber GT when exposed to elevated temperature Temperature,
° C Exposure time, min Nitrile hydratase activity,% R.ruber GT cell suspension Immobilized R.ruber GT cells fifty 10 182 183 thirty 124 160 60 one hundred 120 180 36 59 60 10 29th 56 thirty 9 25 60 four fourteen 180 0.8 3 70 10 2 fourteen thirty 2 fourteen 60 2 fourteen 180 - - 100% - cell nitrile hydratase activity at a temperature of 20 ° C

Table 3
Comparison of the stability of a biocatalyst - immobilized cells of the strain R.ruber GT and cells in suspension with multi-cycle use Conversion Cycles Preservation of nitrile hydratase activity,% R.ruber GT cell suspension Immobilized R.ruber GT cells one one hundred one hundred 2 53 one hundred 3 39 one hundred four eighteen one hundred 5 13 one hundred 6 6 one hundred 7 3 71 8 0 53 9 0 17 10 0 12

Sources used

1. Patent US 4343900. Process for the production of acrylamide using microorganism.

2. Patent of chipboard SU 1811698. Debabov V.G., Voronin S.P., Kozulin S.V., Sinolitsky M.K., Moiseeva T.N., Polyansky A.B., Yanenko A.S., Khorkin A. .A., Bayburdov T.A., Luiksaar I.V., Reshetnikov L.V., Gerasimova T.V. Biotechnological method for producing acrylamide.

3. Patent RU 2077588. Debabov V. G., Voronin S. P., Kozulin S. V., Sinolitsky M. K., Kozulina T. N., Polyansky A. B., Sintin A. A., Yanenko A. .S., Bayburdov T.A., Khorkin A.A., Luiksaar I.V., Reshetnikova L.V., Fedchenko N.N. The method of producing acrylamide.

4. Patent US 6043061. Process for producing amide compound.

5. Patent RU 2196825. Biotechnological method for producing aqueous solutions of acrylamide.

Claims (4)

1. A method of producing an immobilized biocatalyst based on Rhodococcus actinobacteria cells having nitrile hydratase activity, characterized in that the cells are immobilized on carbon carriers by passing a cell suspension through a column filled with a carrier, or by joint centrifugation of cells and carriers, while as carbon carriers use activated carbons of plant and polymeric materials with a porous structure parameters - a total pore volume of 0.4-1.8 cm ³ / g and a volumetric fraction of macro Op 45-92%. 2. A method of obtaining aqueous solutions of amides, including acrylamide and nicotinamide, by hydration of nitriles of carboxylic acids using a biocatalyst, characterized in that they use the immobilized biocatalyst obtained according to claim 1. 3. The method according to claim 2, characterized in that, in order to obtain an aqueous solution of acrylamide with a concentration of up to 55%, acrylonitrile is used as a substrate, with a concentration of the latter up to 20%. 4. The method according to claim 2, characterized in that, in order to obtain an aqueous solution of nicotinamide with a concentration of up to 23%, 3-cyanopyridine is used as a substrate.