RU2460790C2 - Method for immobilisation of l-phenylalanine-ammonium-lyase on magnetic nanoparticles - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a method for immobilisation of L-phenylalanine-ammonium-lyase on magnetic nanoparticles of metal oxides in the presence of the condensing agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

EFFECT: method enables higher specific activity of L-phenylalanine-ammonium-lyase with maintained activity.

6 tbl, 3 ex

Description

The invention relates to biotechnology, and in particular to a method for immobilizing the enzyme L-phenylalanine-ammonium lyase, which biotransforms L-phenylalanine to ammonia and trans-cinnamic acid, on surface-modified magnetic nanoparticles. The invention can be used as therapeutic compositions, as well as to create specialized food products for patients with phenylketonuria.

Immobilized enzymes have a number of obvious advantages over native ones: ease of separation of a heterogeneous biocatalyst from the reaction medium; the continuity of the enzymatic process with the ability to control the rate of the catalyzed reaction and product yield; directed change in the properties of the enzyme (specificity, dependence of catalytic activity on pH and other environmental parameters, stability to denaturing effects); the ability to regulate the catalytic activity of immobilized enzymes by changing the properties of the carrier.

Many technical solutions have been proposed in this area, among which the most widely used are chemical methods for immobilizing enzyme preparations, the essence of which is the covalent binding of biomolecules with an inert carrier modified with reactive functional groups (amino, azido, carboxyl, hydroxyl, etc.) . However, for the most part, these methods of immobilization have several disadvantages, namely the use of expensive materials and reagents for immobilization, the complexity and multi-stage methods.

A known method of immobilization (see US Pat. No. WO 2009120916 (A1), IPC C08K 3/08, claimed March 28, 2008, published January 10, 2009) of bioobjects on polycarbonate nanocomposites, which are metal nanoparticles coated with a polycarbonate layer by polymerization in a reaction mixtures. In this case, polycarbonate nanocomposites are prepared from a mixture of dihydroxy compounds, activated carbonate, a metal precursor and a solvent. Then the reaction mixture is polymerized to form a polycarbonate nanocomposite, which is a polycarbonate matrix with distributed metal particles. In this case, oxides of metals such as gold, platinum, palladium, cobalt, iron, nickel, and magnesium are used as precursors. This method of immobilization is characterized by such disadvantages as the use of expensive metals and the low specific activity of the preparations obtained.

The invention is also known (see Pat. IT No. WO 2009040843 (A2), IPC A61K 47/48, application 04.08.2008, published 02.04.2009), which describes a method for producing inorganic micro- and nanoparticles of metal oxides with controlled porosity used as carriers for biomolecules. This technical solution describes inorganic micellar composites functionalized using various reagents using the graft copolymerization process. This technical solution has the following disadvantages: multi-stage process and low stability of the drugs.

There is also known a method of polymerization immobilization of biological macromolecules (see US Pat. RF No. RU 2216547 (C2), application form 16.10.2001, publ. 20.11.2003), which describes a composite for the attachment of oligonucleotides, proteins and nucleic acids, the structure of which includes active functional groups : - amino, sulfhydryl, etc. As monomers in this technical solution, compounds such as acrylamide, methacrylamide, 2-hydroxyethyl ethyl methacrylate or derivatives of acrylic, methacrylic, cinnamon, cretonic and other unsaturated acids are used. The disadvantages of this invention are as follows: the use of toxic compounds and a narrow range of optimal operating conditions: pH and temperature.

Closest to the claimed invention - the prototype - is a technical solution (see US Pat. No. 0230649 (A1), IPC C12N 11/10, decl. 12.23.1986, publ. 05.08.1987), which consists in the immobilization of L-phenylalanine ammonium lyases by adding polyamine, chitosan and multifunctional compounds containing amino groups such as glutaraldehyde. Examples of the polyamines used in this invention include polyethylene diamine, polyethyleneimine, polydiethylenetriamine, polytriethylenetetramine, polypentaethylene hexamine or polyhexamethylene diamine. In this case, glutaraldehyde functions as a condensing agent, through which covalent bonds are formed between the functional groups of the enzyme and the carrier.

The main disadvantages of this technical solution should be considered a relatively low specific activity of the immobilized drug and a narrow range of operating conditions.

The objective of the technical solution is to increase the specific activity and stability of the immobilized preparation of L-phenylalanine-ammonium lyase due to the chemical interaction of the reactive functional groups of the enzyme molecule with the surface of magnetic nanoparticles, which opens up prospects for the repeated use of the biocatalyst in the technological process and the continuous carrying out of the enzymatic process with the possibility controlling the rate of the catalyzed reaction and product yield. These characteristics of immobilized drugs will reduce their cost. The use of magnetic nanoparticles in the preparation of immobilized enzyme preparations is promising due to the fact that they have a developed active surface and high sorption capacity, as well as being able to approach and bind to a biological object due to sizes comparable to the sizes of cells, viruses, proteins, DNA . The search showed that in the information sources there are no technical solutions associated with the totality of the distinguishing features set forth in the invention.

The technical result is achieved through the use as a carrier for immobilization of magnetic nanoparticles, which are metal oxides. The choice of metal oxides as nanoparticles is due to their wide distribution and low cost. This technical problem is achieved by modifying the surface of magnetic nanoparticles with functional groups, followed by covalent addition of the enzyme L-phenylalanine-ammonium lyase in the presence of a condensing agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

A method of obtaining surface-modified magnetic nanoparticles for immobilization of biological substances includes the following operations. At the first stage, magnetic nanoparticles containing electrophilic ester groups on the surface are obtained by reacting polymethyl methacrylate, the corresponding metal chloride, and diethylene glycol. The second stage consists in the formation of a layer of aminopropyltriethoxysysilane on the surface of nanoparticles due to the aminolysis of electrophilic fragments of the carrier. The obtained nanoparticles serve for subsequent immobilization of enzymes using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

The invention is illustrated by the following examples of specific performance.

Example 1

A mixture of polymethyl methacrylate, iron (III) chloride and diethylene glycol is prepared, heated to 220 ° C with stirring. Then, a solution of sodium hydroxide in diethylene glycol is added to the mixture. After a few minutes, Fe ₃ O ₄ nanoparticles with surface ester groups are formed, which are separated by centrifugation. The resulting magnetic nanoparticles are suitable for immobilizing biological substances through a condensing agent.

In the second stage of obtaining the enzyme preparation, a layer of aminopropyltriethoxysilane is formed on the surface of Fe ₃ O ₄ nanoparticles due to the aminolysis of the electrophilic fragments of the carrier. It is important to avoid the presence of water in the system at this stage, since its presence leads to the hydrolysis of aminopropyltriethoxysilane and the formation of oligomeric silanes. Therefore, in a separate chamber with a volume of 30 cm ^3, Fe ₃ O ₄ nanoparticles (1.0 mg) are coated with gaseous 3-aminopropyltriethoxysilane (1 cm ³ ) at atmospheric pressure and a temperature of 70 ° C for 30 minutes. After cooling, the sample is treated with ethyl alcohol three times 5 cm ³ and dried in air. Next, Fe ₃ O ₄ nanoparticles (1.0 mg) are treated with a 0.5% solution of 3-aminopropyltriethoxysilane (the solvent is a mixture of ethanol: water 1: 1) for one hour at a temperature of 50 ° C. At this stage, a joint polycondensation of silane with trialkoxysilane groups occurs. At the end of the modification process, Fe ₃ O ₄ nanoparticles are washed several times with a water - ethyl alcohol solvent (1: 1) and a suspension of Fe ₃ O ₄ nanoparticles with a concentration of 5 mg / cm ³ in 0.1 M phosphate buffer (pH 7.8) is prepared.

Then, 200 μl of the resulting suspension are incubated with 200 μl of 100 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in 0.1 M phosphate buffer for three hours with continuous stirring at room temperature. At the end of the incubation, the suspension is washed three times with 0.025 M phosphate buffer and a suspension is prepared in 500 μl of 0.1 M phosphate buffer. In the next step, 20 μl of L-phenylalanine ammonium lyase are mixed with 500 μl of 0.1 M phosphate buffer and the resulting solution is incubated with 500 μl of a suspension of Fe ₃ O ₄ nanoparticles in 0.1 M phosphate buffer at 30 ° C for two hours. At the end of the immobilization, the Fe ₃ O ₄ nanoparticles are washed with 0.025 M phosphate buffer solution until the activity in the wash water disappears. The obtained immobilized preparation is stored in the form of a suspension of nanoparticles with a covalently bound enzyme in 1000 μl of 0.1 M phosphate buffer. The physicochemical characteristics of L-phenylalanine-ammonium lyase immobilized on Fe ₃ O ₄ nanoparticles are presented in Table 1. In this case, L-phenylalanine-ammonium-lyase immobilized on unmodified Fe ₃ O ₄ nanoparticles by adsorption is used as a control.

Table 2 presents the data obtained by studying the stability of Fe ₃ O ₄ L-phenylalanine-ammonium lyase immobilized on nanoparticles, compared with the control during storage. From table 2 it follows that the immobilization of L-phenylalanine-ammonium lyase on Fe ₃ O ₄ nanoparticles leads to stabilization of the enzyme. So, after 30 days of storage, L-phenylalanine-ammonium lyase immobilized on Fe ₃ O ₄ nanoparticles retains 65.5% of activity, while 32.9% of activity is retained in the control experiment.

Table 1 Physicochemical parameters of immobilized L-phenylalanine-ammonium lyase Immobilization method The specific activity of the enzyme, units / mg protein The specific activity of the enzyme,% of native Optimum pH Temperature optimum, ° С Immobilization on modified Fe ₃ O ₄ nanoparticles 0.53 60.9 7.5-9.0 30 ± 3 Control experience 0.33 37.9 8.0-9.0 30 ± 1

table 2 Storage stability of L-phenylalanine-ammonium lyase immobilized on Fe ₃ O ₄ nanoparticles compared to the control Duration of storage, day The residual activity of the enzyme,% L-phenylalanine-ammonium lyase immobilized on Fe ₃ O ₄ nanoparticles Control experience 5 98.0 91.5 10 95.6 85.0 fifteen 82,4 69.8 twenty 77.6 57.0 25 72.0 40.5 thirty 65.5 32.9

Example 2

A mixture of polymethyl methacrylate, nickel chloride and diethylene glycol is thoroughly mixed and heated to 180 ° C. A solution of sodium hydroxide in diethylene glycol is added to the reaction mixture. NiO nanoparticles with surface ester groups formed after several minutes are separated by centrifugation. The next step in the preparation of the enzyme preparation is the formation of an aminopropyltriethoxysilane layer due to the aminolysis of the electrophilic fragments of the carrier. For this, in a separate chamber with a volume of 50 cm ^3, NiO nanoparticles (5.0 mg) are covered with gaseous 3-aminopropyltriethoxysilane (2 cm ³ ) at atmospheric pressure and a temperature of 80 ° C for 20 minutes. After cooling, the sample is treated with ethyl alcohol three times 5 cm ³ and dried in air. Next, NiO nanoparticles (1.0 mg) are treated with a 0.5% solution of 3-aminopropyltriethoxysilane (the solvent is a mixture of ethanol: water 1: 1) for one hour at a temperature of 70 ° C. At this stage, a joint polycondensation of silane with trialkoxysilane groups occurs. At the end of the modification process, the NiO nanoparticles are washed several times with a solvent of water - ethyl alcohol (1: 1) and a suspension of NiO nanoparticles with a concentration of 5 mg / cm ³ in 0.1 M phosphate buffer (pH 7.8) is prepared.

Subsequent immobilization of L-phenylalanine-ammonium lyase on NiO nanoparticles by the condensing agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is carried out analogously to example 1. Physico-chemical characteristics of L-phenylalanine-ammonium lyase immobilized on NiO nanoparticles, according to compared with the control are presented in table.3.

Table 4 presents the data obtained by studying the stability of NiO L-phenylalanine-ammonium lyase immobilized on nanoparticles in comparison with the control during storage. The results presented in Table 4 indicate that the immobilization of L-phenylalanine-ammonium lyase on NiO nanoparticles leads to stabilization of the enzyme. So, after 30 days of storage, Ni-L-phenylalanine-ammonium lyase immobilized on nanoparticles retains 64.4% of activity, while 32.9% of activity is retained in the control experiment.

Table 3 Physicochemical parameters of immobilized L-phenylalanine-ammonium lyase Immobilization method The specific activity of the enzyme, units / mg protein The specific activity of the enzyme,% of native Optimum pH Temperature optimum, ° С Immobilization on modified NiO nanoparticles 0.65 74.7 7.3-9.2 30 ± 4 Control experience 0.33 37.9 8.0-9.0 30 ± 1

Table 4 Storage stability of L-phenylalanine-ammonium lyase immobilized on NiO nanoparticles compared to the control Duration of storage, day The residual activity of the enzyme,% L-phenylalanine-ammonium lyase immobilized on NiO nanoparticles Control experience 5 95.0 91.5 10 93.2 85.0 fifteen 80.1 69.8 twenty 74.0 57.0 25 69.5 40.5 thirty 64,4 32.9

Example 3

To obtain surface-modified TiO ₂ nanoparticles, a mixture of titanium (IV) chloride, polymethyl methacrylate and diethylene glycol is prepared, and then it is heated with stirring to 220 ° C. Then, sodium hydroxide in diethylene glycol is added to the reaction mixture. After a few minutes, TiO ₂ nanoparticles are formed, which are separated by centrifugation. The next step is the formation of a layer of aminopropyltriethoxysilane due to the aminolysis of electrophilic fragments of the carrier. For this, in a separate chamber with a volume of 10 cm ^3, TiO ₂ nanoparticles (1.0 mg) are coated with gaseous 3-aminopropyltriethoxysilane (1 cm ³ ) at atmospheric pressure and a temperature of 100 ° C for 60 minutes. After cooling, the sample is treated with ethyl alcohol three times 5 cm ³ and dried in air. Next, TiO ₂ particles (1.0 mg) are treated with a 0.5% solution of 3-aminopropyltriethoxysilane (solvent - ethanol: water 1: 1 mixture) for two hours at a temperature of 90 ° C. At this stage, a joint polycondensation of silane with trialkoxysilane groups occurs. At the end of the modification process, TiO ₂ nanoparticles are washed several times with a water - ethyl alcohol solvent (1: 1) and a suspension of nanoparticles with a concentration of 5 mg / cm ³ in 0.1 M phosphate buffer (pH 7.8) is prepared.

The immobilization of L-phenylalanine-ammonium lyase on TiO ₂ nanoparticles by means of the condensing agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is carried out analogously to example 1. Physicochemical characteristics of L-phenylalanine-ammonium lyase immobilized on TiO ₂ nanoparticles, compared with the control are presented in table.5.

Table 6 presents the data obtained by studying the stability of TiO ₂ L-phenylalanine-ammonium lyase immobilized on nanoparticles, compared with the control during storage. The results presented in table 6 indicate that the immobilization of L-phenylalanine-ammonium lyase on TiO ₂ nanoparticles leads to stabilization of the enzyme. Thus, after 30 days of storage, L-phenylalanine-ammonium lyase immobilized on TiO ₂ nanoparticles retains 75.0% of activity, while 32.9% of activity is retained in the control experiment.

Table 5 Physicochemical parameters of immobilized L-phenylalanine-ammonium lyase Immobilization method The specific activity of the enzyme, units / mg protein The specific activity of the enzyme,% of native Optimum pH Temperature optimum, ° С Immobilization on modified TiO ₂ nanoparticles 0.71 81.6 7.4-9.1 30 ± 4 Control experience 0.33 37.9 8.0-9.0 30 ± 1

Table 6 Storage stability of L-phenylalanine-ammonium lyase immobilized on TiO ₂ nanoparticles compared to the control Duration of storage, day Residual activity,% L-phenylalanine-ammonium lyase immobilized on TiO ₂ nanoparticles Control experience 5 98.7 91.5 10 97.2 85.0 fifteen 90.3 69.8 twenty 85,4 57.0 25 81.5 40.5 thirty 75.0 32.9

Thus, the immobilization of L-phenylalanine-ammonium lyase on surface-modified nanoparticles of metal oxides opens up the prospect of modulating the catalytic properties of the enzyme. This invention has several advantages over the prototype:

- the possibility of using magnetic nanoparticles as an inert carrier, which have a number of advantages over macro objects;

- increase the specific activity and stability of immobilized drugs;

- optimization of the functioning conditions of immobilized drugs;

- simplification and cheaper technology for obtaining immobilized drugs.

The stabilizing effect of metal oxide nanoparticles on L-phenylalanine-ammonium lyase is obviously due to conformational changes in the protein molecule as a result of its interaction with nanoscale objects. The catalytic properties of the obtained immobilized preparation of L-phenylalanine-ammonium lyase allow its wide use in biotechnology.

Claims (1)

A method of immobilizing L-phenylalanine-ammonium lyase on magnetic nanoparticles, which involves covalently bonding enzyme molecules to the surface of a carrier by means of a condensing agent, characterized in that metal oxide nanoparticles act as a carrier for immobilization, and 1-ethyl 3- (3-dimethylaminopropyl) carbodiimide.