Amine dehydrogenase catalytic system and application thereof as well as eutectic solvent and preparation thereof

Abstract

The invention discloses an amine dehydrogenase catalytic system, which comprises a deep eutectic solvent and a buffer solution, the deep eutectic solvent takes choline chloride or betaine as a hydrogen bond acceptor and carboxylic acid ammonium salt as a hydrogen bond donor; the catalytic system is applied to a reaction for synthesizing corresponding chiral amine from ketone, especially beta-position heteroaryl ketone, so that the conversion rate and the yield are remarkably improved. The eutecticevaporate solvent is non-toxic, harmless, easy to operate and easy to degrade, the enzyme activity and stability of amine dehydrogenase and glucose dehydrogenase in the ketoreductive amination process can be successfully improved, optimization of an amine dehydrogenase catalytic system is achieved, and development and application of a green, convenient and low-cost ketoreductive amination reaction method are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme catalysts, in particular to an amine dehydrogenase catalytic system and application thereof, as well as a low eutectic solvent used in the catalytic system and preparation thereof. Background Art

[0002] Chiral amines are a very important type of structural unit, existing in many important compounds and bioactive molecules. About 40% of the chemical drugs approved by the US FDA have chiral amine structures. Common chiral amine synthesis methods include chemical synthesis and biocatalysis. Chemical synthesis often uses metal catalysts for asymmetric synthesis, but this catalytic method often requires harsh reaction conditions and is accompanied by problems such as heavy pollution. Enzyme catalysis in biocatalysis has become a popular research direction for chiral amine synthesis due to its green and environmentally friendly advantages.

[0003] The enzymes involved in the synthesis of chiral amines include transaminases, imine reductases, reductive aminases, monoamine oxidases and amine dehydrogenases. Among them, amine dehydrogenases are a type of enzyme that relies on the coenzyme NAD(P)H for reductive amination. They have become a research hotspot in recent years because they can use cheap ammonia as an ammonia donor and only produce water as a byproduct. Although the research on amine dehydrogenases ranges from genetic modification to immobilized applications, these methods generally have defects such as long cycles, high difficulty, high carrier production costs, and instability. Therefore, finding a green, convenient, and low-cost method to improve the enzymatic activity and stability of amine dehydrogenases and achieve efficient catalysis of chiral amines has become a valuable and challenging technical issue. Summary of the invention

[0004] In order to solve the shortcomings of the existing enzymatic synthesis of chiral amines, the present invention develops an amine dehydrogenase catalytic system, which uses a low eutectic solvent composed of ammonium carboxylate as a hydrogen bond donor to improve the catalytic activity and stability of amine dehydrogenase and glucose dehydrogenase, and is used to catalyze the asymmetric synthesis of chiral amines, thereby achieving efficient catalytic synthesis of chiral amines, coenzyme recycling and high stability.

[0005] To achieve the above object, the present invention provides an amine dehydrogenase catalytic system, comprising a low eutectic solvent and a buffer solution, wherein the low eutectic solvent has choline chloride or betaine as a hydrogen bond acceptor and an ammonium carboxylate as a hydrogen bond donor.

[0006] Deep eutectic solvents (DES) are a new type of green and environmentally friendly solvent developed in recent years. They are composed of hydrogen bond donors and hydrogen bond acceptors in a certain ratio and are a multi-component mixture with a low melting point. The difference between them and ionic liquids is that DES are usually composed of natural substances existing in nature. Therefore, they have the advantages of being non-toxic, biodegradable, economical, and easy to prepare, which are reflected in the fields of carrier preparation, separation and extraction of mixtures, polymer chemistry, biocatalysis, etc.

[0007] The present invention introduces a low eutectic solvent into a chiral amine catalytic synthesis reaction system, selects carboxylic acid ammonium salt, which is different from the commonly used hydrogen bond donor, as a hydrogen bond donor according to the characteristics of amine dehydrogenase, and forms a DES with a hydrogen bond acceptor choline chloride or betaine, and then combines with a buffer solution to form an amine dehydrogenase catalytic system, thereby realizing efficient catalysis of the chiral amine synthesis reaction by amine dehydrogenase.

[0008] As a limitation of the above technical solution, the ammonium carboxylate salt is selected from any one of ammonium formate, ammonium acetate and ammonium propionate.

[0009] As a limitation of the above technical solution, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 4:1 to 1:3, and preferably, the optimal molar ratio is 1:1 to 1:3.

[0010] As a limitation of the above technical solution, the buffer solution is an ammonium chloride-ammonia aqueous solution, and preferably, the pH of the buffer solution is 8.0-10.5.

[0011] As a limitation of the above technical solution, amine dehydrogenase is added to the low eutectic solvent and the buffer solution, and then glucose dehydrogenase is added to form a dual enzyme system; preferably, the added amounts of amine dehydrogenase and glucose dehydrogenase are in a ratio of 1:0.1 to 1:4 based on enzyme activity.

[0012] Further improve the composition and ratio of the low eutectic solvent, the composition and pH of the buffer solution and the dual enzyme system to optimize the activity and stability of the catalytic system.

[0013] Meanwhile, the present invention provides the use of the amine dehydrogenase catalytic system as described above, that is, the catalytic system for synthesizing the corresponding chiral amine from ketone. Preferably, the ketone as the reaction raw material is a heteroaryl ketone at the β position.

[0014] As a limitation of the above technical solution, an amine dehydrogenase catalytic system is used to catalyze 4-fluorophenylacetone to synthesize (R) 4-fluorophenylpropylamine; preferably,

[0015] A reaction system is prepared from 4-fluorophenylacetone, glucose, NADH, ammonium chloride-ammonia buffer solution, a low eutectic solvent, amine dehydrogenase and glucose dehydrogenase, and the reaction is shaken at 20-60° C. to synthesize (R) 4-fluorophenylpropylamine;

[0016] Further, the total amount of the reaction system is 5 mL, the concentration of 4-fluorophenylacetone added in the reaction system is 50-500 mM, the concentration of glucose added is 60-600 mM, the concentration of NADH added is 1 mM, the amount of low eutectic solvent added is 250-750 μL, that is, the amount added is 5-15% v / v by volume fraction, the enzyme activity of amine dehydrogenase added is 8 U, and the enzyme activity of glucose dehydrogenase added is 8 U. A 2M ammonium chloride-ammonia buffer solution is used to quantitatively react the system, and the reaction is shaken at 20-60° C. for 2-12 hours.

[0017] The present invention uses a catalytic system formed by dissolving amine dehydrogenase and glucose dehydrogenase in a DES-buffered solution system for the synthesis of chiral amines from ketones, especially β-heteroaryl ketones, to achieve efficient catalysis of the synthesis reaction, cyclic regeneration of coenzymes in the reaction, and high stability of the double enzymes.

[0018] In addition, the present invention also provides the above-mentioned low eutectic solvent, that is, a low eutectic solvent formed by using choline chloride or betaine as a hydrogen bond acceptor and using ammonium carboxylate salt, especially any one of ammonium formate, ammonium acetate and ammonium propionate as a hydrogen bond donor, which can improve the catalytic activity and stability of amine dehydrogenase and glucose dehydrogenase.

[0019] As a limitation of the above technical solution, the low eutectic solvent is betaine-ammonium acetate, and the molar ratio of betaine to ammonium acetate is 1:2.

[0020] Low eutectic solvents used in the field of biocatalysis are mostly selected from neutral components as hydrogen bond donors, such as urea, glycerol, ethylene glycol, etc. The DES composed of these commonly used hydrogen bond donors has no obvious effect on the conversion rate of the synthesis reaction of β-heteroaryl ketones, and some even have an inhibitory effect. In addition, since the pH of the acidic hydrogen bond donor component has a significant effect on the acidity and alkalinity of the reaction system, thereby affecting the enzyme activity, the low eutectic solvent composed of acidic hydrogen bond donors is rarely used in enzyme catalytic reactions. The present invention innovatively uses ammonium carboxylate as a hydrogen bond donor to form a low eutectic solvent, which can not only improve the dual enzyme activity and stability of amine dehydrogenase and glucose dehydrogenase, but also significantly improve the conversion rate of the synthesis reaction.

[0021] Furthermore, the present invention also provides a method for preparing a low eutectic solvent, which is to mix a hydrogen bond acceptor and a hydrogen bond donor in proportion, and fully stir them at 80 to 90° C. to prepare a stable and uniform solution; if a stable and uniform solution cannot be formed under the above conditions, a small amount of ultrapure water is added during the heating and stirring process.

[0022] Commonly used methods for preparing low eutectic solvents include evaporation, heating and stirring, freeze-drying, grinding, and microwave methods. The present invention adopts the heating and stirring method, which is simple to operate, has high stability, and is conducive to wide application.

[0023] In summary, the present invention provides an amine dehydrogenase catalytic system constructed by using DES as a hydrogen bond donor using ammonium carboxylate, which can simultaneously improve the dual enzyme activity, pH stability, storage stability and thermal stability of amine dehydrogenase and glucose dehydrogenase, thereby significantly improving the conversion rate of enzymatic ketone reductive amination per unit time and per unit enzyme activity. The DES used is non-toxic, harmless, easy to operate and easy to degrade, which is conducive to optimizing the process and reducing production costs.

[0024] The present invention also optimizes the molar ratio, water content and single component of DES, and finally selects the optimal DES betaine-ammonium acetate, successfully improves the enzyme activity and stability of amine dehydrogenase and glucose dehydrogenase in the ketone reduction amination reaction process, strengthens the enzymatic properties of amine dehydrogenase and glucose dehydrogenase, improves the rate of the catalytic reaction, the cofactor circulation capacity and the reaction duration, and uses molecular dynamics simulation and molecular docking to speculate and explain the mechanism, thereby realizing the optimization of the amine dehydrogenase catalytic system, which is beneficial to the development and application of a green, convenient and low-cost ketone reduction amination synthesis method. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 , the enzyme activity of amine dehydrogenase in different types of DES-buffer solution systems;

[0026] Figure 2 , glucose dehydrogenase activity in different types of DES-buffer solution systems;

[0027] Figure 3 , the conversion rate of enzymatic reactions in different types of DES-buffer solution systems;

[0028] Figure 4 , the experimental results of optimizing the single component, molar ratio and usage of DES on the conversion rate of enzymatic reaction (a is single component, b is molar ratio, c is addition amount);

[0029] Figure 5 , Experimental results of the effects of different ammonium salts on the conversion rate of enzymatic reactions;

[0030] Figure 6 , optimization experimental results of enzymatic reaction conditions (a is substrate concentration, b is dual enzyme ratio, c is optimal temperature, d is optimal pH);

[0031] Figure 7 , Determination of stability in DES-buffer solution system (a, b are thermal stability, c is storage stability, d, e are pH stability);

[0032] Figure 8 , RMSD comparison of DES-buffer solution and pure buffer solution in molecular dynamics simulation;

[0033] Fig. 9 , DES-buffer solution and pure buffer solution system RMSF comparison in molecular dynamics simulation;

[0034] Fig.10 , DES-buffer solution and pure buffer solution system in molecular dynamics simulation of secondary structure comparison (a is pure buffer solution, b is DES-buffer solution);

[0035] Fig.11 , DES-buffer solution and pure buffer solution in two systems of internal hydrogen bonding of the target amino acid comparison (a is pure buffer solution, b is DES-buffer solution);

[0036] Fig.12 , contact sites between DES and enzyme molecules (a is the action site between acetic acid molecules and amino acids, b is the action site between betaine and amino acids);

[0037] Fig.13 , the shift of amino acid residues after DES contacts the enzyme molecule (a is the shift of 146-148, b is the shift of 308-310);

[0038] Fig.14 , DES-buffer solution and pure buffer solution system distance comparison after molecular docking (a is the pure buffer system, b is the DES-buffer system). DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0041] Example

[0042] 1. Different types of deep eutectic solvents DES and their preparation.

[0043] 1-1. According to the ratio in Table 1 below, weigh the hydrogen bond donor and hydrogen bond acceptor quantitatively, so that the final mass of the two is greater than 10g, place the weighed materials in a 100mL round-bottom flask, put in a magnetic rod of appropriate size, heat and stir at 80-90°C for 2-6h until a stable and uniform DES is formed, then cool to room temperature, collect in a 50mL plastic tube, and store under dry conditions. If a stable and uniform solution cannot be formed under the above conditions, add a small amount of ultrapure water (about 5-10 drops) during the heating and stirring process until a stable and uniform solution is formed at room temperature. The addition of a small amount of ultrapure water can be regarded as a bridge to maintain the stability between the hydrogen bond donor and the hydrogen bond acceptor, and does not affect the amount of DES used in the experiment.

[0044] Table 1 Composition of different types of deep eutectic solvents

[0045]

[0046]

[0047] Second, the activities of amine dehydrogenase and glucose dehydrogenase (determined by microplate reader) and the conversion rate of ketone reductive amination reaction (determined by gas chromatography GC) in 25 DES were determined to screen out the optimal DES.

[0048] 2-1. Determination of amine dehydrogenase activity

[0049] Amine dehydrogenase consumes the coenzyme NADH to generate NAD when catalyzing the conversion of ketone substrates into chiral amines. + , resulting in a decrease in the absorbance at 340 nm, so the enzyme activity was determined by measuring the initial change rate of the absorbance of NADH at a wavelength of 340 nm using a microplate reader.

[0050] The enzyme activity reaction system is: 0.05mmol substrate 4-fluorophenylacetone, 0.5μmol NADH, 0.05g amine dehydrogenase, 100μL DES (N-1 to N-25 correspond to 25 groups of DES in Experimental Table 1, and no DES is added in the blank control group 0), and ammonium chloride / ammonia water (2.0mol / L, pH10.0) buffer solution is quantified to 1mL, that is, the total enzyme activity reaction system is 1mL, and placed at 37°C.

[0051] The enzyme activity unit is defined as: under the test conditions, the amount of enzyme required to consume 1 μmol NADH per unit time (1 min) is defined as 1 U. The enzyme activities of the amine dehydrogenase and glucose dehydrogenase are determined by measuring the absorbance change at 340 nm with an ELISA reader.

[0052] Results Figure 1The DES-buffered solution composed of common hydrogen bond donors such as ethylene glycol, glycerol, and urea had no improvement or even an inhibitory effect on the enzymatic activity of amine dehydrogenase, while the use of ammonium carboxylate as a hydrogen bond donor improved the enzyme activity to varying degrees. The optimal DES was N-15 betaine-ammonium acetate, with a specific enzyme activity of 17.6 U / mg, which was 1.47 times higher than that of the control group.

[0053] 2-2. Determination of glucose dehydrogenase activity

[0054] Glucose dehydrogenase (GDH) catalyzes the conversion of glucose into gluconic acid, consuming the coenzyme NAD + NADH is generated, so the initial rate of change of absorbance at 340 nm is also measured to determine the enzyme activity.

[0055] The enzyme activity reaction system is: 0.05mmol glucose, 0.5μmol NAD + , 0.01 g GDH, 100 μL DES (N-1 to N-25 correspond to the 25 groups of DES in Experimental Table 1, and nothing is added in the blank control group 0), and ammonium chloride / ammonia water (2.0 mol / L, pH 10.0) buffer solution were quantified to 1 mL, i.e., the total reaction volume was 1 mL, and placed at 37°C.

[0056] The enzyme activity unit is defined as: under the detection conditions, the amount of enzyme required to generate 1 μmol NADH per unit time (1 min) is defined as 1 U.

[0057] Results Figure 2 The enzymatic activity of GDH was enhanced in most of the selected DES-buffer solutions. However, since GDH is mainly involved in the cofactor cycle and is not the main rate-limiting enzyme, it must be comprehensively judged in combination with the enzymatic activity data of amine dehydrogenase. Therefore, N-15 betaine-ammonium acetate was selected with a specific enzyme activity of 30U / mg, which was 1.88 times higher than that of the control group.

[0058] 2-3. Determination of conversion rate of enzymatic ketone reduction amination reaction

[0059] Enzymatic reaction: 0.25mmol 4-fluorophenylacetone, 0.3mmol glucose, 2.5μmol NADH, 500μL DES (N-1 to N-25 correspond to the 25 groups of DES in Experimental Table 1, and no DES was added in the blank control group 0), 2mL amine dehydrogenase enzyme solution (3mg / mL), 1mL glucose dehydrogenase enzyme solution (3mg / mL), and 2M ammonium chloride-ammonia buffer solution at pH 10 were quantified to 5mL, that is, the total reaction system was 5mL, and the reaction was carried out at 37°C and 200rpm on a shaker for 2h. If the reaction pH changed, the reaction pH was adjusted to the optimal condition by adding dilute ammonia.

[0060] Conversion rate determination: 1 mL of the reaction solution was added with an equal volume of ethyl acetate for extraction, and then 100 μL of the upper extract was taken and 10 μL of acetic anhydride was added for derivatization. The prepared sample was then subjected to gas chromatography GC to determine the reaction conversion rate, configuration and ee value.

[0061] Gas chromatography conditions: Agilent J&W CP-Chiralsil-DEXCB column (25m×0.32mm×0.25μm), column temperature program: starting temperature 130℃, heating to 185℃ at a rate of 5℃ / min, and retaining at 185℃ for 6min. The peak appearing at around 1.8min is the substrate 4-fluorophenylacetone; the peak appearing at 6.16min is the product (R)4-fluorophenylpropylamine.

[0062] Results Figure 3 The DES-buffered solution composed of common hydrogen bond donors such as ethylene glycol, glycerol, and urea had no effect on or inhibited the conversion rate of the reaction, while ammonium carboxylates improved the conversion rate of the reaction to varying degrees. The optimal DES was N-15 betaine-ammonium acetate, with a conversion rate of 90%, while the control group had only 60%, an increase of 30%.

[0063] 3. Optimization of DES Betaine-Ammonium Acetate

[0064] 3-1. Comparison of the effects of single-component solvent and DES betaine-ammonium acetate on reaction conversion rate

[0065] Betaine, ammonium acetate, choline chloride and DES betaine-ammonium acetate (molar ratio 1:2) of equal mass were weighed and added to 4 groups of enzymatic reaction systems (the total amount of each reaction system was 5 mL), and the added amount was 10% v / v by volume fraction. At the same time, a blank control group was set as Buffer. The catalytic reaction and detection were carried out according to 2-3 to explore the effect of different solvents on the reaction conversion rate. The results are shown in Figure 4 a, The optimal condition obtained was DES betaine-ammonium acetate.

[0066] 3-2. Effect of the molar ratio of DES betaine to ammonium acetate on the reaction conversion rate

[0067] DES betaine-ammonium acetate with different molar ratios (i.e., 1:1, 1:2, and 1:3) were prepared according to 1-1, and the catalytic reaction and detection were carried out according to 2-3 (the blank control group was Buffer) to explore the effect of the molar ratio of DES betaine-ammonium acetate on the reaction conversion rate. The results are shown in Figure 4 b, the optimal molar ratio obtained is 1:2.

[0068] 3-3. Effect of the amount of DES betaine-ammonium acetate added on the reaction conversion rate

[0069] 5, 7.5, 10, 12.5, and 15% v / v DES were added to 5 groups of enzymatic reaction systems (the total amount of each reaction system was 5 mL), that is, 250, 375, 500, 625, and 750 μL DES betaine-ammonium acetate (molar ratio 1:2) were added, and a blank control group was set as Buffer. The 2-3 catalytic reaction and detection were performed to explore the effect of the concentration of DES betaine-ammonium acetate on the reaction conversion rate. The results are shown in Figure 4 c. The optimal amount of DES added is 12.5% ​​v / v (i.e., the optimal amount added to a 5 mL reaction system is 625 μL).

[0070] 3-4. Compare the effects of single-component solvents such as ammonium iodide, ammonium chloride, and ammonium acetate on the reaction conversion rate, and explore the role of ammonium ions in the reaction

[0071] 625 μL of ammonium iodide, ammonium chloride, and ammonium acetate were added to three reaction systems (the total amount of each reaction system was 5 mL), and a blank control group was set as Buffer. The 2-3 catalytic reaction and detection were performed to explore the effects of different types of ammonium ions on the reaction conversion rate. The results are shown in Figure 5 , with the same increase in ammonium ion concentration, both ammonium iodide and ammonium chloride have an inhibitory effect on the reaction conversion rate, indicating that under ordinary solvent conditions, supplementing the ammonium ion concentration will inhibit the reaction conversion rate. Under ammonium acetate conditions, not only is the ammonium ion concentration increased, but an additional ammonia source is provided for amine dehydrogenase, allowing DES to serve as a co-substrate of the reaction, and the reaction conversion rate can also be improved.

[0072] In summary, the optimization result of DES betaine-ammonium acetate is a molar ratio of 1:2 and an addition amount of 12.5% ​​v / v. Under this optimal DES condition, the conversion rate is increased by about 40% compared with the control group. Subsequent experiments were carried out according to the optimized DES betaine-ammonium acetate molar ratio and addition amount for catalytic reaction.

[0073] 4. Optimization of Enzymatic Reaction Conditions

[0074] 4-1. Determination of the optimal substrate concentration for enzyme-catalyzed reactions

[0075] Different amounts of substrate ketone were added to control the concentrations of substrate ketone in each enzymatic reaction system to be 50, 100, 300, 500, and 1000 mM, respectively, and the corresponding glucose concentrations were 60, 120, 360, 600, and 1200 mM, respectively (the glucose concentration was always kept at 1.2 eq of the substrate concentration). The catalytic reaction was carried out according to the DES betaine-ammonium acetate molar ratio and addition amount optimized in 3-2 and 3-3. Other catalytic reaction conditions and detection were the same as in 2-3. The results are shown in Figure 6 a, To improve the yield of the enzymatic reaction, the substrate ketone concentration of 300 mM was selected for subsequent optimization.

[0076] 4-2. Optimal ratio of two enzymes for enzymatic reactions

[0077] Amine dehydrogenase and glucose dehydrogenase with different enzyme activity ratios were added, which were 1:0.1, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, and 1:4, respectively. The enzymatic reaction was carried out according to the DES betaine-ammonium acetate molar ratio and addition amount optimized in 3-2 and 3-3 and the substrate concentration optimized in 4-1. Other catalytic reaction conditions and detection were the same as in 2-3. The results are shown in FIG. Figure 6 b, The optimal dual enzyme ratio obtained is 1:2.

[0078] 4-3. Determination of the optimal temperature for enzyme-catalyzed reactions

[0079] The catalytic reaction was carried out according to the optimized DES betaine-ammonium acetate molar ratio and addition amount in 3-2 and 3-3. The 10 mL plastic tubes containing each enzymatic reaction system were placed in a water bath shaker at different temperatures (20, 30, 37, 40, 50, 60 ° C) for reaction. According to the conditions optimized in 4-1 and 4-2, other catalytic reaction conditions and detection were the same as in 2-3. The results are shown in Figure 6 c, the optimum temperature obtained was 37°C.

[0080] 4-4. Determination of the optimal pH for enzymatic reactions

[0081] Ammonium chloride-ammonia buffer solutions with different pH values ​​(pH 8.0, 8.5, 9.0, 9.5, 10.0, and 10.5, respectively) and concentrations of 2 mol / L were added to each enzymatic reaction system, and the enzymatic reaction was carried out according to the DES betaine-ammonium acetate molar ratio and addition amount optimized in 3-2 and 3-3. According to the conditions optimized in 4-1, 4-2, and 4-3, other catalytic reaction conditions and detection were the same as in 2-3. The results are shown in Figure 6 d, and the optimum pH was obtained to be 9.5.

[0082] 5. Stability determination of amine dehydrogenase and glucose dehydrogenase in DES-buffer system

[0083] 5-1. Thermal stability

[0084] The experimental group and the control group were set up. In the experimental group, 100U of amine dehydrogenase and 100U of glucose dehydrogenase were weighed and placed in two 50mL centrifuge tubes, and then 1.25mL of DES solution was added to each centrifuge tube and shaken evenly; in the control group, 100U of amine dehydrogenase and 100U of glucose dehydrogenase were weighed and placed in two 50mL centrifuge tubes, and then 1.25mL of ultrapure water was added to each centrifuge tube and shaken evenly to ensure that the protein concentration was consistent with that of the experimental group. The amine dehydrogenase samples of the experimental group and the control group were placed in a 50℃ water bath for continuous static incubation, and the glucose dehydrogenase samples were placed in a 45℃ water bath for continuous static incubation. The amine dehydrogenase was detected every 10h, and the glucose dehydrogenase was detected every 20min. The enzyme activity of each sample was determined according to the methods of 2-1 and 2-2, and the enzyme activity of the first determination was defined as 100%, and the relative enzyme activity was calculated.

[0085] Results Figure 7 a, 7b, amine dehydrogenase can still maintain 71% activity after incubation in betaine-ammonium acetate buffer solution at 50°C for 36 hours, while the control group only retains 26% activity; glucose dehydrogenase can still maintain 70% activity after incubation in betaine-ammonium acetate buffer solution at 45°C for 80 minutes, while the control group only retains 15% activity.

[0086] 5-2. Storage stability

[0087] The experimental group and the control group were set up. In the experimental group, 100U of amine dehydrogenase and 100U of glucose dehydrogenase were weighed and loaded into two 50mL centrifuge tubes, and then 1.25mL of DES solution was added to each centrifuge tube and shaken evenly; in the control group, 100U of amine dehydrogenase and 100U of glucose dehydrogenase were weighed and loaded into two 50mL centrifuge tubes, and then 1.25mL of ultrapure water was added to each centrifuge tube and shaken evenly to ensure that the protein concentration was consistent with that of the experimental group. The amine dehydrogenase samples and glucose dehydrogenase samples of the experimental group and the control group were placed in a 4℃ refrigerated environment for continuous static incubation. Every 2 days, the enzyme activity of each sample was determined according to the methods of 2-1 and 2-2. The enzyme activity measured for the first time was defined as 100%, and the relative enzyme activity was calculated.

[0088] Results Figure 7 c. Amine dehydrogenase can still maintain 63% activity after being stored in betaine-ammonium acetate buffer solution at 4°C for 20 days, while the control group only retains 49% activity; glucose dehydrogenase can still maintain 77% activity after being stored in betaine-ammonium acetate buffer solution at 4°C for 10 days, while the control group only retains 45% activity.

[0089] 5-3. pH stability

[0090] Two experimental groups and two control groups were set up repeatedly, in which 100U amine dehydrogenase and 100U glucose dehydrogenase were weighed and placed in two 50mL centrifuge tubes in the experimental group, and then 1.25mL DES solution was added to each centrifuge tube and shaken evenly; 100U amine dehydrogenase and 100U glucose dehydrogenase were weighed and placed in two 50mL centrifuge tubes in the control group, and then 1.25mL ultrapure water was added to each centrifuge tube and shaken evenly to ensure that the protein concentration was consistent with that of the experimental group. All samples of one experimental group and the control group were placed in a pH 4 citric acid-sodium citrate buffer, and all samples of the other experimental group and the control group were placed in a pH 10 ammonium chloride-ammonia buffer solution for continuous static incubation; every other day, the enzyme activity of each sample was determined according to the method of 2-1 and 2-2, and the enzyme activity measured for the first time was defined as 100%, and the relative enzyme activity was calculated.

[0091] Results Figure 7 d, 7e, amine dehydrogenase can still maintain 52% activity after incubation in betaine-ammonium acetate buffer solution at pH 4 for 14 days, and can still maintain 54% activity after incubation in betaine-ammonium acetate buffer solution at pH 10 for 14 days, while the control group retains 47% and 35% activity, respectively; glucose dehydrogenase can still maintain 66% activity after incubation in betaine-ammonium acetate buffer solution at pH 4 for 4 days, and can still maintain 64% activity after incubation in betaine-ammonium acetate buffer solution at pH 10 for 8 days, while the control group retains 39% and 30% activity, respectively.

[0092] VI. Analysis of experimental data and mechanism speculation and interpretation

[0093] 6-1. Comparison of RMSD of two systems during kinetics

[0094] The Gromacs5.1.5 open source software package was used to analyze the root mean square deviation (RMSD) of atoms in the DES-buffer solution and pure buffer solution systems to reflect whether the system has reached stability, and the size of the RMSD was used to determine the degree of deviation of the structural changes from the initial model. Figure 8 The RMSD value in the pure ammonium chloride-ammonia buffer solution system is higher than that in the DES-buffer solution system. The lower RMSD in the DES-buffer solution system indicates that the addition of DES helps to improve the binding stability with the substrate. The average RMSD in the pure buffer solution system is DES-buffered solution system

[0095] 6-2. Comparison of RMSF of the two systems during the kinetic period

[0096] The Gromacs5.1.5 open source software package was used to analyze the changing trend of the amino acid root mean square fluctuation (RMSF) of proteins in pure buffer solution system and DES-buffer solution system. Fig. 9 In the DES-buffer solution system, the RMSF is generally smaller than that in the pure buffer solution system, indicating that the addition of DES further increases the stability of the protein, while the pure buffer solution system has large fluctuations in the P137-N143 amino acid residue region, indicating that the amino acids in this region are not stable. Therefore, it is speculated that the secondary structure of the protein in this region has undergone significant changes during the 0-50ns simulation process.

[0097] 6-3. Comparison of the secondary structures of the two systems during kinetics

[0098] The changes in the protein secondary structure in the region of residues P137-N143 were analyzed, see Fig.10 In the pure buffer solution system without DES molecules, P137-N143 undergoes a transformation from an α-helix to a ring structure ( Fig.10 a), while this phenomenon was not observed in the DES-buffer solution system ( Fig.10 b). Combining the above viewpoints, the large change in RMSF values ​​in the P137-N143 residue region in the two systems is due to the obvious change in the secondary structure of this region.

[0099] 6-4. Prediction and explanation of the catalytic mechanism of DES improving the activity and stability of amine dehydrogenase

[0100] 6-4-1. Comparison of internal hydrogen bonding of amino acid residues in two systems

[0101] Comparison of the internal hydrogen bonding of the secondary structure of the P137-N143 region in the two systems shows that Fig.11 In pure buffer solution system, this region only forms one hydrogen bond with E302 ( Fig.11 a), while in the DES-buffered solution system, this region forms hydrogen bonds with G144, S146, Q298, D301, and K308 ( Fig.11 b) This multiple hydrogen bond connection maintains the stability of the secondary structure conformation of this segment, resulting in its structure remaining unchanged.

[0102] 6-4-2. Contact between each single component of DES and enzyme molecules and displacement of enzyme molecules in DES-buffer solution system

[0103] The distribution of each single component in DES on the protein surface and its contact with amino acids were observed. Fig.12 , 13 , where ammonium acetate as the HBD part forms a van der Waals contact with V148 ( Fig.12a), which caused the V148 residue to shift by 90°, leading to the relative shift of the S146-S147 segment ( Fig.13 a), thereby making it easier for S146 to form a hydrogen bond with Y140 residue; in addition, A309 and R310 form van der Waals contact and salt bridge interactions with ammonium acetate, respectively ( Fig.12 a), which caused A309 and R319 to shift by 24° and 73°, respectively, and caused K308 to shift by 30° ( Fig.13 b), bringing it closer to the G142 residue and forming a stable hydrogen bond interaction, thereby stabilizing the α-helical structure.

[0104] On the other hand, we observed that a betaine molecule as HBA formed a salt bridge with E138 ( Fig.12 b), which makes betaine directly involved in inhibiting the transition from α helix to loop between P137-N143. The above results also verify the result that ammonium acetate is more effective than betaine in the single-component factor analysis experiment, because ammonium acetate acts on more sites than betaine.

[0105] 6-5. Comparison of distances between key sites in two systems in molecular docking

[0106] Molecular docking was used to analyze the distances between substrate small molecules and NADH and key amino acids in the two systems. The results are shown in Fig.14 , after comparison, it can be seen that the DES-buffer solution system ( Fig.14 a) The distance between the substrate and NADH-C4, D115-COOH and K80-NH2 is significantly smaller than that of the pure buffer solution system ( Fig.14 b), where the distance between the substrate and NADH-C4 is Shorten to The distance between the substrate and D115-COOH is Shorten to The distance between the substrate and the catalytic site K80 is determined by Shorten to It is nearly twice as much. It can be seen that in the DES-buffer solution system, the substrate can bind more tightly to the cofactor and catalytic residues.

[0107] In summary, the beneficial effects of the present invention are specifically shown as follows:

[0108] (1) Amine dehydrogenase and glucose dehydrogenase both have better enzyme activities in DES-buffered solution.

[0109] The specific enzyme activity of amine dehydrogenase in betaine-ammonium acetate-buffer solution was 17.6 U / mg, which was 1.47 times higher than the specific enzyme activity of 12 U / mg in pure buffer solution under the same conditions.

[0110] The specific enzyme activity of glucose dehydrogenase in betaine-ammonium acetate-buffered solution was 30 U / mg, which was 1.88 times higher than the specific enzyme activity of 16 U / mg in pure buffered solution under the same conditions.

[0111] (2) The ketone reductive amination reaction has a higher conversion rate in DES-buffered solution.

[0112] The conversion rate of the ketone reductive amination reaction carried out in the betaine-ammonium acetate-buffered solution reached 90%, which was 30% higher than the conversion rate of 60% in the pure buffer solution under the same conditions.

[0113] (3) Amine dehydrogenase and glucose dehydrogenase have better pH stability, thermal stability and storage stability in DES-buffered solution.

[0114] Amine dehydrogenase can still maintain 52% of its activity after incubation in betaine-ammonium acetate buffer solution at pH 4 for 14 days, and can still maintain 54% of its activity after incubation in betaine-ammonium acetate buffer solution at pH 10 for 14 days; while under the same conditions, when incubated in pure buffer solutions at pH 4 and 10 for 14 days, 47% and 35% of their activities are retained respectively.

[0115] Glucose dehydrogenase can still maintain 66% activity after incubation in betaine-ammonium acetate buffer solution at pH 4 for 4 days, and can still maintain 64% activity after incubation in betaine-ammonium acetate buffer solution at pH 10 for 8 days; while the control group only retained 39% and 30% activity respectively.

[0116] Amine dehydrogenase can still maintain 71% activity when incubated in betaine-ammonium acetate buffer solution at 50°C for 36 hours; while under the same conditions, it can only retain 26% activity when incubated in pure buffer solution at 50°C for 36 hours.

[0117] Glucose dehydrogenase can still maintain 70% activity when incubated in betaine-ammonium acetate buffer solution at 45°C for 80 minutes; while under the same conditions, it can only retain 15% activity when incubated in pure buffer solution at 45°C for 80 minutes.

[0118] Amine dehydrogenase can still maintain 63% activity when stored in betaine-ammonium acetate buffer solution at 4°C for 20 days; while it only retains 49% activity when stored in pure buffer solution at 4°C for 20 days.

[0119] Glucose dehydrogenase can still maintain 77% activity when stored in betaine-ammonium acetate buffer solution at 4°C for 10 days; while it only retains 45% activity when stored in pure buffer solution at 4°C for 10 days.

[0120] (4) Betaine-ammonium acetate can also be used as a co-substrate for catalytic reactions

[0121] The betaine and ammonium acetate components in the low eutectic solvent can not only improve the enzyme activity and stability, but the ammonium ions in the ammonium acetate can also serve as a co-substrate in the catalytic reaction, thereby providing an additional ammonia source for amine dehydrogenase without affecting the enzyme activity.

[0122] (5) The catalytic mechanism of the low eutectic solvent was reasonably speculated and explained by molecular dynamics simulation and molecular docking. Most of the existing mechanisms are judged from the connection distance between the substrate small molecule and the catalytic site, while the mechanism speculated in this paper covers that the addition of betaine-ammonium acetate affects the deviation direction of local amino acid residues inside the protein. This deviation adjusts the local secondary structure, thereby bringing the substrate closer to the active site of the enzyme.

Claims

1. An amine dehydrogenase catalytic system, characterized in that: The invention comprises a low eutectic solvent and a buffer solution. The low eutectic solvent uses choline chloride or betaine as a hydrogen bond acceptor and uses carboxylic acid ammonium salt as a hydrogen bond donor.

2. The amine dehydrogenase catalytic system according to claim 1, characterized in that: The ammonium carboxylate salt is selected from any one of ammonium formate, ammonium acetate and ammonium propionate.

3. The amine dehydrogenase catalytic system according to claim 1, characterized in that: The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 4:1 to 1:3, preferably, the optimal molar ratio is 1:1 to 1:

3.

4. The amine dehydrogenase catalytic system according to claim 1, characterized in that: The buffer solution is an ammonium chloride-ammonia aqueous solution. Preferably, the pH of the buffer solution is 8.0-10.

5.

5. The amine dehydrogenase catalytic system according to claim 1, characterized in that: Amine dehydrogenase is added to a low eutectic solvent and a buffer solution, and then glucose dehydrogenase is added to form a dual enzyme system; preferably, the added amount of amine dehydrogenase and glucose dehydrogenase is in a ratio of 1:0.1 to 1:4 based on enzyme activity.

6. Use of the amine dehydrogenase catalytic system according to any one of claims 1 to 5, characterized in that: The invention is used as a catalytic system for synthesizing the corresponding chiral amine from ketone. Preferably, the raw ketone of the reaction is a heteroaryl ketone at the β position.

7. The use of the amine dehydrogenase catalytic system according to claim 6, characterized in that: The amine dehydrogenase catalytic system is used to catalyze the synthesis of (R) 4-fluorophenylpropylamine from 4-fluorophenylacetone; preferably, A reaction system is prepared from 4-fluorophenylacetone, glucose, NADH, ammonium chloride-ammonia buffer solution, a low eutectic solvent, amine dehydrogenase and glucose dehydrogenase, and the reaction is shaken at 20-60° C. to synthesize (R) 4-fluorophenylpropylamine.

8. A deep eutectic solvent according to any one of claims 1 to 5, characterized in that: The low eutectic solvent formed by using choline chloride or betaine as hydrogen bond acceptor and ammonium carboxylate as hydrogen bond donor can improve the catalytic activity and stability of amine dehydrogenase and glucose dehydrogenase.

9. The deep eutectic solvent according to claim 8, characterized in that: The low eutectic solvent is betaine-ammonium acetate, and the molar ratio of betaine to ammonium acetate is 1:

2.

10. A preparation of the deep eutectic solvent according to claim 8, characterized in that: The hydrogen bond acceptor and the hydrogen bond donor are mixed in proportion, and stirred thoroughly at 80-90° C. to prepare a stable and uniform solution.

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