Method for immobilization of biocatalysts and use in synthesis of chiral pesticides

Abstract

The present application relates to the technical field of organic synthesis, in particular to a kind of immobilization method of biological catalyst and application in the synthesis of chiral pesticide.The immobilization method includes the following steps: uniformly mixing sodium alginate solution and sodium carboxymethyl cellulose solution to obtain carrier solution; biological catalyst is dissolved in buffer to prepare enzyme solution, then enzyme solution and carrier solution are fully mixed; mixed solution is added dropwise into calcium chloride solution, and gel microspheres are formed; gel microspheres are added into epichlorohydrin solution for crosslinking, and immobilized biological catalyst is obtained.The present application improves the shortcomings of poor stability, low loading rate and unstable selectivity of traditional immobilized carrier formed by sodium alginate and sodium carboxymethyl cellulose by adding epichlorohydrin for crosslinking, improves the stability of immobilized enzyme, realizes the repeated use of immobilized enzyme, greatly reduces the use cost of enzyme preparation, and is suitable for application and popularization in production practice.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic synthesis, and particularly relates to a method for immobilizing a biological catalyst and application thereof in synthesis of chiral pesticides. BACKGROUND

[0002] With the increasing demand for optical purity of chiral drugs, pesticides and fine chemicals, enzyme-catalyzed synthesis of chiral compounds has become an important research direction in the field of green chemistry due to its high enantioselectivity, mild conditions and environmental friendliness. In particular, esterases are widely used in the kinetic resolution of chiral alcohols and acids, asymmetric synthesis and esterification / acylation reactions due to their good specificity and catalytic efficiency for fatty acid ester substrates. However, free enzymes have poor operational stability, are difficult to recycle and reuse, and other problems, which seriously limit their application in industrial reaction processes.

[0003] In order to improve the operational stability, thermal stability and reusability of enzymes, enzyme immobilization technology is usually used to fix enzymes on the surface or inside of carriers by physical embedding, covalent bonding or adsorption. This not only can significantly improve the anti-inactivation ability of enzymes, but also can regulate the spatial conformation of enzymes, thereby affecting their stereoscopic recognition ability to substrates. The commonly used immobilization methods include adsorption, embedding, cross-linking and covalent binding. Embedding is a commonly used method for immobilization. By embedding enzymes in a polymer network, the aggregation of enzymes can be reduced, thereby reducing the risk of enzyme inactivation and maintaining high catalytic activity. For example, polymers such as polyacrylamide are often used to embed enzymes. The network formed by these polymers can limit the free movement of enzymes, reduce the interaction between enzyme molecules, and avoid aggregation and denaturation of enzymes. However, embedding method also has certain limitations, such as the high molecular matrix may produce additional restrictions on the active site of the enzyme, affecting the access of the substrate and the reaction rate.

[0004] Cross-linking method connects enzyme molecules together through chemical bonds to form a stable network structure, thereby improving the thermal stability and mechanical stability of the enzyme. For example, glutaraldehyde cross-linking is a commonly used cross-linking method, which can react with functional groups such as amino groups on enzyme molecules to form a cross-linked network. However, the cross-linking process may have some impact on the active center of the enzyme, and the activity and selectivity of the enzyme need to be balanced by optimizing the cross-linking conditions. Rodrigues, R. C., et al. (2013) mentioned that by interfacial activation, lipase was immobilized on hydrophobic carriers (such as octyl sepharose, octadecyl Sepabeads), which showed higher enantioselectivity in the kinetic resolution of racemic alcohols and esters. In contrast, the use of carriers with ion exchange function or glutaraldehyde activation for immobilization often leads to a decrease or even reversal of chiral selectivity. Therefore, there is an urgent need for an immobilization method of biocatalysts that can improve the reusability of enzymes while ensuring that their substrate selectivity is not affected. SUMMARY

[0005] The present application is to overcome the defects of the prior art, that is, the poor stability, low activity and easy change of chiral selectivity of the enzyme fixed by the existing immobilization method of biocatalysts, and provides an immobilization method of biocatalysts and its application in the synthesis of chiral pesticides.

[0006] To achieve the above-mentioned purpose of the application, the following technical solutions are used:

[0007] In a first aspect, the present application discloses a method for immobilizing biocatalysts, comprising the following steps:

[0008] S1, uniformly mix a sodium alginate solution and a sodium carboxymethyl cellulose solution to obtain a carrier solution;

[0009] S2, dissolve the biocatalyst in a buffer solution to prepare an enzyme solution, and then mix the enzyme solution and the carrier solution to obtain a mixed solution in which the enzyme is adsorbed on the immobilized carrier;

[0010] S3, add the mixed solution dropwise into a calcium chloride solution to form a gel microsphere;

[0011] S4, cross-link the gel microsphere in an epichlorohydrin solution to obtain an immobilized biocatalyst.

[0012] The immobilization strategy has a significant impact on the chiral selectivity of the enzyme. For example, the esterase immobilized on a hydrophobic carrier (such as octyl sepharose) can induce its conformation to tend to an open state, thereby enhancing the recognition ability of (S)-alcohol substrate; and the use of cross-linking fixation (such as glutaraldehyde) can change the spatial configuration of the enzyme active center through multi-point bonding, thereby achieving the improvement or adjustment of selectivity.

[0013] Sodium alginate (SA) is a natural polysaccharide extracted from brown algae, which is non-toxic and non-immunogenic, and is suitable for food, medicine and other fields with high biological safety requirements. The extraction process is mature, the price is low, and it is suitable for large-scale industrial application. The carboxyl and hydroxyl groups in the molecular chain can interact with metal ions or organic molecules to form a stable gel network. Sodium carboxymethyl cellulose (CMC-Na) is a cheap and easily soluble cellulose derivative with excellent water solubility, which can form a high-viscosity solution and has thickening, suspending and bonding functions. It is often used with sodium alginate as a composite carrier to enhance the stability of the gel network. However, on the one hand, sodium carboxymethyl cellulose itself is easy to break and has high hardness after film formation, and although the tensile strength can be improved after being combined with sodium alginate, the elongation at break is significantly reduced, leading to increased brittleness and easy cracking under stress; on the other hand, after sodium alginate and sodium carboxymethyl cellulose form an immobilized carrier, the enzyme molecules still have a certain degree of freedom inside the carrier, and changes in external reaction conditions or the action of substrate molecules can easily cause changes in enzyme conformation, making the active site not accurately match the specific enantiomer of chrysanthemic acid precursor, resulting in unstable selectivity.

[0014] Therefore, the present application uses sodium alginate SA as an embedding agent to form a composite immobilized carrier SA-CMC-NA with sodium carboxymethyl cellulose, thereby improving the temperature tolerance, pH tolerance, and catalytic stability of the esterase, and using epichlorohydrin (ECH) for cross-linking, changing the ionic interaction between the composite carriers to covalent interaction, thereby greatly improving the low mechanical strength of SA as an immobilized material; at the same time, forming a covalent cross-linking between the carrier and the enzyme, limiting the disordered changes in enzyme conformation and the freedom of relative displacement, and the active site is more inclined to the optimal shape and direction, thereby more stably maintaining its precise chiral structure to effectively distinguish the enantiomers of chrysanthemic acid precursor. The covalently cross-linked immobilized enzyme formed by the present application can ensure that the selectivity of the enzyme remains relatively stable during long-term reactions, and the catalytic efficiency and substrate selectivity are maintained at a high level. Compared with traditional immobilization methods, the present application ensures the activity of the enzyme while improving the loading rate of the enzyme, forming an immobilized enzyme with improved temperature tolerance, pH tolerance, catalytic stability, selectivity stability, and reusability.

[0015] In addition, in order to improve the immobilization effect, the gel microspheres can be placed at 4°C for 20 minutes in step S3 to form a preliminary cross-linking network and obtain a preliminarily immobilized esterase; the immobilized esterase is washed with a buffer solution to remove unbound free enzymes, and then step S4 is performed. In practice, epichlorohydrin can be diluted with glycine-NaOH buffer solution before use, and then step S4 is performed.

[0016] Further, the concentration of the sodium alginate solution in step S1 is 1-2% (w / v). In practice, to ensure the stability of the immobilization environment, the SA can be dissolved in the glycine-NaOH buffer solution, and stirred magnetically until completely dissolved to prepare the sodium alginate solution.

[0017] Further, the concentration of the sodium alginate solution in step S1 is 1-2% (w / v). In practice, to ensure the stability of the immobilization environment, the SA can be dissolved in the glycine-NaOH buffer solution, and stirred magnetically until completely dissolved to prepare the sodium alginate solution.

[0018] Further, the concentration of the sodium alginate solution in step S1 is 1-2% (w / v). In practice, to ensure the stability of the immobilization environment, the SA can be dissolved in the glycine-NaOH buffer solution, and stirred magnetically until completely dissolved to prepare the sodium alginate solution. 2+ The CaCl2 solution in step S3 has a concentration of 3-5% (w / v). Since Ca2+ in the CaCl2 solution promotes the ionic crosslinking of the SA, thereby forming the immobilized enzyme carrier microspheres, the concentration of CaCl2 is also an important factor affecting the immobilization effect.

[0019] Further, the concentration of the sodium alginate solution in step S1 is 1-2% (w / v). In practice, to ensure the stability of the immobilization environment, the SA can be dissolved in the glycine-NaOH buffer solution, and stirred magnetically until completely dissolved to prepare the sodium alginate solution.

[0020] Further, in step S3, the particle size of the gel microspheres is 50-100 um.

[0021] Further, in step S2, the buffer solution is a sodium hydroxide solution of glycine.

[0022] In a second aspect, the application also discloses an application of the immobilization method of the biological catalyst in the synthesis of chiral pesticides.

[0023] In a third aspect, the application discloses an immobilized enzyme for producing enantiomeric chrysanthemic acid, which comprises a carboxylic esterase mutant, the nucleotide sequence of which is shown in SEQ NO. 1, and the amino acid sequence of which is shown in SEQ NO. 2; and the carboxylic esterase mutant is immobilized by the immobilization method of the biological catalyst.

[0024] In the actual production process of chrysanthemic acid, one of the commonly used raw materials, methyl chrysanthemate, has poor water solubility, so in the process of esterase catalytic reaction, methyl chrysanthemate that has not reacted in time interacts with protein to disperse into small droplets to form a milk emulsion, thereby causing emulsification. In the process of long-time reaction, as the esterase that catalyzes gradually partially deactivates, more and more unreacted methyl chrysanthemate is discharged with the product. The emulsification phenomenon not only represents a relatively low reaction conversion rate, but also seriously affects the service life of the ultrafiltration membrane in the subsequent ultrafiltration membrane filtration process, greatly increases the production cost, and affects the production efficiency. The esterase used in the present application is a carboxylic acid esterase mutant from a laboratory, which is formed into an immobilized biological catalyst through the above immobilization method, and through subsequent experimental verification, it has good thermal stability, pH stability, catalytic stability, selectivity stability and repeatability, meeting the needs of industrial application.

[0025] In a fourth aspect, the present application discloses an immobilized enzyme for producing 4-(methyl hydroxy phosphorly)-2-carbonyl butyric acid (PPO), comprising a D-amino acid oxidase mutant, the amino acid sequence of which is shown as SEQ NO. 3; the D-amino acid oxidase mutant is immobilized by the above-mentioned immobilization method of biological catalyst. Through subsequent experimental verification, the immobilization method of the present application can also be well applied in the production of PPO by D-PPT (glufosinate-ammonium) catalyzed by D-amino acid oxidase (DAAO).

[0026] Therefore, the present application has the following beneficial effects:

[0027] (1) The present application improves the shortcomings of the traditional sodium alginate and sodium carboxymethyl cellulose forming an immobilized carrier, such as poor stability, low loading rate and unstable selectivity, by adding an appropriate amount of epichlorohydrin for crosslinking, thereby improving the stability of the immobilized enzyme and realizing the repeated use of the immobilized enzyme.

[0028] (2) The immobilized enzyme prepared by the carboxylic acid esterase mutant and the D-amino acid oxidase mutant has good effects in catalytic stability and repeatability, and also has good storage stability, greatly reducing the use cost of enzyme preparations, and is suitable for application and promotion in production practice. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The figure is a relationship diagram of the SA concentration in Example 2 of the present application and the relative residual activity of the immobilized enzyme of the present application.

[0030] Figure 2 The figure is a relationship diagram of the CMC-Na concentration in Example 2 of the present application and the relative residual activity of the immobilized enzyme of the present application.

[0031] Figure 3Figure for the relationship between CaCl2 concentration in Example 3 of the present application and the residual activity of the immobilized enzyme of the present application.

[0032] Figure 4 Figure for the relationship between ECH concentration in Example 4 of the present application and the residual activity of the immobilized enzyme of the present application.

[0033] Figure 5 Figure for the storage stability test of the immobilized esterase of Example 7 of the present application.

[0034] Figure 6 Figure for the comparison of the conversion rate of the immobilized esterase of Example 8 of the present application and the free esterase.

[0035] Figure 7 Figure for the comparison of the emulsification degree of the immobilized esterase of Example 9 of the present application and the free esterase.

[0036] Figure 8 Figure for the reuse test of the immobilized esterase of Example 10 of the present application.

[0037] Figure 9 Figure for the comparison of the conversion rate of the immobilized amino acid oxidase of Example 11 of the present application and the free esterase.

[0038] Figure 10 Figure for the reuse test of the immobilized amino acid oxidase of Example 12 of the present application. DETAILED DESCRIPTION

[0039] The present application will be further described in conjunction with specific examples. Those skilled in the art will be able to implement the present application based on these descriptions. In addition, the examples of the present application involved in the following descriptions are generally only a part of the examples of the present application, rather than all the examples. Therefore, all the other examples obtained by those skilled in the art based on the examples in the present application without making creative efforts shall belong to the scope of protection of the present application. The relative residual activity of the enzyme in the following examples is calculated by the following formula: relative residual activity = (immobilized enzyme activity / maximum value of immobilized enzyme activity under relative conditions) x 100%. The enzyme activity unit is defined as: the amount of enzyme required for producing 1 micromole of dextro-trans-chrysanthemic acid per minute under the conditions of 45°C and pH 10, which is defined as one enzyme activity unit, U. The specific enzyme activity is defined as the number of activity units per gram of cells, U / g. The standard conditions for detecting the esterase enzyme activity are as follows: 400 g / L of methyl chrysanthemate is added in a 100 mL reaction system, 45°C, pH 10 100 mM phosphate buffer is used as the reaction medium, the reaction is carried out under the conditions of 800 rpm for 10 min, the sample is treated and analyzed by HPLC.

[0040] Example 1: Preparation of immobilized esterase (Est@SA-CMC-Na-ECH)

[0041] First, a SA solution with a certain concentration is added to a CMC-Na solution in a certain proportion, and mixed thoroughly by a magnetic stirrer to form a SA-CMC-Na mixed solution. An esterase with a concentration of 50 U / ml (based on the total mass of SA+CMC-Na) is mixed with the carrier solution at an enzyme addition amount of 50 U / g, and stirred for one hour to allow the enzyme to be fully adsorbed, and then the mixed solution is added dropwise into a 5% (w / v) CaCl2 solution to form gel microspheres (the measured particle size is 50-100 um), which are placed at 4°C for one hour for fixation, and then washed with deionized water for three times to remove the unreacted Ca 2+ After the solidification is completed, 0.35% (w / v) of an ECH solution is added to crosslink the Est immobilized carrier, and the crosslinked immobilized carrier is obtained after being kept at 25°C with constant temperature and oscillation at 120 rpm for one hour. The immobilized carrier is washed with deionized water for three times to remove the unreacted ECH, and the immobilized esterase for producing dextro-trans-chrysanthemic acid according to the present application is obtained.

[0042] Example 2: Selection of the concentrations of sodium alginate and sodium carboxymethyl cellulose

[0043] ECH can be affinity substituted with the hydroxyl group of SA and the carboxyl group of CMC-Na to form ether bond (C-O-C), thereby increasing the strength of the immobilized carrier. The ratio of SA and CMC-Na in the mixed gel can affect the state of the mixed gel. Too low concentration can result in insufficient mechanical strength, and too high concentration can inhibit enzyme activity.

[0044] SA solution with a concentration of 0.5-2.0% (w / v) was prepared using 0.1 M glycine-NaOH buffer at pH 8.0. 4 ml of SA solution with different concentrations was added with 1 ml of CMC-Na solution with a concentration of 0.2-1% (w / v), and stirred vigorously for 2 h. Ultrasonic was used to remove bubbles. The esterase solution with a concentration of 50 U / ml was mixed with the carrier solution at an enzyme addition amount of 50 U / g, and stirred for 1 h. Then, the mixed solution was sucked into a syringe, and added dropwise into a CaCl2 solution with different concentrations for solidification. After solidification, the unreacted Ca 2+ was removed by washing with deionized water for three times. A certain concentration of ECH solution was added for crosslinking, and the crosslinked carrier beads were obtained after 1 h. The obtained immobilized esterase was washed with deionized water, and the operation was repeated three times to sufficiently remove the excess ECH solution. The obtained immobilized esterase was placed in a refrigerator at 4°C for standby.

[0045] As shown in Figure 1 , the SA concentration can affect the relative residual activity of the immobilized esterase. When the SA concentration is 1-2% (w / v), the relative residual activity of the immobilized enzyme is higher, and when the SA concentration is 1.5% (w / v), the relative residual activity of the immobilized enzyme reaches the highest. As shown in Figure 2 , the CMC-Na concentration can affect the relative residual activity of the immobilized enzyme. When the CMC-Na concentration is 0.2-0.6% (w / v), the relative residual activity of the immobilized enzyme is higher, and when the CMC-Na concentration is 0.4% (w / v), the relative residual activity of the immobilized enzyme reaches the highest.

[0046] Example 3: Selection of calcium chloride concentration

[0047] The Est@SA-CMC-Na gel was prepared according to the experimental steps of Example 1, and the Est@SA-CMC-Na gel was sucked into a syringe and added dropwise into a CaCl2 solution with a concentration of 2-6% (w / v) prepared with distilled water at a certain speed for solidification. When the concentration of the CaCl2 solution was 2% (w / v), the strength of the immobilized gel was low, and the material was broken. After solidification, the unreacted Ca 2+ was removed by washing with deionized water for three times. A certain amount of ECH solution was added for activation after solidification. The activated carrier beads were obtained, and the above operation was repeated three times to sufficiently remove the excess ECH solution. The obtained immobilized esterase was placed in a refrigerator at 4°C for standby.

[0048] As shown in Figure 3 The CaCl2 concentration can affect the relative residual activity of the immobilized esterase. When the CaCl2 concentration is 3-5% (w / v), the relative residual activity of the immobilized enzyme is higher. When the CaCl2 concentration is 4% (w / v), the relative residual activity of the immobilized enzyme reaches the highest.

[0049] Example 4: Selection of epichlorohydrin concentration

[0050] First, prepare an ECH solution with a concentration of 0.2-1% (w / v) using pure water. Second, add the immobilized carrier prepared in the above experiment to the same volume of ECH solution with different concentrations in sequence and crosslink for 1 h. Finally, wash the activated carrier beads with deionized water, repeat the above operation 3 times, and remove the excess ECH solution to obtain the immobilized enzyme, which is stored in a 4°C refrigerator for standby.

[0051] As shown in Figure 4 When the concentration of the epichlorohydrin solution is 0.2-0.6% (w / v), the relative residual activity of the immobilized enzyme is higher. When the concentration of the epichlorohydrin solution is 0.4% (w / v), the relative residual activity of the immobilized enzyme reaches the highest.

[0052] Example 5: Optimization of Est@SA-CMC-Na-ECH preparation by orthogonal experiment

[0053] According to the single-factor experiment results of the above examples, select the SA concentration, CMC-Na concentration, CaCl2 solution concentration, and ECH solution concentration of the immobilized carrier Est@SA-CMC-Na-ECH, take the relative residual activity of the immobilized esterase as the evaluation index, design an orthogonal experiment, the orthogonal experiment factor level is shown in Table 1, and the experimental results are shown in Table 2.

[0054] Table 1: Orthogonal experiment factor level for optimization of Est@SA-CMC-Na-ECH preparation conditions

[0055]

[0056] Table 2: Orthogonal experiment results and analysis of Est@SA-CMC-Na-ECH preparation condition optimization

[0057]

[0058] As shown in Table 2, the optimal preparation process of the immobilized esterase is A2B2C2D2 (0.3% ECH, 0.3% CMC-Na, 1.0% SA, 4% CaCl2), corresponding to the experimental number 2 of 83.4%.

[0059] Example 6: Enzymatic property evaluation of immobilized esterase

[0060] Take 4 mL of 1.0% (w / v) SA solution, add 1 mL of 0.3% (w / v) CMC-Na solution, mix thoroughly with a magnetic stirrer, to form a SA-CMC-Na mixed carrier solution. Mix 50 U / ml of esterase (based on the total mass of SA+CMC-Na) with the carrier solution at an enzyme addition amount of 50 U / g, stir for one hour to allow the enzyme to be fully adsorbed, then add the mixture dropwise to a 4% (w / v) CaCl2 solution to form gel microspheres (measured particle size 50-100 um), and let stand at 4°C for one hour, then rinse with deionized water three times to remove unreacted Ca 2+ After the solidification is complete, immerse the gel microspheres in 0.3% (w / v) ECH solution to crosslink the Est immobilized carrier, and obtain the crosslinked immobilized carrier after constant temperature at 25°C with 120 rpm oscillation for one hour. Rinse with deionized water three times to remove unreacted ECH, and obtain the immobilized esterase Est@SA-CMC-Na-ECH.

[0061] Take Est@SA-ECH as the control group, and Est@SA-CMC-Na and Est@SA-CMC-Na-ECH as the experimental groups, wherein Est@SA-ECH is prepared by directly mixing sodium alginate solution with esterase solution, then solidifying with CaCl2 solution and crosslinking with ECH; Est@SA-CMC-Na is not crosslinked with ECH, and the rest of the steps are the same as Est@SA-CMC-Na-ECH. The BCA kit and liquid phase detection are used to detect the loading rate and enzyme activity of Est, respectively. First, for the enzyme loading rate, the protein content of the Est initial solution, filtrate, carrier residual liquid and washing liquid is detected, and the protein amount is obtained by comparison with the standard curve. The mass of the protein loaded on the scaffold is calculated using the difference between the initial protein content in the Est solution and the remaining protein content in the filtrate, carrier residual liquid and washing liquid. The relative residual activity of the enzyme is calculated according to the liquid phase detection results, and the test results are shown in Table 3. The formula for evaluation is: enzyme loading rate = [P1-(P2-P3)-P4] / P1 x 100%;

[0062] P1 is the initial protein content of the Est solution; P2 is the protein content of the Est filtrate; P3 is the residual protein content of the carrier; and P4 is the protein content of the washing liquid.

[0063] Table 3: Comparison of the enzymatic properties of immobilized enzymes

[0064] Sample Enzyme loading rate (%) Enzyme relative residual activity (%) Product e.e value Est@SA-ECH 81.45 85.43 99% Est@SA-CMC-Na 83.79 82.97 99% Est@SA-CMC-Na-ECH 89.33 84.26 99%

[0065] The results are shown in Table 3, the enzyme loading rate of Est@SA-ECH is 81.45%, the enzyme loading rate of Est@SA-CMC-Na is 83.79%, the enzyme loading rate of Est@SA-CMC-Na-ECH is 89.33%, the addition of CMC-Na effectively increases the loading site of Est, and Est@SA-CMC-Na-ECH still maintains 84.26% of the enzyme activity.

[0066] Example 7: Storage stability test of immobilized esterase

[0067] The Est@SA-CMC-Na-ECH prepared according to the method of Example 6 was added to a reaction system containing 60 mL of glycine-NaOH buffer solution and 40 mL of methyl d- chrysanthemate solution (the amount of immobilized esterase was 200 mM protein based on protein loading), and the reaction was started in a 45°C incubator. The Est@SA-CMC-Na-ECH was filtered out, and the supernatant was separated. The Est@SA-CMC-Na-ECH was stored, the buffer was washed 3 times, and was stored at 4°C. The corresponding substances were added to the incubation system again at 1, 3, 6, 9, 15, 20, 25, and 30 days, respectively, for re-catalysis. The supernatant solution of each time was taken to a 1.5 mL EP tube, extracted with ethyl acetate three times, filtered, and then detected for product concentration by high performance liquid chromatography in three parallel groups. Finally, the amount of product was calculated according to the product standard curve to characterize the catalytic stability of the immobilized enzyme. The experimental results are shown in Table 4, and the residual activity of Est@SA-CMC-Na-ECH remained at about 30% on the 30th day. Figure 5

[0068] Example 8: Comparison of conversion rates of immobilized esterase and free esterase

[0069] The Est@SA-CMC-Na-ECH prepared according to the method of Example 6 was added to a reaction system containing 60 mL of glycine-NaOH buffer solution and 40 mL of methyl d- chrysanthemate solution (the amount of immobilized esterase was 200 mM protein based on protein loading), and the reaction was started in a 45°C incubator. The amount of protein of the immobilized enzyme was calculated according to the loading rate, and a free enzyme solution was prepared with 60 mL of glycine-NaOH buffer solution to have the same protein amount as the immobilized enzyme. After adding 40 mL of substrate solution, the reaction was started in a 45°C incubator. Samples were taken after 1, 3, 6, 9, 12, 18, and 24 h of reaction, extracted with ethyl acetate three times, filtered, and then detected for product concentration by high performance liquid chromatography in three parallel groups. The experimental results are shown in Table 5, and the immobilized enzyme and the free enzyme can reach a conversion rate of 45% after 14 h of reaction. Figure 6

[0070] Example 9: Comparison of emulsification degree after reaction of immobilized esterase and free esterase​​

[0071] The experimental procedure of Example 8 was repeated, and the absorbance was detected at a wavelength of 600 nm using a spectrophotometer to determine the degree of emulsification of the reaction system. The results are shown in Table 2. Figure 7 As shown in Table 2, the absorbance of Est@SA-CMC-Na-ECH at 600 nm remained at about 0.2 after 24 h of reaction.

[0072] Example 10: Test of reusability of the immobilized esterase

[0073] The experimental procedure of Example 8 was repeated, except that after 20 min of incubation, the Est@SA-CMC-Na-ECH was filtered out, the supernatant was separated and subjected to high-performance liquid chromatography detection, the immobilized enzyme was retained, the buffer was washed 3 times, and the corresponding substances were directly added to the incubation system for re-catalysis. The reusability of the immobilized enzyme was characterized by detecting the product concentration after 30 cycles of re-catalysis. Figure 8 As shown in Table 3, the Est@SA-CMC-Na-ECH retained more than 75% of the enzyme activity after 30 cycles of reuse.

[0074] Example 11: Comparison of conversion rates of immobilized amino acid oxidase (DAAO) and free enzyme

[0075] The experimental procedure of Example 1 was repeated, except that the esterase was replaced by amino acid oxidase (DAAO) to prepare immobilized DAAO (DAAO@SA-CMC-Na-ECH). The DAAO@SA-CMC-Na-ECH was added to 100 mL of phosphate buffer solution and the reaction was started in a 35°C incubator. The protein mass of the immobilized enzyme was calculated according to the loading rate, and a free enzyme solution with the same protein mass as the immobilized enzyme was prepared using 100 mL of phosphate buffer solution. After the addition of 50 mM D, L-PPT, the reaction was started in a 35°C incubator. Samples were taken after 1, 3, 6, 9, 12, 18, and 24 h of reaction, 10 μL of 6 M HCl was added to terminate the reaction, and the filtrate was subjected to high-performance liquid chromatography detection in triplicate. The experimental results are shown in Table 4. Figure 9 As shown in Table 4, the immobilized enzyme and the free enzyme both achieved a conversion rate of 99% for D-PPT after 6 h of reaction.

[0076] Example 12: Test of reusability of the immobilized amino acid oxidase

[0077] The experimental procedure of Example 11 was repeated, except that after incubation for 20 min, the DAAO (DAAO@SA-CMC-Na-ECH) was removed by filtration, the supernatant was separated and subjected to HPLC detection, the immobilized enzyme was retained, the buffer was washed 3 times, and the corresponding substances were directly added to the incubation system for re-catalysis. The reusability of the immobilized enzyme was characterized by repeating the above process 30 times and detecting the product concentration of each of the 30 times. The results are shown in Table 3. Figure 10 As shown in Table 3, the DAAO (DAAO@SA-CMC-Na-ECH) still maintained more than 65% of the enzyme activity after being reused 30 times.

[0078] Example 13: Activity and selectivity test of esterase immobilized by different cross-linking agents

[0079] Referring to the method of Example 1, the cross-linking agent was replaced by GA glutaraldehyde, Genipin, polyethyleneimine (PEI) or carbodiimide (EDC / NHS) to prepare esterase immobilized by different cross-linking agents. The enzymatic property of the immobilized esterase was evaluated according to the method of Example 6. The results are shown in Table 4.

[0080] Table 4: Comparison of enzymatic properties of esterase immobilized by different cross-linking agents

[0081]

[0082] The activity and selectivity of esterase immobilized by different cross-linking agents are shown in Table 4. Among them, Est@SA-CMC-Na-polyethyleneimine (PEI) disintegrated during the reaction, Est@SA-CMC-Na-Genipin disintegrated during the 14th cycle when the reusability was detected, and Est@SA-CMC-Na-carbodiimide (EDC / NHS) leaked enzyme when the reusability was detected.

[0083] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solution to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for immobilizing a biocatalyst, characterized in that, Includes the following steps: S1. Mix sodium alginate solution and sodium carboxymethyl cellulose solution evenly to obtain a carrier solution; S2. Dissolve the biocatalyst in buffer solution to prepare an enzyme solution, and then mix the enzyme solution with the carrier solution to obtain a mixture in which the enzyme is adsorbed on the immobilized carrier. S3. Add the mixture dropwise to the calcium chloride solution to form gel microspheres; S4. The gel microspheres are added to an epichlorohydrin solution for cross-linking to obtain an immobilized biocatalyst, which is an immobilized enzyme.

2. The method for immobilizing a biocatalyst according to claim 1, characterized in that: The concentration of sodium alginate solution in step S1 is 1~2 w / v.

3. The method for immobilizing a biocatalyst according to claim 1, characterized in that: The concentration of the sodium carboxymethyl cellulose solution in step S1 is 0.2~0.6 w / v.

4. The method for immobilizing a biocatalyst according to claim 1, characterized in that: The concentration of the calcium chloride solution in step S3 is 3~5 w / v.

5. The method for immobilizing a biocatalyst according to claim 1, characterized in that: In step S4, the concentration of the epichlorohydrin solution is 0.2~0.6 w / v.

6. The method for immobilizing a biocatalyst according to claim 1, characterized in that: In step S3, the particle size of the gel microspheres is 50~100um.

7. The method for immobilizing a biocatalyst according to claim 1, characterized in that: In step S2, the buffer solution is a sodium hydroxide solution of glycine.

8. The application of a method for immobilizing a biocatalyst as described in any one of claims 1-7 in the synthesis of chiral pesticides.

9. An immobilized enzyme for the production of enantiomeric chrysanthemic acid, prepared by the immobilization method of the biocatalyst as described in any one of claims 1-7, characterized in that: The enzyme includes a carboxylesterase mutant, the nucleotide sequence of which is shown in SEQ NO.1 and the amino acid sequence of which is shown in SEQ NO.

2.

10. An immobilized enzyme for the production of 4-(methylhydroxyphosphoryl)-2-carbonylbutyric acid, prepared by the immobilization method of the biocatalyst as described in any one of claims 1-7, characterized in that: The enzyme includes a D-amino acid oxidase mutant, the amino acid sequence of which is shown in SEQ NO.3.

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