A dicarboxylic acid modifier, a synthesis method thereof and application thereof in modifying recombinant trypsin

Abstract

The application discloses a dicarboxylic acid modifier, a synthesis method thereof and application of the dicarboxylic acid modifier in modifying recombinant trypsin, wherein the dicarboxylic acid modifier is 2-(3-carboxylpropylamido)-5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoic acid, the synthesis method takes mono-tert-butyl succinate and 1-glutamic acid tert-butyl ester as raw materials, and successfully prepares the dicarboxylic acid modifier (Suc-Glu-OSU) through amidation, glutamic acid side chain activation, re-amidation and tert-butyl ester deprotection. Suc-Glu-OSU is used as the modifier, the activated ester group (-OSU) at the end of Suc-Glu-OSU is covalently combined with lysine epsilon-NH2 of recombinant trypsin, a stable amide bond is formed, and modified recombinant trypsin TPS-Suc-Glu-OSU is obtained. The application reduces the occurrence of self-cleavage through the effects of steric hindrance shielding of a self-cleavage site, hydrophobic interaction strengthening of a stable conformation and the like, thereby enhancing the stability of the recombinant trypsin, and further widening the application of the recombinant trypsin in the industrial or scientific research field.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and more specifically to a dicarboxylic acid modifier, its synthesis method, and its application in modifying recombinant trypsin. Background Technology

[0002] Trypsin, due to its strong substrate specificity and high hydrolysis efficiency, is widely used in leather softening, food processing, clinical diagnostics, and biochemical detection, with a large market demand. However, the purity of trypsin obtained through traditional methods of extracting trypsin from mammalian pancreas is difficult to guarantee, impurities are unstable, and there is a risk of endogenous viral contamination. Recombinant trypsin, expressed as inclusion bodies in *E. coli*, is industrially produced using fermentation technology and then refolded. This allows for the industrial-scale production of recombinant trypsin, avoiding the limitations of simple extraction methods, such as raw material sourcing constraints, viral and other microbial contamination, and contamination by other enzymes.

[0003] In addition, due to the inherent mechanism of trypsin's action, recombinant trypsin still undergoes inactivation by cleaving the carboxyl terms of its arginine and lysine residues, resulting in unavoidable autolysis and affecting its stability and hydrolysis efficiency. During production, storage, and use, trypsin's activity rapidly declines or even becomes inactive due to its own mediated degradation, increasing production costs, shortening shelf life, and reducing efficiency, thus limiting its application range. Therefore, improving trypsin's resistance to autolysis and maintaining its stability has significant industrial value. Chemical modification, as a relatively simple and effective method for improving enzyme performance, has always attracted widespread attention. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a dicarboxylic acid modifier, its synthesis method, and its application in modifying recombinant trypsin. The active ester group (-OSU) at the end of the self-synthesized dicarboxylic acid modifier (Suc-Glu-OSU) undergoes a nucleophilic substitution reaction with the lysine residue ε-NH2 on the surface of recombinant trypsin to form a stable amide bond, thereby achieving covalent modification.

[0005] The technical solution adopted by this invention to solve the technical problem is: a dicarboxylic acid modifier, wherein the dicarboxylic acid modifier is 2-(3-carboxypropamido)-5-[(2,5-dioxopyrrolidone-1-yl)oxy]-5-oxovalerate, and its chemical structural formula is as follows: .

[0006] The present invention further discloses a method for synthesizing a dicarboxylic acid modifier. In the method, monotert-butyl succinate and 1-glutamic acid tert-butyl ester are used as raw materials, and the dicarboxylic acid modifier 2-(3-carboxypropamido)-5-[(2,5-dioxopyrrolidone-1-yl)oxy]-5-oxovalerate is synthesized by amidation, activation of glutamic acid side chain, re-amidation, and deprotection of tert-butyl ester.

[0007] Further, the amidation is specifically performed as follows: at 15~25 °C, monotert-butyl succinate, N-hydroxysuccinimide (HOSU) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) are added sequentially to reaction flask one, followed by the addition of acetonitrile. The reaction is stirred under nitrogen protection to obtain the first intermediate product, which requires no post-treatment and can be directly used in the next reaction.

[0008] Furthermore, during the amidation process, the molar ratio of monotert-butyl succinate, N-hydroxysuccinimide (HOSU), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) is 1:1.1~1.8:1.1~1.8.

[0009] Further, the activation of the glutamic acid side chain specifically involves: adding 1-glutamic acid tert-butyl ester and sodium carbonate aqueous solution to reaction flask two at 15-25°C, stirring to obtain a pale yellow clear solution; transferring the solution in reaction flask two to the amidated first intermediate product, stirring the reaction for 18-24 h, adjusting the pH of the system to 4-5; extracting and combining the organic phases, washing, drying, and concentrating the filtrate obtained by vacuum filtration to obtain the second intermediate product.

[0010] Furthermore, during the activation of the glutamic acid side chain, the molar ratio of 1-glutamic acid tert-butyl ester to the first intermediate is 1:1.1~1.8. Further, the reamidation specifically involves: at 15-25 °C, adding the second intermediate product with activated glutamic acid side chain, N-hydroxysuccinimide (HOSU), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) sequentially to reaction flask three, followed by the addition of acetonitrile; the molar ratio of the second intermediate product, N-hydroxysuccinimide (HOSU), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) is 1:1.1-1.8:1.1-1.8; after the reaction is completed under nitrogen protection, the organic phases are extracted and combined, washed, dried, and the filtrate obtained by vacuum filtration is concentrated to obtain the third intermediate product.

[0011] Further, the deprotection of the tert-butyl ester specifically involves: adding the re-amidated third intermediate to reaction flask four at 15-25 °C, followed by adding trifluoroacetic acid and stirring the reaction at 15-25 °C; the concentration of the third intermediate in the trifluoroacetic acid is 0.15-0.25 g / mL; after completion, the reaction system is concentrated, and excess trifluoroacetic acid is removed; then, after dissolving in ethyl acetate, the mixture is placed at -5 °C, petroleum ether is added, and a white solid precipitates. The solid is then filtered to obtain the crude target product, 2-(3-carboxypropamido)-5-[(2,5-dioxopyrrolidone-1-yl)oxy]-5-oxovalerate.

[0012] Furthermore, the present invention also discloses the application of a dicarboxylic acid modifier in the modification of recombinant trypsin, wherein the dicarboxylic acid modifier is 2-(3-carboxypropamido)-5-[(2,5-dioxopyrrolidone-1-yl)oxy]-5-oxovalerate, as described in any one of claims 1 to 87.

[0013] Furthermore, the dicarboxylic acid modifier is used to modify recombinant trypsin specifically by: under alkaline conditions, the lysine residue ε-NH2 on the surface of recombinant trypsin is deprotonated, and the dicarboxylic acid modifier undergoes a nucleophilic substitution reaction with the ε-NH2 through its terminal activated ester group (-OSU) to form a stable amide bond, thereby achieving covalent modification.

[0014] Furthermore, during the modification process, phosphate buffer solution with pH=7.8 was first added to the flask, and stirring was started. Recombinant trypsin was then added to the solution. After dissolution, the pH value was lowered to 5.0-6.0, and the pH was adjusted to 7.0-8.0 with triethylamine. The dicarboxylic acid modifier was dissolved in DMF solution at a molar ratio of 20:1 to 50:1 and added dropwise to the recombinant trypsin reaction solution. The pH of the reaction system was maintained at 7.0-8.0 with triethylamine. After the addition was complete, the reaction was stirred at room temperature for 1-2 hours to obtain the modified recombinant trypsin.

[0015] The beneficial effects of the present invention are as follows: Compared with the prior art, the present invention provides a dicarboxylic acid modifier, its synthesis method and its application in modifying recombinant trypsin. Under alkaline conditions, the lysine residue ε-NH2 on the surface of recombinant trypsin is deprotonated, and Suc-Glu-OSU undergoes a nucleophilic substitution reaction with the ε-NH2 through its terminal activated ester group (-OSU) to form a stable amide bond, thereby achieving covalent modification.

[0016] Based on the action and autolysis mechanisms of trypsin, hydrophilic or hydrophobic groups are used to modify the lysine and N-terminal amino sites of recombinant trypsin to perform single-site or multi-site modifications. By steric hindrance shielding of self-cleavage sites and hydrophobic interactions to enhance stable conformation, the occurrence of autolysis is reduced, thereby enhancing the stability of recombinant trypsin and further broadening its application in industrial or scientific research fields. Attached Figure Description

[0017] Figure 1 This is an HPLC chromatogram of mono-tert-butyl succinate, the raw material used in this invention.

[0018] Figure 2 This is the HPLC chromatogram of the first intermediate product in this invention.

[0019] Figure 3 This is the H-NMR spectrum of the first intermediate product in this invention.

[0020] Figure 4 This is the LC-MS chromatogram of the first intermediate product in this invention, where [M+H-56] + =216.

[0021] Figure 5 This is the HPLC chromatogram of the second intermediate product in this invention.

[0022] Figure 6 This is the H-NMR spectrum of the second intermediate product in this invention.

[0023] Figure 7 This is the LC-MS chromatogram of the second intermediate product in this invention, where [M+H-56-55] + =248.

[0024] Figure 8 This is the HPLC chromatogram of the third intermediate product in this invention.

[0025] Figure 9 This is the H-NMR spectrum of the third intermediate product in this invention.

[0026] Figure 10 This is the LC-MS chromatogram of the third intermediate product in this invention, where [M+H-56-55] + =345.1.

[0027] Figure 11 This is the H-NMR spectrum of the target product in this invention.

[0028] Figure 12 This is the LC-MS chromatogram of the target product in this invention, where [M+H] + =345.1.

[0029] Figure 13 The stability of recombinant trypsin and Suc-Glu-OSU modified enzyme at 37℃ was measured.

[0030] Figure 14 The results of SDS-PAGE analysis of unreduced (a) and reduced (b) Suc-Glu-OSU modified recombinant trypsin are shown. Detailed Implementation

[0031] The present invention will be further illustrated below with specific embodiments. However, these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] Example 1. Synthesis of dicarboxylic acid modifier (Suc-Glu-OSU) The synthetic route is shown in the figure below. Using mono-tert-butyl succinate and 1-glutamic acid tert-butyl ester as raw materials, the target dicarboxylic acid modifier 2-(3-carboxypropamido)-5-[(2,5-dioxopyrrolidone-1-yl)oxy]-5-oxovaleric acid, which can also be named N-succinyl-L-glutamic acid-γ-monoester-N-hydroxysuccinimide ester (Suc-Glu-OSU), is synthesized through amidation, activation, re-amidation, and deprotection.

[0033]

[0034] The specific synthesis process is as follows: (1) Synthesis of the first intermediate product CP-1-02 At 15–25 °C, 10 g of mono-tert-butyl succinate CP-1-01, 7.9 g of HOSU, and 13.2 g of EDCI were added sequentially to a reaction flask, followed by 50 mL of MeCN. The mixture was stirred for 2 h under nitrogen protection. The reaction was monitored by TLC (PE:EA = 3:1). Upon completion, the first intermediate (CP-1-02) was obtained, which required no further processing and could be used directly in the next reaction. The obtained first intermediate was subjected to HPLC, ¹H-NMR, and LC-MS ([M+H-56]). + =216) characterization, such as Figures 2-4 As shown, the formation of the first intermediate product (CP-1-02) was confirmed.

[0035] (2) Synthesis of the second intermediate product CP-1-03

[0036] At 15–25 °C, 9 g of 1-glutamic acid tert-butyl ester was added to reaction flask II, followed by 45 mL of 10% sodium carbonate aqueous solution. The mixture was stirred for 30 min, and the reaction system was a pale yellow and clear state. The solution in reaction flask II was transferred to reaction flask I by constant dropping, and the reaction was stirred at 15–25 °C for 18–24 h. The reaction was monitored by TLC (PE:EA = 2:1). After the reaction was completed, 2 mol / L hydrochloric acid was added to adjust the pH to 4–5. 50 mL of ethyl acetate was added for extraction, and the organic phase was separated twice. The organic phases were combined, washed twice with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 40 °C to obtain the crude second intermediate product CP-1-03. The obtained second intermediate product was subjected to HPLC, H-NMR and LC-MS ([M+H-56-55)). + =248) characterization, such as Figures 5-7 As shown, the formation of the second intermediate (CP-1-03) was confirmed.

[0037] (3) Synthesis of the third intermediate product CP-1-04

[0038] At 15–25 °C, the second intermediate CP-1-03 (10 g), HOSU (3.8 g), and EDCI (6.4 g) were added sequentially to reaction flask III, followed by 50 mL of MeCN. The mixture was stirred for 3 h under nitrogen protection at 15–25 °C. The reaction was monitored by TLC (PE:EA:TEA = 1:1:0.05). After the reaction was complete, 50 mL of ethyl acetate was added for extraction, and the organic phase was separated twice. The combined organic phases were washed twice with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 40 °C to obtain the crude third intermediate CP-1-04. The obtained third intermediate was subjected to HPLC, ¹H-NMR, and LC-MS ([M+H-56-55]). + =345.1) characterization, such as Figures 8-10 As shown, the formation of the third intermediate (CP-1-04) was confirmed.

[0039] (4) Synthesis of the target product CP-1-05

[0040] CP-1-04 (10 g) was added to reaction flask four at 15–25 °C, followed by trifluoroacetic acid (50 mL). The mixture was stirred at 15–25 °C for 2 h. After the reaction was complete, the reaction system was concentrated, and the trifluoroacetic acid was removed under vacuum at 35 °C. Then, 50 mL of ethyl acetate was added to dissolve the solid. The mixture was then placed at -5 °C, and 100 mL of petroleum ether was added. A white solid precipitated, which was filtered to obtain the crude target product (Suc-Glu-OSU). The obtained target product was subjected to ¹H NMR and LC-MS ([M+H)) analysis. + =345.1) characterization, such as Figures 11-12 As shown, the formation of the target product Suc-Glu-OSU was confirmed.

[0041] 2. Modification of recombinant trypsin by dicarboxylic acid modifier (Suc-Glu-OSU) (1) The principle of Suc-Glu-OSU modification Under alkaline conditions, the lysine residues ε-NH2 on the surface of recombinant trypsin are deprotonated. Suc-Glu-OSU undergoes a nucleophilic substitution reaction with the ε-NH2 through its terminal activated ester group (-OSU), forming a stable amide bond and achieving covalent modification.

[0042] (2) Suc-Glu-OSU modification method The lysine ε-NH2 of recombinant porcine trypsin was modified with a dicarboxylic acid modifier (Suc-Glu-OSU) with a molecular weight of 344.27 Da. The modifier (Suc-Glu-OSU) was added at a molar ratio of 45:1 to the recombinant trypsin, and the resulting recombinant trypsin was designated TPS-Suc-Glu-OSU.

[0043] Add 15 mL of phosphate buffer solution with pH 7.8 to the flask, start stirring, weigh 200 mg of recombinant porcine trypsin and add it to the solution. After dissolving, the pH value drops to 5.56. Adjust the pH to 7.0-8.0 with triethylamine. Weigh 129 mg of the side chain and dissolve it in 1 mL of LDM solution. Add the solution dropwise to the reaction solution and adjust the pH to 7.0-8.0 with triethylamine. After the addition is complete, stir at room temperature for 1.5 h.

[0044] 3. Determination of protein concentration Protein concentration was determined spectrophotometrically according to pharmacopoeia standards. 20 μL of the post-modification concentrate was diluted to 1 mL with 1 mmol / L HCl. Using a UV-Vis spectrophotometer, with the 1 mmol / L HCl solution as a blank control, the absorbance of the diluted solution at 280 nm was measured, and the protein concentration was calculated.

[0045] Protein concentration calculation formula:

[0046] Where: f is the dilution factor; 1.36 is the absorption coefficient of 1 mg / L recombinant trypsin at 280 nm in this buffer.

[0047] 4. Determination of Suc-Glu-OSU modified trypsin protein concentration, enzyme activity, and specific activity. The modified enzyme (TPS-Suc-Glu-OSU) and the unmodified control enzyme (TPS-CK) were diluted to a final concentration of 5 mg / mL with 67 mmol / L phosphate buffer (pH 7.6) and incubated at 37°C in the dark. Samples were taken at 0, 2, 4, 6, 8, and 26 h to determine protein concentration, enzyme activity, and specific activity. The results are shown in Table 1.

[0048] Table 1. Data on protein concentration, enzyme activity, and specific activity of recombinant trypsin before and after Suc-Glu-OSU modification.

[0049] Analysis of Table 1 shows that after modifying trypsin with Suc-Glu-OSU, the specific activity of the modified trypsin TPS-Suc-Glu-OSU decreased by 56.0% compared to the unmodified enzyme, indicating that the modification process significantly inhibited catalytic activity. This phenomenon may be due to the hygroscopic nature of Suc-Glu-OSU, leading to incomplete drying and residual organic solvents causing partial enzyme inactivation. Alternatively, the dicarboxylic acid structure of Suc-Glu-OSU may introduce an additional negative charge, interfering with the electrostatic complementarity of the substrate-binding pocket (S1 pocket), or it may hinder substrate (BAEE) from entering the active site through steric hindrance.

[0050] 5. Changes in the stability of trypsin modified with Suc-Glu-OSU After incubation at 37℃ for different times, the relative activities of trypsin before and after the Suc-Glu-OSU modification reaction were measured. The results are shown in Table 2 and... Figure 13As shown, after 8 h of incubation, the relative activity of the modified enzyme TPS-Suc-Glu-OSU was 4.1 times that of the unmodified enzyme; after 26 h of incubation, the modified enzyme still retained 59.94% of its activity, while the control group retained only 3.93%. The dicarboxylic acid group of Suc-Glu-OSU helps increase the negative charge density on the enzyme surface, thereby preventing positively charged autolysis sites (such as Lys131 and Arg122) from being recognized and cleaved by neighboring enzyme molecules through electrostatic repulsion. Simultaneously, it may enhance the rigidity of the random coil region (residues 112Lys-138Thr) through a hydrogen bond network, reducing the exposure of autolysis-sensitive sites and thus enhancing the stability of trypsin. Compared with other modification strategies, the stability improvement effect is better than N-terminal single-site modification, but lower than multi-site PEG modification. This strategy is suitable for industrial scenarios where catalytic efficiency requirements are low but long-term stability is needed.

[0051] Table 2. Stability data of recombinant modified enzymes before and after Suc-Glu-OSU modification at 37℃

[0052] 6. SDS-PAGE electrophoresis results of Suc-Glu-OSU modified trypsin In the stability assay of Suc-Glu-OSU modified trypsin, the recombinant trypsin samples incubated at 37℃ for different times were separated by SDS-PAGE, and the resulting bands were as follows: Figure 14 As shown.

[0053] from Figure 14 As can be seen, the self-degradation products of both the unmodified enzyme and the modified enzyme increased with the increase of incubation time. However, after the modification with the dicarboxylic acid modifier, the self-degradation products of the obtained recombinant trypsin sample were less than those of the unmodified enzyme, which is consistent with the effect of Suc-Glu-OSU modification on improving the stability of recombinant trypsin.

[0054] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention, and the patent protection scope of the present invention should be defined by the claims.

Claims

1. A dicarboxylic acid modifier, characterized in that, The dicarboxylic acid modifier is 2-(3-carboxypropionamido)-5-[(2,5-dioxopyrrolid-1-yl)oxy]-5-oxovaleric acid, and its chemical structural formula is as follows: .

2. A method for synthesizing a dicarboxylic acid modifier, characterized in that: In the synthesis method, monotert-butyl succinate and tert-butyl glutamate are used as raw materials. The dicarboxylic acid modifier 2-(3-carboxypropamido)-5-[(2,5-dioxopyrrolidone-1-yl)oxy]-5-oxovaleric acid is synthesized by amidation, activation of glutamate side chain, re-amidation and deprotection of tert-butyl ester.

3. The method for synthesizing a dicarboxylic acid modifier as described in claim 2, characterized in that, The amidation is specifically performed as follows: at 15-25 °C, mono-tert-butyl succinate, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are added sequentially to reaction flask one, followed by the addition of acetonitrile. The reaction is stirred under nitrogen protection to obtain the first intermediate product, which requires no post-processing and can be directly used in the next step of the reaction.

4. The method for synthesizing a dicarboxylic acid modifier as described in claim 3, characterized in that: During the amidation process, the molar ratio of monotert-butyl succinate, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:1.1~1.8:1.1~1.

8.

5. The method for synthesizing a dicarboxylic acid modifier as described in claim 3, characterized in that, The activation of the glutamic acid side chain is specifically as follows: at 15-25°C, 1-glutamic acid tert-butyl ester and sodium carbonate aqueous solution are added to reaction flask II, and after stirring, a pale yellow clear solution is obtained; the solution in reaction flask II is transferred to the amidated first intermediate product, and after stirring for 18-24 hours, the pH of the system is adjusted to 4-5; the organic phases are extracted and combined, washed, dried, and the filtrate obtained by vacuum filtration is concentrated to obtain the second intermediate product; During the activation of the glutamic acid side chain, the molar ratio of 1-glutamic acid tert-butyl ester to the first intermediate is 1:1.1~1.

8.

6. The method for synthesizing a dicarboxylic acid modifier as described in claim 5, characterized in that, The reamidation specifically involves adding the second intermediate product with activated glutamic acid side chain, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide sequentially to reaction flask three at 15-25 °C, followed by the addition of acetonitrile. The molar ratio of the second intermediate product, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:1.1-1.8:1.1-1.

8. After the reaction is completed under nitrogen protection, the organic phases are extracted and combined, washed, dried, and the filtrate obtained by vacuum filtration is concentrated to obtain the third intermediate product.

7. The method for synthesizing a dicarboxylic acid modifier as described in claim 6, characterized in that, The deprotection of tert-butyl ester specifically involves: adding the re-amidated third intermediate to reaction flask four at 15-25 °C, followed by the addition of trifluoroacetic acid; the concentration of the third intermediate in trifluoroacetic acid is 0.15-0.25 g / mL; stirring the reaction at 15-25 °C; concentrating the reaction system after the reaction is complete; removing excess trifluoroacetic acid; then dissolving the mixture in ethyl acetate; placing it at -5 °C; adding petroleum ether; precipitating a white solid; and filtering to obtain the crude target product 2-(3-carboxypropamido)-5-[(2,5-dioxopyrrolidone-1-yl)oxy]-5-oxovalerate.

8. The application of a dicarboxylic acid modifier in the modification of recombinant trypsin, characterized in that: The dicarboxylic acid modifier is 2-(3-carboxypropamido)-5-[(2,5-dioxopyrrolidone-1-yl)oxy]-5-oxovaleric acid, as described in any one of claims 1 to 7.

9. The application of the dicarboxylic acid modifier as described in claim 8 in the modification of recombinant trypsin, characterized in that, The dicarboxylic acid modifier is used to modify recombinant trypsin specifically by: under alkaline conditions, the lysine residue ε-NH2 on the surface of recombinant trypsin is deprotonated, and the dicarboxylic acid modifier undergoes a nucleophilic substitution reaction with the ε-NH2 through its terminal activated ester group -OSU to form a stable amide bond, thereby achieving covalent modification.

10. The application of the dicarboxylic acid modifier as described in claim 9 in the modification of recombinant trypsin, characterized in that: During the modification process, phosphate buffer solution with pH=7.8 was first added to the flask, and stirring was started. Recombinant trypsin was then added to the solution. After dissolution, the pH value was lowered to 5.0-6.0, and the pH value was adjusted to 7.0-8.0 with triethylamine. The dicarboxylic acid modifier was dissolved in DMF solution at a molar ratio of 20:1 to 50:1 and added dropwise to the recombinant trypsin reaction solution. The pH value of the reaction system was maintained at 7.0-8.0 with triethylamine. After the addition was completed, the reaction was stirred at room temperature for 1-2 hours to obtain the modified recombinant trypsin.

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