Chemical modifier and method for preparing pepsin by using chemical modifier

Abstract

The invention relates to the technical field of pepsin preparation, in particular to a chemical modifier and a method for preparing pepsin through the chemical modifier, a novel composite modifier Mal-PEG-CMC is synthesized, and the modifier with good water solubility and high coupling rate is obtained by optimizing the molecular weight of PEG, the molar ratio of PEG to maleic anhydride, the substitution degree of CMC and the coupling ratio; the chemically modified pepsin is prepared based on the modifier, and the modified pepsin is obtained through the steps of pepsin purification, sulfydryl exposure through DTT pretreatment, targeted modification reaction and purification. The performance of the modified pepsase is remarkably improved, the pepsase is suitable for animal source and recombinant expression pepsase, the application bottleneck of natural enzyme is effectively broken through, and the pepsase has important value in the fields of food processing, medicine and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of pepsin preparation, in particular to a chemical modifier and a method for preparing pepsin therefrom. Background Art

[0002] As an important aspartic protease, pepsin is widely used in food processing, medicine, and biotechnology. However, natural pepsin has the following technical bottlenecks, such as poor stability, poor stability, and catalytic efficiency is restricted by the environment.

[0003] Existing chemical modification technologies mostly use single-functional modifiers, such as polyethylene glycol (PEG) and succinic anhydride. Although they can improve stability to a certain extent, they have problems such as random modification sites, low activity retention rate (usually less than 60%), and single function. For example, although traditional PEG modification can improve thermal stability, the pH range of application is still limited to pH 2.0-4.0. Although single carboxymethyl chitosan modification can broaden pH adaptability, it will cause the enzyme activity to decrease by more than 40%. Therefore, it is of great significance to develop a new chemical modifier with high activity, wide pH adaptability and high thermal stability for the preparation of pepsin. Summary of the Invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a chemical modifier and a method for preparing pepsin.

[0005] To achieve the above object, the present invention provides the following technical solution: a chemical modifier, wherein the modifier is Mal-PEG-CMC, and the synthesis method of Mal-PEG-CMC comprises the following steps: S1: Maleimidation of PEG: Dissolve PEG with a molecular weight of 1000-5000 Da in dichloromethane, add maleic anhydride at a molar ratio of PEG to maleic anhydride of 1:1.2-2.0, then add DCC and DMAP, and stir at 25°C under nitrogen for 6-10 hours. The reaction solution is filtered, precipitated, centrifuged, and dried to obtain maleimide-PEG, namely Mal-PEG. S2: Coupling of Mal-PEG and CMC: Dissolve carboxymethyl chitosan (CMC) with a degree of substitution of 0.6-1.0 in deionized water and adjust the pH to 5.5-6.5. Add Mal-PEG and DCC obtained in S1 at a mass ratio of Mal-PEG to CMC of 1:1-3:1, and stir at 25-35°C for 10-14 hours. The reaction solution is dialyzed through a dialysis bag with a molecular weight cutoff of 5000Da for 60-84 hours and freeze-dried to obtain the Mal-PEG-CMC modifier.

[0006] The present invention also provides a method for preparing pepsin using a chemical modifier, comprising the following steps: Step 1: Pepsin purification: Take the pepsin raw material, extract it with pH 2.0-3.0 acid buffer, centrifuge it, and purify it with a chromatography column to obtain a pure pepsin product with a specific activity ≥ 2500 U / mg; Step 2: Enzyme pretreatment: Dissolve pure pepsin in 0.05-0.15 M acetate buffer (pH 2.5-3.5), add dithiothreitol (DTT) at a final concentration of 0.5-2.5 mM, react at 25-35°C for 15-60 minutes, and remove DTT via a desalting column. Step 3: Modification reaction: Add the above-mentioned Mal-PEG-CMC modifier to the pretreated enzyme solution at a molar ratio of 1:2-15 between enzyme and modifier, and react at 25-35°C and pH 4.0-4.5 for 1-3 hours; Step 4: Purification: The reaction solution is terminated, ultrafiltered, dialyzed, and freeze-dried to obtain modified pepsin powder.

[0007] Compared with the prior art, the present invention has the following beneficial effects: 1. The Mal-PEG-CMC composite modifier synthesized in this invention achieves a 92% maleimide substitution rate and a 90% coupling rate by optimizing the PEG molecular weight, maleic anhydride molar ratio, and CMC substitution degree, while also exhibiting excellent water solubility. Compared to traditional monofunctional modifiers, this composite modifier combines the enhanced thermal stability of PEG with the broadened pH adaptability of CMC, avoiding the significant decrease in activity or monofunctionality often associated with single modifications, laying the foundation for efficient enzyme modification.

[0008] 2. DTT pretreatment exposes thiol groups, achieving targeted modification sites and reducing nonspecific modification, resulting in enzyme activity retention exceeding 95%. Furthermore, a step-by-step modification approach resulted in a more uniform distribution of modification sites on the enzyme surface, achieving an 80% modification rate within 5Å of the active center, extending the half-life at 60°C to 1.5-2.5 hours, and maintaining activity at ≥85% after 30 days of storage at 4°C, addressing the core bottleneck of natural pepsin's poor stability.

[0009] 3. The pH adaptability of the modified pepsin is significantly improved, with a relative activity of ≥70% at pH 6.0 and ≥60% at pH 7.0, which is much higher than that of the unmodified enzyme and the traditional PEG-modified enzyme. This breaks through the limitation that natural pepsin only works in a strongly acidic environment and can efficiently catalyze in a neutral to slightly acidic environment.

[0010] 4. The modification method of this invention is applicable to both animal-derived and recombinantly expressed pepsin. The modified bovine enzyme has a half-life of 1.9 hours at 60°C and 78% activity at pH 6.0. The recombinant enzyme has a half-life of 1.8 hours and 75% activity at pH 6.0, both significantly outperforming the unmodified enzyme, thus resolving the traditional method's dependence on enzyme source.

[0011] 5. In practical applications, the modified enzyme exhibited higher catalytic performance: in the soy protein hydrolysis experiment, the degree of hydrolysis reached 35% after 4 hours of reaction at pH 6.0 and 50°C; in the meat tenderizing experiment, the shear force dropped from 7.8 kgf in the blank group to 3.2 kgf, proving that it can play an efficient role in food processing and other scenarios, solving the problem that the catalytic efficiency of natural enzymes is restricted by the environment.

[0012] In summary, the present invention achieves high activity retention, high thermal stability, wide pH adaptability, and strong universality of pepsin through composite modifier design, targeted modification, and step-by-step optimization, effectively breaking through the bottleneck of existing technologies and having important application value in food processing, medicine, and other fields. DETAILED DESCRIPTION

[0013] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0014] Example 1: Synthesis of the novel modifier Mal-PEG-CMC, optimization of different PEG molecular weights and CMC substitution degrees (1) Prepare in advance PEG with molecular weight of 1000Da, 2000Da, and 5000Da, containing terminal hydroxyl groups, carboxymethyl chitosan CMC (degree of substitution 0.6, 0.8, and 1.0), maleic anhydride, N,N'-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), dichloromethane (DCM), and anhydrous ethanol; (2) PEG maleimidation: 10 g of PEG of 1000 Da, 2000 Da, and 5000 Da were dissolved in 50 mL of DCM. Maleic anhydride was added at a molar ratio of PEG: maleic anhydride = 1:1.2, 1:1.5, and 1:2.0. 1.2 g of DCC and 0.1 g of DMAP were then added. The mixture was stirred at 25 °C under nitrogen for 8 h. (3) The reaction solution was filtered through a 0.22 μm filter membrane, and 10 volumes of anhydrous ethanol were added to the filtrate for precipitation. The filtrate was allowed to stand at 4°C for 12 hours, and the precipitate was collected by centrifugation at 8000 rpm for 10 minutes. The precipitate was then vacuum dried to obtain maleimide-PEGMal-PEG. From the above, we know that when the PEG molecular weight is 2000Da and the PEG:maleic anhydride ratio is 1:1.5, the maleimide substitution rate is the highest at 92%, and this condition is selected for the subsequent reaction; (4) Conjugation of Mal-PEG and CMC: 5 g of CMC with substitution degree of 0.6, 0.8, or 1.0 was dissolved in 100 mL of deionized water and the pH was adjusted to 6.0. Mal-PEG 2000 Da and 0.5 g of DCC were added at a mass ratio of Mal-PEG:CMC = 1:1, 2:1, or 3:1, and the reaction was stirred at 30 °C for 12 h. (5) The reaction solution was dialyzed for 72 hours using a dialysis bag with a molecular weight cutoff of 5000 Da, with deionized water replaced every 6 hours, and freeze-dried to obtain the Mal-PEG-CMC modifier.

[0015] When the CMC substitution degree is 0.8 and Mal-PEG:CMC=2:1, the coupling rate reaches 90% and is verified by nuclear magnetic resonance hydrogen spectrum. The water solubility of the modifier is best, dissolving at 25℃>30g / 100mL, so this condition is selected.

[0016] Example 2: Preparation of a novel chemically modified pepsin (1) Purify pepsin by homogenizing pig gastric mucosa, adjusting the pH to 2.5 with 1M HCl, extracting at 4°C for 2 hours, centrifuging at 12000 rpm for 20 minutes, and collecting the supernatant. Purify the supernatant by passing it through a Sephadex G-75 column, using 0.05M acetic acid buffer at pH 2.5 as the eluent. Collect the active peak and lyophilize to obtain pure pepsin. The specific activity of pure pepsin is ≥3000 U / mg. (2) Enzyme pretreatment: 1 g of pure pepsin was dissolved in 100 mL of 0.1 M acetate buffer, pH 3.0, and 0.02 g, 0.05 g, and 0.1 g of dithiothreitol (DTT, final concentration 0.5 mM, 1.25 mM, and 2.5 mM) were added, respectively. The reaction was carried out at 30°C for 15 min, 30 min, and 60 min, respectively, to reduce the disulfide bonds and expose the sulfhydryl groups. (3) DTT was removed by PD-10 desalting column, and the amount of thiol exposure was determined by Ellman's reagent method.

[0017] Results: When the DTT concentration was 1.25 mM and the reaction time was 30 min, the exposed sulfhydryl groups reached 6.2 per enzyme molecule, while that was 1.8 per enzyme molecule without DTT. The enzyme activity retention rate was >95%, so this condition was selected. (4) Modification reaction: Mal-PEG-CMC modifier was added to the pretreated enzyme solution. The modifier was synthesized according to the optimal conditions of Example 1. The enzyme:modifier molar ratio was set to 1:2, 1:5, 1:8, 1:10, and 1:15. The reaction was carried out at 35°C and pH 4.0 for 1 h, 2 h, and 3 h, respectively. (5) After the reaction, the pH was adjusted to 7.0 with 0.1 M NaOH to terminate the reaction. The product was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da at an operating pressure of 0.1 MPa. The retentate was collected and dialyzed with 0.05 M acetic acid buffer at pH 3.0 for 48 hours. The product was freeze-dried to obtain the modified pepsin powder.

[0018] Example 3: Optimization of the step-by-step modification method (1) The first step of modification: The pepsin solution pretreated in Example 2 was taken at 1 g / 100 mL, and Mal-PEG-CMC 2000 Da was added at a ratio of enzyme:modifier = 1:4. The reaction was carried out at 25° C. and pH 4.0 for 1 hour to reduce nonspecific modification by using a low reaction temperature. (2) Second step modification: Add Mal-PEG-CMC to the reaction solution at a ratio of 1:4 enzyme:modifier, raise the temperature to 35°C, adjust the pH to 4.5, and continue the reaction for 1 hour to improve the modification efficiency of sites around the active center. (3) Purification: As in step 3 of Example 2, pepsin was modified step by step; (4) Results: After step-by-step modification, the modified sites on the surface of the enzyme molecule were more evenly distributed. Mass spectrometry analysis showed that the modification rate within 5Å of the active center reached 80%, and the half-life at 60°C was extended to 2.2 hours, which was 10% higher than that of the one-step method. The relative activity at pH 6.0 reached 85%.

[0019] Example 4: Modification Effects of Pepsin from Different Sources (1) Modification of bovine pepsin: According to the optimal conditions of Example 2, wherein the enzyme:modifier = 1:8, 35°C, pH 4.0, the bovine pepsin was modified, and the pepsin specific activity was 2800 U / mg, and the performance was tested; (2) Recombinant pepsin modification: The recombinant human pepsin expressed in Escherichia coli with a pepsin specific activity of 2500 U / mg was modified in the same way and the performance was tested; (3) Results: The half-life of the bovine modified enzyme at 60°C was 1.9 hours, and the relative activity at pH 6.0 was 78%; the half-life of the recombinant modified enzyme at 60°C was 1.8 hours, and the relative activity at pH 6.0 was 75%. Both were significantly better than the unmodified enzyme, with a half-life of <0.3 hours and an activity of <20% at pH 6.0. This demonstrates that this method is applicable to pepsin from different sources.

[0020] Example 5: Application scenario testing (1) Soy protein hydrolysis experiment: At pH 6.0 and 50°C, 10% soy protein solution was treated with the modified enzyme of the present invention, the unmodified enzyme, and the comparative example 2, and the degree of hydrolysis was measured; (2) Results: After 4 hours of reaction, the DH of the modified enzyme of the present invention reached 35%, while that of the unmodified enzyme was only 12%, and that of the PEG-modified enzyme was 22%; (3) Meat tenderization experiment: 200 U / g of modified enzyme was applied to the surface of beef, placed at 25°C for 4 hours, and the shear force was measured; (4) Results: The shear force of the modified enzyme treatment group was 3.2 kgf, the unmodified enzyme group was 5.1 kgf, and the blank group was 7.8 kgf, which proved that the modified enzyme still maintained high tenderizing ability under neutral and mild conditions.

[0021] Comparative Example 1: Unmodified pepsin (1) The purified pepsin prepared in Example 2 was directly tested for performance.

[0022] Comparative Example 2: Traditional PEG-modified pepsin (1) Monofunctional methoxy PEG, mPEG, 2000 Da was used as the modifying agent, and the reaction was carried out at an enzyme:mPEG ratio of 1:8 at 35°C and pH 4.0 for 2 hours. The remaining steps were the same as in Example 2.

[0023] Comparative Example 3: Single CMC modified pepsin (1) CMC was activated with EDC, with a carboxyl activation rate of 70%, at an enzyme:CMC ratio of 1:8, at 35°C and pH 4.0 for 2 hours. The remaining steps were the same as in Example 2.

[0024] Comparative Example 4: Mal-PEG-chitosan non-carboxymethylated modified pepsin (1) Chitosan was used to replace CMC. Mal-PEG-chitosan modifier was synthesized according to the method of Example 1. The reaction was carried out at an enzyme:modifier ratio of 1:8. The remaining steps were the same as Example 2. Then, the pH responsiveness was compared.

[0025] Comparative Example 5: Random modification method without pretreatment step (1) The DTT pretreatment in Example 2 was omitted, and pepsin was directly modified with Mal-PEG-CMC, with an enzyme:modifier ratio of 1:8. The remaining steps were the same, and the necessity of thiol-targeted modification was then verified.

[0026] Performance tests were performed on Examples 2, 3, 4 and Comparative Examples 1, 2, 5. See Table 1 for performance test results.

[0027] Table 1 is the performance test results of the embodiments and comparative examples

[0028] It should be noted that Example 4A in Table 1 refers to the performance test results of the modified bovine pepsin in Example 4, and Example 4B refers to the performance test results of the modified recombinant pepsin in Example 4.

[0029] The following conclusions can be drawn from the performance test results in Table 1: (1) In terms of specific activity, the specific activities of Examples 2 and 3 were significantly higher than those of Comparative Examples 2 and 5. Although slightly lower than those of the unmodified enzyme, they still maintained a high activity retention rate. Among them, the specific activity of the step-by-step modification method was the best, indicating that the modification method of the present invention can effectively reduce activity loss; (2) In terms of thermal stability, the half-lives of Examples 2, 3, and 4 were much longer than those of Comparative Examples 1, 2, and 5. The half-life of the step-by-step modification was 10% higher than that of the one-step modification, indicating that the modification of the present invention significantly enhanced the thermal stability of pepsin, and the step-by-step modification further optimized the stability. (3) In terms of pH adaptability, at pH 6.0, the relative activities of Examples 2, 3, and 4 were much higher than those of Comparative Examples 1, 2, and 5; at pH 7.0, the relative activities of Examples 2 and 3 were also significantly higher than those of Comparative Examples 1, 2, and 5, indicating that the modified pepsin of the present invention has a wider pH range of application, especially in neutral to acidic environments. The pH adaptability of the step-by-step modification is the best. (4) In terms of storage stability, the retention rates of Examples 2, 3, and 4 were significantly higher than those of Comparative Examples 1, 2, and 5, indicating that the modified enzymes were more stable in activity when stored at low temperatures, and the step-by-step modification method had the best storage stability.

[0030] (5) Substrate affinity: The Km values ​​of Examples 2 and 3 are lower than those of Comparative Examples 1, 2, and 5. The smaller the Km value, the higher the affinity of the enzyme for the substrate. This indicates that the modified pepsin of the present invention has a stronger ability to bind to the substrate, and the affinity of the step-by-step modification is the best. (6) Catalytic efficiency: The hydrolysis degree of Examples 2, 3, and 4 is significantly higher than that of Comparative Examples 1, 2, and 5. The hydrolysis degree of the step-by-step modification is the highest, indicating that the modified pepsin of the present invention has a higher catalytic hydrolysis efficiency for substrates in practical applications.

[0031] In summary, the data in Table 1 show that the modification method of the present invention, especially the step-by-step method, has significant advantages over unmodified enzymes, traditional PEG-modified enzymes, and randomly modified enzymes without pretreatment in terms of activity retention, thermal stability, pH adaptability, storage stability, substrate affinity, and catalytic efficiency.

[0032] The performance test in Table 1 does not include the data of Example 1 and Comparative Examples 3 and 4. The main reason is related to the experimental purpose, test focus and data integrity of each part. The specific explanation is as follows: (1) The core of Example 1 is to optimize the synthesis conditions of the novel modifier Mal-PEG-CMC, ultimately obtaining a modifier with good water solubility and high coupling rate. Its test indicators are the physical and chemical properties of the modifier itself, such as "maleimide substitution rate", "coupling rate", and "water solubility", rather than the performance of the modified pepsin. Table 1 focuses on the "performance comparison of modified pepsin". Example 1 does not involve enzyme modification and performance testing, so it is not included. (2) Comparative Example 3 is “single CMC modified pepsin”. The above clearly states its core defect: “single carboxymethyl chitosan modification causes enzyme activity to decrease by more than 40%”. Its design purpose is to highlight the advantages of the “composite modifier (Mal-PEG-CMC)” in the present invention by comparing the key defect of “significantly decreased activity”. It does not require complete performance data support, so it is not included in Table 1; (3) Comparative Example 4 is “Mal-PEG-chitosan non-carboxymethylation modification”, the purpose of which is to “compare the differences in pH responsiveness”, that is, it only focuses on the pH adaptability indicator, and does not mention other performance data such as specific activity, half-life, and storage stability; due to its limited testing dimensions and its core function of verifying the importance of “carboxymethylation” to pH adaptability rather than a comprehensive comparison of enzyme performance, it was not included in Table 1, which requires a comprehensive comparison of multiple indicators.

[0033] This article highlights the significant advantages and outstanding effects of the novel chemically modified pepsin and its preparation method by comparing the examples with the comparative examples, as follows: Example 1: A novel Mal-PEG-CMC modifier was synthesized by optimizing the PEG molecular weight, the PEG to maleic anhydride molar ratio, the CMC substitution degree, and the Mal-PEG to CMC mass ratio. This modifier exhibited a maleimide substitution rate of 92%, a coupling rate of 90%, and excellent water solubility, far exceeding the modification efficiency and physicochemical properties of the PEG in Comparative Example 2 and the CMC in Comparative Example 3, laying the foundation for efficient enzyme modification. In Example 2, sulfhydryl groups were exposed by DTT pretreatment, and targeted modification was achieved by combining optimized modification ratios and conditions. Compared with Comparative Example 1, the half-life at 60°C was extended from 0.25 h to 2.0 h, the relative activity at pH 6.0 was increased from 15% to 82%, and the activity retention rate after storage at 4°C for 30 days was increased from 45% to 90%. Compared with Comparative Example 2, the specific activity was increased from 2100 U / mg to 2850 U / mg, and the activity at pH 7.0 was increased from 30% to 65%. Compared with Comparative Example 5, the half-life was extended from 1.0 h to 2.0 h, and the activity at pH 6.0 was increased from 45% to 82%, demonstrating that targeted modification can efficiently retain activity and improve stability. Example 3 uses low-temperature preliminary modification and elevated-temperature precise modification, performed step by step, to reduce nonspecific modification and improve the efficiency of modification around the active center. Compared with Example 2, the enzyme surface modification sites are more uniform, the modification rate within 5Å of the active center reaches 80%, the half-life at 60°C is extended from 2.0h to 2.2h, the activity at pH 6.0 is increased from 82% to 85%, and the degree of soy protein hydrolysis is increased from 35% to 37%, further optimizing the performance of the enzyme. Example 4 is applicable to both bovine pepsin and recombinant human pepsin. The modified bovine enzyme has a half-life of 1.9 hours at 60°C and an activity of 78% at pH 6.0; the recombinant enzyme has a half-life of 1.8 hours and an activity of 75% at pH 6.0, both significantly better than the unmodified enzyme, demonstrating the universal applicability of this method to pepsin from different sources. Example 5 still maintains high catalytic efficiency under neutral to slightly acidic conditions; after 4 hours of hydrolysis of soy protein, the hydrolysis degree reaches 35%, which is significantly higher than that of the unmodified enzyme and the PEG-modified enzyme; during meat tenderization, the shear force is reduced from 7.8 kgf in the blank group to 3.2 kgf, which is better than that of the unmodified enzyme group, proving that the modified enzyme has better application effect in food processing.

[0034] Through the design of new modifiers, targeted modification and step-by-step optimization, the thermal stability, pH adaptability and activity retention rate of pepsin have been significantly improved compared with traditional methods and unmodified enzymes. It is also applicable to enzymes from different sources and exhibits more efficient catalytic performance in scenarios such as food processing, solving the bottleneck of poor stability of natural enzymes and activity being restricted by the environment.

[0035] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A chemical modifier, characterized in that The modifier is Mal-PEG-CMC, and the synthesis method of Mal-PEG-CMC comprises the following steps: S1: Maleimidation of PEG: Dissolve PEG with a molecular weight of 1000-5000 Da in dichloromethane, add maleic anhydride at a molar ratio of PEG to maleic anhydride of 1:1.2-2.0, then add DCC and DMAP, and stir at 25°C under nitrogen for 6-10 hours. The reaction solution is filtered, precipitated, centrifuged, and dried to obtain maleimide-PEG, namely Mal-PEG. S2: Coupling of Mal-PEG and CMC: Dissolve carboxymethyl chitosan (CMC) with a degree of substitution of 0.6-1.0 in deionized water and adjust the pH to 5.5-6.

5. Add Mal-PEG and DCC obtained in S1 at a mass ratio of Mal-PEG to CMC of 1:1-3:1, and stir at 25-35°C for 10-14 hours. The reaction solution is dialyzed through a dialysis bag with a molecular weight cutoff of 5000Da for 60-84 hours and freeze-dried to obtain the Mal-PEG-CMC modifier.

2. A chemical modifier according to claim 1, characterized in that The molecular weight of PEG in S1 is 1500-3000 Da, and the molar ratio of PEG to maleic anhydride is 1:1.4-1.

6.

3. A chemical modifier according to claim 2, characterized in that, The degree of CMC substitution in S2 is 0.7-0.9, and the mass ratio of Mal-PEG to CMC is 1.5:1-2.5:

1.

4. A method for preparing pepsin using the chemical modifier according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: Step 1: Pepsin purification: Take the pepsin raw material, extract it with pH 2.0-3.0 acid buffer, centrifuge it, and purify it with a chromatography column to obtain a pure pepsin product with a specific activity ≥ 2500 U / mg; Step 2: Enzyme pretreatment: Dissolve pure pepsin in 0.05-0.15 M acetate buffer (pH 2.5-3.5), add dithiothreitol (DTT) at a final concentration of 0.5-2.5 mM, react at 25-35°C for 15-60 minutes, and remove DTT via a desalting column. Step 3: Modification reaction: Add the Mal-PEG-CMC modifier according to claim 1 to the pretreated enzyme solution at a molar ratio of 1:2-15 between enzyme and modifier, and react at 25-35° C. and pH 4.0-4.5 for 1-3 hours; Step 4: Purification: The reaction solution is terminated, ultrafiltered, dialyzed, and freeze-dried to obtain modified pepsin powder.

5. The method according to claim 4, characterized in that The modification reaction in step three adopts step-by-step modification: in the first step, the enzyme and modifier are reacted at a molar ratio of 1:3-5 at 25°C and pH 4.0 for 0.5-1.5 hours; in the second step, the modifier is added at the same molar ratio, the temperature is raised to 30-35°C, the pH is adjusted to 4.3-4.7, and the reaction is continued for 0.5-1.5 hours.

6. The method according to claim 5, characterized in that The pepsin raw material in step 1 is animal-derived pepsin, including one of porcine-derived, bovine-derived and recombinantly expressed pepsin.

7. The method according to claim 6, characterized in that Animal-derived pepsin includes recombinant human pepsin expressed in Escherichia coli.

8. The method according to claim 4, characterized in that In step 2, the final concentration of DTT is 1.0-1.5 mM, and the reaction time is 20-40 minutes.

9. The method according to claim 4, characterized in that In step 3, the molar ratio of enzyme to modifier is 1:6-10, the reaction temperature is 30-35° C., and the reaction time is 1.5-2.5 hours.

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