A chemical modifier and a method for preparing pepsin thereto.

Abstract

This invention relates to the field of pepsin preparation technology, specifically to a chemical modifier and a method for preparing pepsin. A novel composite modifier, Mal-PEG-CMC, is synthesized. By optimizing the molecular weight of PEG, the molar ratio of PEG to maleic anhydride, the degree of substitution of CMC, and the coupling ratio, a modifier with good water solubility and high coupling rate is obtained. Based on this modifier, chemically modified pepsin is prepared. The modified pepsin is obtained through pepsin purification, DTT pretreatment to expose thiol groups, targeted modification reaction, and purification steps. The modified pepsin exhibits significantly improved performance and is suitable for animal-derived and recombinant expression of pepsin, effectively overcoming the application bottleneck of natural enzymes and possessing significant value in food processing, pharmaceuticals, and other fields.

Description

Technical Field

[0001] This invention relates to the field of pepsin preparation technology, specifically to a chemical modifier and a method for preparing pepsin. Background Technology

[0002] Pepsin, an important aspartic protease, is widely used in food processing, medicine, and biotechnology. However, natural pepsin faces several technical bottlenecks, such as poor stability and environmentally dependent catalytic efficiency.

[0003] Existing chemical modification techniques mostly employ single-function modifiers, such as polyethylene glycol (PEG) and succinic anhydride. While these can improve stability to some extent, they suffer from problems such as random modification sites, low activity retention (usually <60%), and limited functionality. For example, although traditional PEG modification can improve thermal stability, its applicable pH range is still limited to pH 2.0-4.0. While single carboxymethyl chitosan modification can broaden pH adaptability, it can lead to a decrease in enzyme activity of more than 40%. Therefore, developing a novel chemical modifier that combines high activity, wide pH adaptability, and high thermal stability for the preparation of pepsin is of great significance. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a chemical modifier and a method for preparing pepsin therefrom.

[0005] To achieve the above objectives, 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 includes the following steps:

[0006] S1: Maleimide 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 the reaction at 25°C for 6-10 hours under nitrogen protection; filter, precipitate, centrifuge and dry the reaction solution to obtain maleimide-PEG, which is Mal-PEG;

[0007] 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. Stir and react at 25-35℃ for 10-14 hours. Dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 5000 Da for 60-84 hours and freeze-dry to obtain the Mal-PEG-CMC modifier.

[0008] This invention also provides a method for preparing pepsin using chemical modifiers, comprising the following steps:

[0009] Step 1: Pepsin purification: Take the pepsin raw material, extract it with acid buffer solution at pH 2.0-3.0, centrifuge, and purify it by column chromatography to obtain pure pepsin with a specific activity ≥2500U / mg;

[0010] Step 2: Enzyme pretreatment: Dissolve pure pepsin in 0.05-0.15M acetate buffer (pH 2.5-3.5), add dithiothreitol (DTT) to a final concentration of 0.5-2.5mM, react at 25-35℃ for 15-60 minutes, and remove DTT using a desalting column.

[0011] Step 3: Modification reaction: Add the Mal-PEG-CMC modifier described above to the pretreated enzyme solution at a molar ratio of enzyme to modifier of 1:2-15, and react for 1-3 hours at 25-35℃ and pH 4.0-4.5.

[0012] Step 4: Purification: The reaction solution is terminated, ultrafiltered, dialyzed, and freeze-dried to obtain modified pepsin powder.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] 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 exhibiting excellent water solubility. Compared to traditional single-function modifiers, this composite modifier combines the advantages of improved thermal stability of PEG with the broadened pH adaptability of CMC, avoiding the significant decrease in activity or limited functionality caused by single modification, thus laying the foundation for efficient enzyme modification.

[0015] 2. By exposing thiol groups through DTT pretreatment, targeted modification sites are achieved, reducing non-specific modifications and resulting in an enzyme activity retention rate >95%. Further, a stepwise modification method is employed to achieve a more uniform distribution of modification sites on the enzyme molecule surface, with a modification rate of 80% within 5 Å of the active site. The half-life at 60℃ is extended to 1.5-2.5 hours, and the activity retention rate is ≥85% after 30 days of storage at 4℃, overcoming the core bottleneck of poor stability in natural pepsin.

[0016] 3. The modified pepsin exhibits significantly improved pH adaptability, with relative activity ≥70% at pH 6.0 and ≥60% at pH 7.0, which is far higher than that of unmodified enzymes and traditional PEG-modified enzymes. This breaks through the limitation that natural pepsin can only function in strongly acidic environments, and can catalyze efficiently in neutral to slightly acidic environments.

[0017] 4. The modification method of this invention is applicable to both animal-derived pepsin and recombinant expressed pepsin. After modification, the bovine enzyme has a half-life of 1.9 hours at 60℃ and an activity of 78% at pH 6.0, while 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, thus solving the problem of strong dependence on enzyme source in traditional methods.

[0018] 5. In practical applications, the modified enzyme exhibits higher catalytic performance: In the soybean protein hydrolysis experiment, the degree of hydrolysis reached 35% after 4 hours of reaction at pH 6.0 and 50℃; in the meat tenderization experiment, the shear force decreased from 7.8 kgf in the blank group to 3.2 kgf, proving that it can play a high-efficiency role in food processing and other scenarios, and solving the problem that the catalytic efficiency of natural enzymes is limited by the environment.

[0019] In summary, this invention, through the design of composite modifiers, targeted modification, and stepwise optimization, simultaneously achieves high activity retention, high thermal stability, wide pH adaptability, and strong versatility of pepsin, effectively breaking through the bottlenecks of existing technologies and having significant application value in food processing, medicine, and other fields. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1: Synthesis of a novel modifier, Mal-PEG-CMC, and optimization of different PEG molecular weights and CMC substitution degrees.

[0022] (1) Prepare PEG with molecular weights of 1000 Da, 2000 Da, and 5000 Da, containing terminal hydroxyl groups, carboxymethyl chitosan (CMC) with substitution degrees of 0.6, 0.8, and 1.0, maleic anhydride, N,N'-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), dichloromethane (DCM), and anhydrous ethanol in advance;

[0023] (2) PEG maleimide: Take 10g of PEG and dissolve it in 50ml of 1000Da, 2000Da and 5000Da respectively in 50ml of LDCM. Add maleic anhydride according to the molar ratio of PEG: maleic anhydride = 1:1.2, 1:1.5 and 1:2.0. Then add 1.2g of DCC and 0.1g of DMAP. Stir and react for 8 hours at 25℃ under nitrogen protection.

[0024] (3) The reaction solution was filtered through a 0.22 μm filter membrane, and 10 times the volume of anhydrous ethanol was added to the filtrate to precipitate it. The precipitate was collected by centrifugation at 8000 rpm for 10 min and then vacuum dried to obtain maleimide-PEGMal-PEG.

[0025] As shown above, when the molecular weight of PEG is 2000 Da and the ratio of PEG to maleic anhydride is 1:1.5, the maleimide substitution rate is the highest at 92%. This condition was selected for subsequent reactions.

[0026] (4) Coupling of Mal-PEG with CMC: Dissolve 5g of CMC with a degree of substitution of 0.6, 0.8 and 1.0 in 100mL of deionized water and adjust the pH to 6.0. Add Mal-PEG2000Da and 0.5g DCC in a mass ratio of Mal-PEG:CMC=1:1, 2:1 and 3:1. Stir at 30℃ for 12 hours.

[0027] (5) The reaction solution was dialyzed for 72 hours through a dialysis bag with a molecular weight cutoff of 5000 Da, and deionized water was used. The solution was changed every 6 hours and then freeze-dried to obtain the Mal-PEG-CMC modifier.

[0028] When the degree of CMC substitution is 0.8 and Mal-PEG:CMC = 2:1, the coupling rate reaches 90% and has been verified by 1H NMR spectrum. The modifier has the best water solubility, with a solubility of >30g / 100mL at 25℃. This condition is selected.

[0029] Example 2: Preparation of a novel chemically modified pepsin

[0030] (1) To purify pepsin, take pig gastric mucosa homogenate, adjust the pH to 2.5 with 1M HCl, extract at 4℃ for 2 hours, centrifuge at 12000rpm×20min to collect the supernatant, and pass it through a Sephadex G-75 column with 0.05M acetate buffer, pH 2.5 as the elution buffer. After purification, collect the active peak, freeze dry to obtain pure pepsin. The specific activity of pure pepsin is ≥3000U / mg.

[0031] (2) Enzyme pretreatment: Dissolve 1g of pure pepsin in 100mL of 0.1M acetate buffer, pH 3.0, and add 0.02g, 0.05g, and 0.1g of dithiothreitol (DTT, final concentrations of 0.5mM, 1.25mM, and 2.5mM, respectively). React at 30℃ for 15min, 30min, and 60min, respectively, to reduce disulfide bonds and expose thiol groups.

[0032] (3) DTT was removed by PD-10 desalting column and the thiol exposure was determined by Ellman reagent method.

[0033] Results: When the DTT concentration was 1.25 mM and the reaction time was 30 min, the sulfhydryl group exposure reached 6.2 per enzyme molecule, compared to 1.8 without DTT, and the enzyme activity retention rate was >95%. This condition was selected.

[0034] (4) Modification reaction: Add Mal-PEG-CMC modifier to the pretreated enzyme solution. The modifier was synthesized under the optimal conditions in 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℃ and pH 4.0 for 1h, 2h, and 3h, respectively.

[0035] (5) After the reaction was completed, the pH was adjusted to 7.0 with 0.1M NaOH to terminate the reaction. The mixture was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10000 Da at an operating pressure of 0.1 MPa. The retentate was collected and dialyzed with 0.05M acetate buffer at pH 3.0 for 48 hours. The modified pepsin powder was then freeze-dried.

[0036] Example 3: Step-by-step modification method optimization

[0037] (1) First step of modification:

[0038] Take the pepsin solution pretreated in Example 2, 1 g / 100 mL, add Mal-PEG-CMC2000 Da of enzyme:modifier = 1:4, react at 25 °C and pH 4.0 for 1 hour to reduce non-specific modification by using low reaction temperature;

[0039] (2) Second step of modification:

[0040] Add Mal-PEG-CMC with an enzyme:modifier ratio of 1:4 to the reaction solution, heat 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.

[0041] (3) Purification: Same as step 3 in Example 2, modify pepsin step by step;

[0042] (4) Results: After stepwise modification, the modification sites on the enzyme molecule surface are more evenly distributed. Mass spectrometry analysis showed that the modification rate within 5 Å of the active center reached 80%, the half-life at 60℃ was extended to 2.2 hours, which is 10% higher than that of the one-step method, and the relative activity at pH 6.0 reached 85%.

[0043] Example 4: Modification effects of pepsin from different sources

[0044] (1) Bovine pepsin modification: Bovine pepsin was modified under the optimal conditions of Example 2, wherein the enzyme:modifier = 1:8, 35°C, and pH 4.0, and the specific activity of pepsin was 2800 U / mg. The performance was tested.

[0045] (2) Modification of recombinant pepsin: The recombinant human pepsin expressed by Escherichia coli with a pepsin specific activity of 2500 U / mg was modified in the same way and its performance was tested.

[0046] (3) Results: The bovine modified enzyme had a half-life of 1.9 hours at 60℃ and a relative activity of 78% at pH 6.0; the recombinant modified enzyme had a half-life of 1.8 hours at 60℃ and a relative activity of 75% at pH 6.0. 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, proving that this method is applicable to pepsin from different sources.

[0047] Example 5: Application Scenario Testing

[0048] (1) Soybean protein hydrolysis experiment: Under the conditions of pH 6.0 and 50℃, 10% soybean protein solution was treated with the modified enzyme of this invention, the unmodified enzyme, and Comparative Example 2, and the degree of hydrolysis was determined.

[0049] (2) Results: After 4 hours of reaction, the modified enzyme DH of the present invention reached 35%, the unmodified enzyme only 12%, and the PEG-modified enzyme 22%;

[0050] (3) Meat tenderizing experiment: 200 U / g of the modified enzyme was applied to the surface of the beef and placed at 25°C for 4 hours. The shear force was then measured.

[0051] (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 proves that the modified enzyme still maintains its high tenderizing ability under neutral and mild conditions.

[0052] Comparative Example 1: Unmodified pepsin

[0053] (1) Take the purified pepsin from Example 2 and perform performance testing directly.

[0054] Comparative Example 2: Traditional PEG-modified pepsin

[0055] (1) Using monofunctional methoxy PEG, mPEG, 2000 Da as a modifier, the reaction was carried out at 35°C and pH 4.0 for 2 hours with enzyme: mPEG = 1:8. The remaining steps were the same as in Example 2.

[0056] Comparative Example 3: Single CMC-modified pepsin

[0057] (1) Activate CMC with EDC, with a carboxyl activation rate of 70%, and react at 35°C and pH 4.0 for 2 hours with enzyme:CMC = 1:8. The remaining steps are the same as in Example 2.

[0058] Comparative Example 4: Mal-PEG-chitosan non-carboxymethylated modified pepsin

[0059] (1) Replace CMC with chitosan and synthesize Mal-PEG-chitosan modifier according to the method of Example 1. React according to enzyme:modifier = 1:8. The remaining steps are the same as in Example 2. Then compare the differences in pH response.

[0060] Comparative Example 5: Random Modification Method with No Preprocessing Step

[0061] (1) The DTT pretreatment in Example 2 was omitted, and pepsin was directly modified with Mal-PEG-CMC with enzyme:modifier = 1:8. The remaining steps were the same, and the necessity of thiol-targeted modification was verified.

[0062] Performance tests were conducted on Examples 2, 3, and 4 and Comparative Examples 1, 2, and 5, and the results are shown in Table 1.

[0063] Table 1 shows the performance test results of the examples and comparative examples.

[0064]

[0065] It should be noted that in Table 1, Example 4A refers to the performance test results of bovine pepsin modification in Example 4, and Example 4B refers to the performance test results of recombinant pepsin modification in Example 4.

[0066] The following conclusions can be drawn from the performance test results in Table 1:

[0067] (1) In terms of specific activity, the specific activities of Examples 2 and 3 are significantly higher than those of Comparative Examples 2 and 5. Although they are slightly lower than those of the unmodified enzyme, they still maintain a high activity retention rate. Among them, the specific activity of the stepwise modification method is the best, indicating that the modification method of the present invention can effectively reduce the loss of activity.

[0068] (2) Regarding thermal stability, the half-life of Examples 2, 3, and 4 is much longer than that of Comparative Examples 1, 2, and 5. The half-life of the stepwise modification is 10% longer than that of the one-step modification, indicating that the modification of the present invention significantly enhances the thermal stability of pepsin, and the stepwise modification further optimizes the stability.

[0069] (3) Regarding pH adaptability, under pH 6.0 conditions, the relative activities of Examples 2, 3, and 4 are much higher than those of Comparative Examples 1, 2, and 5; under pH 7.0 conditions, Examples 2 and 3 are 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 applicability, especially in a neutral to slightly acidic environment, and the stepwise modification method has the best pH adaptability.

[0070] (4) Regarding 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 at low temperatures and the stepwise modification method had the best storage stability.

[0071] (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 pepsin modified in this invention has a stronger binding ability to the substrate, and the stepwise modification method has the best affinity.

[0072] (6) Regarding catalytic efficiency: The degree of hydrolysis of Examples 2, 3 and 4 is significantly higher than that of Comparative Examples 1, 2 and 5. The degree of hydrolysis of the stepwise modification is the highest, indicating that the modified pepsin of the present invention has a higher catalytic hydrolysis efficiency for substrates in practical applications.

[0073] In summary, the data in Table 1 show that the modification method of the present invention, especially the stepwise method, has significant advantages over unmodified enzymes, traditional PEG-modified enzymes, and random-modified enzymes without pretreatment in terms of activity retention, thermal stability, pH adaptability, storage stability, substrate affinity, and catalytic efficiency.

[0074] Table 1 does not include data from Example 1 and Comparative Examples 3 and 4. The main reason for this is related to the experimental objectives, testing focus, and data completeness of each part, as explained below:

[0075] (1) The core of Example 1 is to optimize the synthesis conditions of the novel modifier Mal-PEG-CMC, and finally obtain a modifier with good water solubility and high coupling rate. The test index is the physicochemical 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 "performance comparison of modified pepsin". Example 1 does not involve enzyme modification and performance testing, so it was not included.

[0076] (2) Comparative Example 3 is “single CMC modified pepsin”, and its core defect is clearly stated above: “single carboxymethyl chitosan modification will lead to a decrease in enzyme activity of more than 40%”. Its design purpose is to highlight the advantages of the “composite modifier (Mal-PEG-CMC)” in this invention by contrasting the key defect of “significant decrease in activity”. It does not require complete performance data support, so it is not included in Table 1;

[0077] (3) Comparative Example 4 is “Mal-PEG-chitosan non-carboxymethylation modification”, the purpose of which is to “compare the differences in pH response”, that is, to focus only on the pH adaptability indicator and not to mention other performance data such as specific activity, half-life, and storage stability. Because its test dimensions are limited and its core function is to verify the importance of “carboxymethylation” to pH adaptability rather than to comprehensively compare enzyme performance, it was not included in Table 1, which requires comprehensive comparison of multiple indicators.

[0078] This article highlights the significant advantages and outstanding effects of the novel chemically modified pepsin and its preparation method through comparisons of examples and comparative examples, as follows:

[0079] Example 1 synthesized a novel Mal-PEG-CMC modifier by optimizing the molecular weight of PEG, the molar ratio of PEG to maleic anhydride, the degree of substitution of CMC, and the mass ratio of Mal-PEG to CMC. This modifier achieved a maleimide substitution rate of 92% and a coupling rate of 90%, exhibiting excellent water solubility that far surpasses the modification efficiency and physicochemical properties of PEG in Comparative Example 2 and CMC in Comparative Example 3, laying the foundation for highly efficient modified enzymes.

[0080] Example 2 exposed thiol groups through DTT pretreatment, and combined with optimized modification ratios and conditions, achieved targeted modification. 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 increased from 15% to 82%, and the activity retention rate after 30 days of storage at 4°C increased from 45% to 90%. Compared with Comparative Example 2, the specific activity increased from 2100 U / mg to 2850 U / mg, and the activity at pH 7.0 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 increased from 45% to 82%, demonstrating that targeted modification can efficiently retain activity and improve stability.

[0081] Example 3 employs a step-by-step approach of initial low-temperature modification followed by precise temperature-induced modification, reducing non-specific modifications and improving the modification efficiency of sites surrounding the active center. Compared to Example 2, the enzyme surface modification sites are more uniform, the modification rate within 5 Å of the active center reaches 80%, the half-life at 60℃ is extended from 2.0 h to 2.2 h, the activity at pH 6.0 is increased from 82% to 85%, and the degree of hydrolysis of soybean protein is increased from 35% to 37%, further optimizing enzyme performance.

[0082] Example 4 is applicable to both bovine pepsin and recombinant human pepsin. After modification, the bovine enzyme has a half-life of 1.9 h at 60 °C and an activity of 78% at pH 6.0; the recombinant enzyme has a half-life of 1.8 h and an activity of 75% at pH 6.0, both of which are significantly better than the unmodified enzyme, proving that this method is universally applicable to pepsin from different sources.

[0083] Example 5 shows that the enzyme maintained high catalytic efficiency under neutral to slightly acidic conditions. After 4 hours of hydrolysis of soybean protein, the degree of hydrolysis reached 35%, which was significantly higher than that of the unmodified enzyme and the PEG-modified enzyme. In meat tenderization, the shear force decreased from 7.8 kgf in the blank group to 3.2 kgf, which was better than that of the unmodified enzyme group, proving that the modified enzyme has a better effect in food processing.

[0084] Through novel modifier design, targeted modification, and stepwise optimization, this method significantly improves the thermal stability, pH adaptability, and activity retention of pepsin compared to traditional methods and unmodified enzymes. It is applicable to enzymes from different sources and exhibits more efficient catalytic performance in food processing and other scenarios, thus solving the bottlenecks of poor stability and environmentally restricted activity of natural enzymes.

[0085] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only to 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 includes the following steps: S1: Maleimide 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 the reaction at 25°C for 6-10 hours under nitrogen protection; filter, precipitate, centrifuge and dry the reaction solution to obtain maleimide-PEG, which is 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. Stir and react at 25-35℃ for 10-14 hours. Dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 5000 Da for 60-84 hours and freeze-dry to obtain the Mal-PEG-CMC modifier.

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

5. 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 is carried out in steps: First, the enzyme and the modifier are reacted at a molar ratio of 1:3-5 at 25℃ and pH 4.0 for 0.5-1.5 hours; Second, the modifier is added in the same molar ratio, the temperature is raised to 30-35℃, 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 mentioned in step one is animal-derived pepsin, including porcine, bovine, and recombinantly expressed pepsin.

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

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

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