Carboxylic esterase lyophilization protectant, method of making and use thereof

Abstract

The application discloses a carboxylic esterase lyophilization protective agent, a preparation method and application thereof, and belongs to the technical field of bioengineering. The carboxylic esterase lyophilization protective agent is a single-component polyvinylpyrrolidone; wherein, the polyvinylpyrrolidone has a viscosity-average molecular weight Mv of 10000-15000000; a K value of 17-90; when used for lyophilization protection of carboxylic esterase, the carboxylic esterase lyophilized powder can effectively improve the stability of the carboxylic esterase lyophilized powder at normal temperature, prevent the purity and activity of the carboxylic esterase lyophilized powder from decreasing, and the lyophilized powder cake is complete, easy to be powdered, the enzyme powder resuspension is clear and transparent, the enzyme activity recovery rate is high, and the carboxylic esterase is convenient for being applied to different application scenarios.

Description

Technical Field

[0001] This invention relates to a carboxylesterase freeze-drying protectant, its preparation method, and its application, belonging to the field of bioengineering technology. Background Technology

[0002] SGNH family esterases are unique members of the esterase family. Their primary sequence contains highly conserved serine, glycine, asparagine, and histidine residues in regions I, II, III, and V, respectively, making them valuable for applications in daily chemicals, pharmaceuticals, and food processing. Chinese patent CN 120866273 A discloses an SGNH family esterase SH2 screened from a Tibetan hot spring environment. This recombinant esterase exhibits good thermal stability and broad pH adaptability. However, as a biomacromolecule, this enzyme still faces cold chain issues during storage and transportation under normal temperature liquid conditions, significantly increasing transportation costs and the risk of enzyme activity degradation. Long-term storage also leads to enzyme activity degradation, limiting the product's shelf life and severely restricting its industrial production and commercial application. Therefore, developing a stable formulation suitable for SGNH family esterase SH2 is of great significance.

[0003] Freeze-drying is currently one of the mainstream methods for improving the long-term stability of protein preparations. By pre-freezing the enzyme solution at low temperature and then removing moisture through vacuum sublimation, a dry enzyme powder preparation can be obtained, significantly extending the shelf life of the enzyme and facilitating transportation and use. However, the freeze-drying process itself can cause multiple types of damage to enzyme molecules, mainly including: mechanical damage caused by ice crystal formation, solute aggregation due to the freeze-concentration effect, and irreversible changes in protein conformation during dehydration. To mitigate these damages, a freeze-drying protectant needs to be added before freeze-drying to maintain the enzyme activity after freeze-drying and reconstitution.

[0004] Currently, commonly used freeze-drying protectants in existing technologies mainly include sugars (such as sucrose, trehalose, and maltodextrin), polyols (such as sorbitol and mannitol), macropolymers (such as polyethylene glycol, gelatin, and polyvinylpyrrolidone), and proteins (such as bovine serum albumin). Among these, sugars are widely used as protein freeze-drying protectants due to their excellent glass-forming ability and ability to substitute for hydrogen bonds in proteins. However, existing technologies generally suffer from the following problems: First, sugar protectants at high concentrations can easily cause the collapse of the freeze-dried cake structure, affecting the product's appearance and reconstitution performance; second, some protectants and enzyme solutions exhibit compatibility issues before freeze-drying, manifesting as increased turbidity or phase separation, affecting the homogeneity of the formulation; third, existing protectant systems largely rely on empirical screening, lacking systematic research on the compatibility between the protectant's molecular structure and the enzyme's surface characteristics, leading to unstable protective effects.

[0005] Existing research indicates that lyophilization protectants primarily maintain the native conformation of enzymes by forming hydrogen bonds with the enzyme surface through a "water substitution" mechanism or by forming a rigid glassy state to encapsulate the enzyme molecule and restrict its movement. However, the effectiveness of protectants is not universal; their compatibility with enzyme surface characteristics directly determines the level of protection efficiency. For enzymes like carboxylesterases, which have typical α / β hydrolases with folded structures, the compatibility between their surface characteristics and protectants is even more complex. On the one hand, the active site of carboxylesterases is mainly composed of a conserved catalytic triplet (Ser-His-Asp) and a precisely arranged oxygen anion well (such as N90 / G50) composed of amide hydrogens in the main chain. The overall framework is compact, with a densely packed hydrophobic core, resulting in low conformational plasticity. Existing protectants (such as trehalose and sucrose) mainly stabilize enzyme molecules through hydrogen bond substitution and glassy solidification, but if they are not well-suited to the enzyme surface, they can cause a series of specific damages. For example, small-molecule protectants (such as glycerol) can freely diffuse into open active pockets, where their hydroxyl groups form non-natural hydrogen bonds with catalytic serine or histidine, directly interfering with the charge relay network. Secondly, if the spatial distribution of the protectant's hydroxyl groups is misaligned with the hydrogen bond sites of polar residues on the enzyme surface, it can induce localized strain on the rigid backbone. While this strain does not lead to global denaturation, it is sufficient to cause the main chain amide hydrogens of the oxygen anion well to deviate from their correct orientation, losing their ability to stabilize the negative charge of the transition state. Furthermore, if the glassy matrix formed by the protectant after dehydration is too brittle (e.g., crystalline mannitol), the mechanical stress generated by its shrinkage can directly tear the delicate hydrogen bond network in the oxygen anion well region; if the matrix is ​​too soft (e.g., low molecular weight PEG), it cannot restrict the vibration of enzyme molecules, leading to the exposure of hydrophobic patches on the surface and causing irreversible aggregation. In addition, the uneven distribution of charge on the surface of carboxylesterases makes them particularly sensitive to localized microenvironment pH changes caused by freeze-concentration effects. If the protectant cannot provide suitable electrostatic shielding or preferential hydration, the ion interaction network on the enzyme surface may be disrupted, leading to irreversible structural rearrangement.

[0006] Therefore, the development of lyophilized formulations targeting carboxylesterases urgently requires a lyophilization protection agent screening strategy that can balance high enzyme activity recovery, good lyophilized cake form, rapid reconstitution, and system stability. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a carboxylesterase lyophilization protectant, its preparation method, and its application. This carboxylesterase uses a single-component polyvinylpyrrolidone as a lyophilization protectant, which can effectively improve the stability of the lyophilized carboxylesterase powder at room temperature and prevent its purity and activity from decreasing. Furthermore, the lyophilized powder cake is intact, easy to form into powder, and the enzyme powder resuspension is clear and transparent with a high enzyme activity recovery rate.

[0008] To achieve the above objectives, the following technical solution is provided: The first objective of this invention is to provide a carboxylesterase lyophilization protectant, which is a single-component polyvinylpyrrolidone.

[0009] In one embodiment, the polyvinylpyrrolidone has a viscosity-average molecular weight Mv of 10,000 to 15,000,000 and a K value of 17 to 90; preferably, the viscosity-average molecular weight Mv of 10,000 to 400,000 and the K value of 17 to 60; more preferably, the viscosity-average molecular weight Mv of 270,000 to 400,000 and the K value of 54 to 64.8, i.e., PVPK60.

[0010] In one embodiment, the amount of polyvinylpyrrolidone used is 20-100 g / L; preferably 40-100 g / L; more preferably 40-80 g / L; and even more preferably 40-60 g / L.

[0011] In one embodiment, the carboxylesterase is derived from Chinese patent CN 120866273 A.

[0012] A second objective of this invention is to provide the application of the aforementioned lyophilization protectant in the preparation of carboxylesterase lyophilized powder.

[0013] A third objective of this invention is to provide a carboxylesterase lyophilized powder, which is obtained by mixing a carboxylesterase and the aforementioned lyophilization protectant, followed by lyophilization.

[0014] In one embodiment, the water content of the carboxylesterase lyophilized powder is 1.0~6.0%.

[0015] A fourth objective of this invention is to provide a method for preparing the above-described carboxylesterase lyophilized powder, the method comprising: After mixing the carboxylesterase stock solution with the freeze-drying protectant described above, the solution is sterilized by filtration membrane. The filtrate is then subjected to pre-freezing, primary drying, and secondary drying in sequence to obtain the final product.

[0016] In one embodiment, the size of the filter membrane is 0.22 μm.

[0017] In one embodiment, the pre-freezing temperature is -40 to -60°C, and the time is 3 to 5 hours.

[0018] In one embodiment, the vacuum degree of the primary drying is 6-10 Pa, and the drying is carried out by a step-by-step heating method. Specifically, the temperature is maintained at -40 to -50°C for 2-3 hours, and then the temperature is increased by 10-15°C within 30 minutes in a 10°C gradient heating mode. After the temperature is constant, the current temperature is maintained and the vacuum is applied for 2-5 hours until the temperature reaches 0°C, so as to ensure that the ice crystals sublimate at a low temperature.

[0019] In one embodiment, the secondary drying process maintains a vacuum of 3-5 Pa, raises the temperature to 5-10 °C, and holds for 2 hours. If the sample condition is good, a gradient heating mode of 5 °C is maintained: the temperature is increased by 10-15 °C within 30 minutes, and after the temperature is constant, the current temperature is maintained and a vacuum is applied for 2.5 hours until the temperature reaches 30 °C.

[0020] The fifth objective of this invention is to provide the application of the above-described carboxylesterase lyophilized powder in toothpaste whitening or clothing cleaning.

[0021] Beneficial effects: The carboxylesterase lyophilization protectant provided by this invention has a single component. When used for the lyophilization protection of carboxylesterase, it can effectively improve the stability of carboxylesterase lyophilized powder at room temperature and prevent its purity and activity from decreasing. Furthermore, the lyophilized powder cake is intact, easy to form into powder, and the enzyme powder resuspension is clear and transparent with a high enzyme activity recovery rate. This makes it convenient for carboxylesterase to be used in different application scenarios. Attached Figure Description

[0022] Figure 1 This is a comparison diagram of the resuspended enzyme solution after lyophilization in Scheme 4 and Scheme 11 in Example 1; Figure 2 Image of freeze-dried cakes containing PVP of different molecular weights and carboxylesterase stock solution. Figure 3 This is a comparison chart of the resuspended enzyme solution after lyophilization in schemes 12-16. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the 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. The specific embodiments described below further illustrate the present invention.

[0024] The source of raw materials involved in this invention: Sucrose, maltodextrin, trehalose, sorbitol, lactose, polyethylene glycol-335 (PEG-335), PEG-4000, PEG-6000, PEG-12000, polyvinylpyrrolidone (PVP), cross-linked polyvinylpyrrolidone (XL-10); Polyvinylpyrrolidone (PVP) is differentiated by calculating its K value based on polymer molecular weight and aqueous solution viscosity. The polyvinylpyrrolidone (PVP) used in this invention is classified into the following types: PVP K17: K value 15.3-18.36, viscosity-average molecular weight Mv 10000-16000; PVP K25: K value 22.5-27.0, viscosity-average molecular weight Mv 30000-40000; PVP K30: K value 27-32.4, viscosity-average molecular weight Mv 45000-58000; PVP K60: K value 54-64.8, viscosity-average molecular weight Mv 270,000-400,000; PVP K90: K value 81-97.2, viscosity-average molecular weight Mv 1,000,000-1,500,000.

[0025] Triacetin (glyceryl triacetate) and 6% hydrogen peroxide solution were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; other commonly used reagents were purchased from Sinopharm Group.

[0026] The recombinant carboxylesterase strain is a recombinant SGNH family esterase SH2 strain screened from the hot spring environment in Tibet, as disclosed in patent publication number CN 120866273 A. The carboxylesterase pure enzyme liquid was obtained according to the chromatographic purification scheme disclosed in the scheme. A Tris buffer system was added to the pure carboxylesterase liquid to maintain the target protein at an enzyme activity concentration of 3000~5000 U / mL in a 10 mM Tris buffer system (pH 7.4) to protect the target protein, i.e., the carboxylesterase stock solution.

[0027] The testing method involved in this invention: 1. Calculation of carboxylesterase activity recovery rate Using the total enzyme activity (A1) of the carboxylesterase stock solution, freeze-drying mixtures were prepared according to different formulations of freeze-drying protectants. After freeze-drying according to the freeze-drying procedure, freeze-dried cakes were obtained. The carboxylesterase freeze-dried cakes were pulverized using a pulverizer, and the specific activity of the enzyme powder was measured. The total enzyme activity (A2) of the freeze-dried enzyme powder was calculated based on the weight of the obtained enzyme powder. The activity recovery rate of the carboxylesterase was calculated using the following formula; Freeze-drying recovery rate =

[0028] Example 1 A method for lyophilization protection of carboxylesterase, comprising the following: (1) Mix the carboxylesterase stock solution with the following mass-volume fractions; Scheme 1 - Sucrose: Add 10wt% sucrose solution to carboxylesterase stock solution at a volume ratio of 5%, mix well, and then sterilize through a 0.22μm filter to obtain sample 1 to be freeze-dried; Option 2 - Trehalose: Add trehalose to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 2 to be freeze-dried; Scheme 3 - Maltodextrin: Add maltodextrin to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 3 to be freeze-dried; Scheme 4 - PVP K60: Add PVP K60 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 4 to be freeze-dried. Scheme 5 - Sorbitol: Add sorbitol to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 5 to be lyophilized; Scheme 6 - Lactose: Add lactose to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 6 to be freeze-dried; Scheme 7 - PEG-335: Add PEG-335 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 7 to be lyophilized; Scheme 8 - PEG-4000: Add PEG-4000 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 8 to be lyophilized; Scheme 9 - PEG-6000: Add PEG-6000 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 9 to be lyophilized; Scheme 10 - PEG-12000: Add PEG-12000 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 10 to be freeze-dried. Scheme 11-XL-10: Add XL-10 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 11 to be freeze-dried; (2) Freeze-drying procedure for carboxylesterase Pre-freezing stage: Set the shelf temperature to At 50℃, once the temperature of the shelf plate has stabilized at this temperature, place the sample to be freeze-dried in the shelf plate and keep it for 3-4 hours to allow the sample to completely solidify and form a stable skeleton. Primary drying stage: Maintain a vacuum of 6–10 Pa and maintain the shelf temperature at [temperature missing]. 45℃, maintain this state for 2 hours; if the sample condition is good, maintain the plate temperature in a 10℃ gradient heating mode: increase the temperature by 10℃ within 30 minutes, and after the temperature is constant, maintain the current temperature and evacuate for 2 hours until the temperature reaches 0℃, so as to ensure that the ice crystals sublimate at low temperature. Secondary drying stage: Maintain a vacuum of 3-5 Pa, raise the temperature of the plate to 5°C, and hold for 2 hours; if the sample is in good condition, maintain the plate temperature at 5°C in a gradient heating mode: increase the temperature by 10°C within 30 minutes, and after the temperature is constant, maintain the current temperature and apply vacuum for 2.5 hours until the temperature reaches 30°C. At this time, the gradual heating removes the tightly bound water on the solid surface, so that the final water content of the product is 4%.

[0029] Example 2 A method for lyophilization protection of carboxylesterase, comprising the following: (1) Polyvinylpyrrolidone of different molecular weights was mixed with carboxylesterase stock solution according to the following mass-volume fractions; Scheme 12-PVP K17: Add PVP K17 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 12 to be freeze-dried. Scheme 13-PVP K25: Add PVP K25 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 13 to be freeze-dried. Scheme 14 - PVP K30: Add PVP K30 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 14 to be freeze-dried. Scheme 15 - PVP K60: Add PVP K60 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 15 to be freeze-dried. Scheme 16 - PVP K90: Add PVP K90 to the carboxylesterase stock solution at a mass-volume ratio of 5% (50 g / L), mix well, and then sterilize through a 0.22 μm filter to obtain sample 16 to be freeze-dried. (2) Freeze-drying procedure for carboxylesterase Pre-freezing stage: Set the shelf temperature to At 50℃, once the temperature of the shelf plate has stabilized at this temperature, place the sample to be freeze-dried in the shelf plate and keep it for 3-4 hours to allow the sample to completely solidify and form a stable skeleton. Primary drying stage: Maintain a vacuum of 6–10 Pa and maintain the shelf temperature at [temperature missing]. 45℃, maintain this state for 2 hours; if the sample condition is good, maintain the plate temperature in a 10℃ gradient heating mode: increase the temperature by 10℃ within 30 minutes, and after the temperature is constant, maintain the current temperature and evacuate for 2 hours until the temperature reaches 0℃, so as to ensure that the ice crystals sublimate at low temperature. Secondary drying stage: Maintain a vacuum of 3-5 Pa, raise the temperature of the plate to 5°C, and hold for 2 hours; if the sample is in good condition, maintain the plate temperature at 5°C in a gradient heating mode: increase the temperature by 10°C within 30 minutes, and after the temperature is constant, maintain the current temperature and apply vacuum for 2.5 hours until the temperature reaches 30°C. At this time, the gradual heating removes the tightly bound water on the solid surface, so that the final water content of the product is 4%.

[0030] Results Analysis 1. Evaluation of the appearance of different types of lyophilization protectants and carboxylesterases after lyophilization. The results are shown in Table 1: Table 1. Appearance of different types of lyophilization protectants and carboxylesterases after lyophilization

[0031] Table 1 shows that sucrose, maltodextrin, and PEG cannot form cakes after freeze-drying. However, trehalose, polyvinylpyrrolidone, and lactose, when used as freeze-drying preservatives, can form a homogeneous cake with the carboxylesterase stock solution, easily turning into powder. But trehalose and lactose rapidly absorb moisture after powdering, adhering to the container surface and causing significant enzyme powder loss. Furthermore, the highly hygroscopic nature of freeze-dried enzyme powder necessitates strict packaging requirements, as it easily absorbs moisture during storage and transportation, affecting product quality. Therefore, even though trehalose and lactose can help carboxylesterase freeze-dry into a complete cake, they are not the optimal choice.

[0032] 2. Evaluation of resuspension status and enzyme activity recovery rate after lyophilization of different types of freeze-drying protectants The lyophilized samples were resuspended in pure water, and the state of the resuspended enzyme solution was observed and evaluated. The activity was also tested, and the results are shown in Table 2. It was observed that, except for XL-10, which showed obvious precipitation and stratification upon resuspending the lyophilized enzyme solution, the enzyme solutions of other lyophilization protectants were all clear and transparent after lyophilization. Figure 1 ).

[0033] Table 2. Statistics on resuspending state and enzyme activity recovery rate of lyophilized samples

[0034] Based on the results of different types of freeze-drying protectants in terms of cake-forming and powder-forming properties, as well as enzyme activity recovery rates, polyvinylpyrrolidone was selected from a large number of freeze-drying protectants and showed outstanding performance in terms of carboxylesterase freeze-drying cake-forming, powder-forming and enzyme activity recovery rates.

[0035] 3. Evaluation of the lyophilization protection effect of polyvinylpyrrolidone (PVP) with different molecular weights on carboxylesterases PVP can be categorized into a series of products, such as PVP K17, PVP K25, PVP K30, PVP K60, and PVP K90, based on the K value calculated according to the polymer molecular weight and aqueous solution viscosity. To analyze the effect of PVP K value on the lyophilization efficiency of carboxylesterase, the lyophilization of PVP with K values ​​between 17 and 90 and the carboxylesterase stock solution was evaluated. The results are shown in Table 3. Figure 2 As shown; Table 3. Appearance of polyvinylpyrrolidone and carboxylesterase stock solutions of different molecular weights after freeze-drying

[0036] 4. Evaluation of enzyme resuspension status and enzyme activity recovery rate after lyophilization of PVP with different molecular weights and carboxylesterases Table 4. Statistics on resuspending state and enzyme activity recovery rate of lyophilized samples

[0037] The results show that when the K value of PVP is below 60, the freeze-dried cake exhibits better cake formation with carboxylesterase, and the more complete and uniform the freeze-dried cake becomes as the K value increases. When the freeze-dried powder was resuspended with the enzyme solution, the resuspended enzyme solution of PVP K25 showed a stable turbid state, but it did not precipitate or separate upon standing, and this did not affect the enzyme solution performance. Experimental data indicate that PVP with a K value between 17 and 60 exhibits a high enzyme activity recovery rate after freeze-drying with carboxylesterase, ranging from 70% to 90%, with PVP K60 showing a recovery rate as high as approximately 97%. However, the enzyme activity recovery rate of PVP K90 after freeze-drying with carboxylesterase is only 55%, which may be related to the phase separation that occurs after resuspending the enzyme solution.

[0038] 5. Evaluation of the amount of PVP K60 used during lyophilization of carboxylesterase stock solution The amount of PVP used was evaluated by adding 1-10% (w / v) of PVP K60 during the lyophilization process of carboxylesterase. The results are shown in Table 5. In the lyophilization process of carboxylesterase, the amount of PVP used is controlled between 4% and 8%, which is a better amount and can obtain a higher enzyme activity recovery rate and a better lyophilized cake state.

[0039] Table 5. Evaluation of the optimal amount of PVP K60 used during lyophilization of carboxylesterase stock solution

[0040] In summary, this invention, based on a carboxylesterase, screened 12 lyophilization protectants from three major categories (sugars, polyols, and polymers) for their lyophilization protection effects on carboxylesterase using four indicators: lyophilized cake state, powder formation, enzyme resuspension state, and total enzyme activity recovery rate. Polyvinylpyrrolidone (PVP) was found to be highly promising for lyophilization protection of carboxylesterase. Further screening revealed that PPVP with a K value controlled between 17 and 60 exhibited good lyophilization effects on carboxylesterase, achieving enzyme activity recovery rates of 70% to 98%. Based on these findings, the optimal dosage of PPVP in the lyophilization process of carboxylesterase was determined to be 4% to 8% by weight / volume.

[0041] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A carboxylesterase lyophilization protectant, characterized in that, It is a single-component polyvinylpyrrolidone.

2. The carboxylesterase lyophilization protectant according to claim 1, characterized in that, The polyvinylpyrrolidone has a viscosity-average molecular weight (Mv) of 10,000 to 15,000,000 and a K value of 17 to 90.

3. The carboxylesterase lyophilization protectant according to claim 1, characterized in that, The polyvinylpyrrolidone has a viscosity-average molecular weight (Mv) of 10,000 to 400,000 and a K value of 17 to 60.

4. The carboxylesterase lyophilization protectant according to claim 1, characterized in that, The polyvinylpyrrolidone has a viscosity-average molecular weight (Mv) of 270,000-400,000 and a K value of 54-64.8, i.e., PVPK60.

5. The carboxylesterase lyophilization protectant according to claim 1, characterized in that, The amount of polyvinylpyrrolidone used is 20~100 g / L.

6. The carboxylesterase lyophilization protectant according to claim 1, characterized in that, The carboxylesterase mentioned is derived from Chinese patent CN 120866273 A.

7. The use of the carboxylesterase lyophilization protectant according to any one of claims 1 to 7 in the preparation of carboxylesterase lyophilized powder.

8. A lyophilized powder of carboxylesterase, characterized in that, The carboxylesterase lyophilized powder is obtained by mixing carboxylesterase and the carboxylesterase lyophilization protectant according to any one of claims 1 to 7, and then lyophilizing the mixture.

9. A method for preparing the lyophilized carboxylesterase powder according to claim 8, characterized in that, The method includes: After mixing the carboxylesterase stock solution with the carboxylesterase lyophilization protectant according to any one of claims 1 to 7, the mixture is sterilized by filtration membrane, and the filtrate is subjected to pre-freezing, primary drying, and secondary drying in sequence to obtain the final product.

10. The use of the carboxylesterase lyophilized powder according to claim 8 in toothpaste whitening or clothing cleaning.

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