A method for preparing millet lipase

Abstract

This invention discloses a method for preparing millet lipase, belonging to the fields of bioengineering and deep processing of agricultural products. The method uses millet flour as raw material, extracts a crude enzyme solution using a buffer solution, and further constructs a polyethylene glycol-inorganic salt aqueous two-phase system to achieve targeted distribution of the millet lipase, completing rapid enrichment and preliminary purification. Subsequently, polyethylene glycol and inorganic salts are removed from the system by ultrafiltration or dialysis, and molecular sieve chromatography is used for fine purification when necessary to obtain a high specific activity millet lipase preparation. Experimental results show that compared with the traditional ammonium sulfate fractionation precipitation method, the method of this invention significantly shortens the processing time, improves the recovery rate and purification fold of the lipase, and has mild operating conditions and good reproducibility, demonstrating good potential for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and deep processing technology of agricultural products, and specifically relates to a method for preparing millet lipase. Background Technology

[0002] Millet, also known as foxtail millet, is one of my country's important traditional coarse grain crops, with a widespread cultivation and consumption base in northern regions. Millet is rich in lipids, proteins, and various bioactive components, but it is prone to quality deterioration during processing and storage. Lipid hydrolysis and oxidation are the main causes of millet aging, rancidity, and flavor decline. Studies have shown that endogenous lipases in millet can catalyze lipid hydrolysis during storage, generating free fatty acids, which in turn promotes lipid oxidation reactions, significantly impacting the quality stability of millet.

[0003] On the other hand, compared with microbial lipases, plant-derived lipases typically possess unique substrate specificity and position selectivity, showing potential application value in structural lipid preparation, functional oil modification, and food flavor improvement. Therefore, systematic research on millet lipases and the development of efficient extraction and purification methods suitable for their characteristics are of great significance for a deeper understanding of the lipid degradation mechanism in millet and for expanding its applications.

[0004] Currently, the extraction and purification of plant lipases mainly employ ammonium sulfate fractionation, organic solvent precipitation, and multi-step column chromatography. Ammonium sulfate precipitation is a time-consuming process, typically requiring multiple salting-outs and prolonged dialysis, making rapid separation difficult. Organic solvent precipitation can easily cause conformational changes in the enzyme protein, leading to reduced enzyme activity, and also poses solvent residue and environmental safety risks. While fine purification methods based on various chromatographic media can yield high-purity enzyme preparations, their high equipment costs and low throughput make them unsuitable for large-scale preparation or industrial applications.

[0005] Aqueous two-phase extraction (APE) technology has been widely used in the separation and purification of biomolecules in recent years due to its advantages such as mild operating conditions, water as the main component of the system, large throughput, and ease of scale-up. Previous studies have reported the use of polyethylene glycol-inorganic salt aqueous two-phase systems for the separation and enrichment of some plant or microbial lipases, but systematic studies on lipase extraction are still relatively few.

[0006] Because millet raw materials contain a high proportion of prolysins and unique pigments such as carotenoids, these substances easily co-partition with lipases or form stable emulsions in conventional extraction and aqueous two-phase systems. This leads to difficulties in phase separation, low lipase recovery rates, insufficient purity, and even affects the accurate determination of enzyme activity. Therefore, existing general lipase extraction or aqueous two-phase extraction processes are not directly applicable to the efficient preparation of millet lipase.

[0007] In summary, there is an urgent need to develop an extraction and purification method that is tailored to the characteristics of millet matrix, can achieve efficient enrichment of lipase under mild conditions, effectively remove impurities, and maintain high specific activity, so as to meet the needs of basic research and potential applications of millet lipase. Summary of the Invention

[0008] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0009] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0010] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing millet lipase.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing millet lipase, comprising: extraction of crude enzyme solution: millet raw material is crushed, and then a buffer solution is added for extraction, and the supernatant is taken to obtain crude enzyme solution; aqueous two-phase extraction: polyethylene glycol (PEG) and inorganic salt are mixed to construct an aqueous two-phase system, the crude enzyme solution is added, the mixture is allowed to stand and separate into layers, and the lower phase is taken; post-treatment: the lower phase of the collected extract is subjected to impurity removal to remove most of the salt and polyethylene glycol, thereby obtaining millet lipase solution.

[0012] In a preferred embodiment of the method described in this invention, the buffer solution is a phosphate buffer or a Tris-HCl buffer with a pH of 7 to 9.

[0013] As a preferred embodiment of the method described in this invention, the mass-to-volume ratio of the pulverized millet raw material to the buffer solution is (1~2) g: 5 mL.

[0014] In a preferred embodiment of the method described in this invention, the polyethylene glycol has a molecular weight of 4000-8000.

[0015] In a preferred embodiment of the method described in this invention, the inorganic salt is selected from one or more of phosphates, sulfates, and citrates.

[0016] In a preferred embodiment of the method described in this invention, the inorganic salt is selected from ammonium sulfate.

[0017] As a preferred embodiment of the method described in this invention, wherein: in the aqueous two-phase system, the mass fraction of polyethylene glycol (PEG) in the aqueous two-phase system is 12%~14%, and the mass fraction of ammonium sulfate in the aqueous two-phase system is 16%~18%.

[0018] In a preferred embodiment of the method described in this invention, the volume ratio of the crude enzyme solution to the aqueous two-phase system is 1:3~5.

[0019] As a preferred embodiment of the method described in this invention, the treatment of the millet lipase solution further includes isocratic elution using a molecular sieve, collecting the elution peaks with lipase activity, and obtaining a purified enzyme solution.

[0020] As a preferred embodiment of the method described in this invention, the method further includes a drying step, wherein the refined enzyme solution is subjected to vacuum freeze-drying to obtain millet lipase powder.

[0021] Beneficial effects of this invention: (1) This invention constructs a mild separation environment based on a polyethylene glycol-inorganic salt aqueous two-phase system, which allows millet lipase to undergo directional distribution in the system and preferentially accumulate in the salt phase. This effectively reduces the interference of co-distribution of impurities such as lipids, alcohol-soluble proteins, and pigments in the millet raw material, achieving simultaneous enrichment and preliminary purification of lipase. By rationally controlling the molecular weight of polyethylene glycol and the composition ratio of the two phases, the content of impurity proteins can be significantly reduced in a single step, and the specific activity of lipase can be improved, achieving unexpected purification results.

[0022] (2) The method of the present invention is carried out in an aqueous system without the use of organic solvents, which avoids the destruction of the spatial conformation and catalytic activity of enzyme proteins by organic solvents or high-intensity salting-out conditions, and is conducive to maintaining the natural structure and biological activity of millet lipase. The specific activity of the obtained lipase preparation is significantly higher than that of the traditional ammonium sulfate fractionation precipitation method and direct chromatography purification method, and has a better activity retention effect.

[0023] (3) This invention integrates the enrichment and preliminary purification process of lipase into the aqueous two-phase extraction step, which reduces the cumbersome operations such as multiple salting out, repeated dialysis and multi-stage chromatography in traditional processes, significantly shortens the overall processing cycle, improves experimental efficiency and process stability, and is suitable for laboratory scale-up and continuous processing needs.

[0024] (4) After pretreatment with a two-phase system, the content of impurities, pigments and colloidal substances in the target enzyme solution is significantly reduced, which creates more favorable loading conditions for subsequent chromatographic purification, making the elution peak more concentrated and the separation resolution higher, thereby reducing the chromatographic medium load, improving the stability and repeatability of the fine purification steps, and reducing purification costs.

[0025] (5) The present invention has good adaptability to the type of extraction buffer system, pH range and system composition. It can maintain stable phase separation behavior and high lipase recovery efficiency under different conditions. The process has good reproducibility and certain process robustness.

[0026] (6) The present invention uses water as the main separation medium. The polyethylene glycol and inorganic salts used are commonly used and safe separation materials. The process is green and has a low environmental burden. It has the potential to be further expanded in food science research and related processing fields. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 The images are SDS-PAGE electrophoresis diagrams after different purification steps, showing the protein distribution in the solution. From left to right, they are marker, crude enzyme solution (Example 1), enzyme solution after aqueous two-phase extraction (Example 1), and enzyme solution after chromatography (Example 7).

[0028] Figure 2 The protein standard marker bands selected in this invention are compared with the SDS-PAGE electrophoresis results to determine the molecular weight of different bands.

[0029] Figure 3 This is a molecular sieve chromatography purification diagram from Example 7 of the present invention. There are three distinct peaks: the leftmost peak represents high molecular weight protein, the middle peak represents medium molecular weight protein, and the rightmost peak represents low molecular weight protein. The middle peak contains the target lipase. Detailed Implementation

[0030] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0031] All raw materials used in this invention are commercially available products.

[0032] Example 1 A method for preparing millet lipase includes the following steps: (1) Preparation of crude enzyme solution: Take fresh millet, crush it through a 50-mesh sieve, add 0.05 mol / L phosphate buffer (pH 8.0) at a material-to-liquid ratio of 1:5, stir and extract at 4℃ for 2 hours, centrifuge at 8000 r / min for 20 min, and collect the supernatant as crude enzyme solution.

[0033] (2) Aqueous two-phase extraction: Weigh a certain amount of PEG4000 and ammonium sulfate respectively, add deionized water to dissolve them, and construct an aqueous two-phase system so that the final mass fraction of PEG in the system is 14% and the mass fraction of ammonium sulfate is 16%.

[0034] (3) Phase separation and recovery: Add the crude enzyme solution to the above system (the volume ratio of crude enzyme solution to aqueous two-phase system is 1:4). Mix evenly at 10~30℃ and let stand for 30 min to separate the phases.

[0035] The enzyme activity of the crude enzyme solution was measured to be 63.57 nmol / min / mg using the BCA protein concentration assay kit and the lipase kit. The lipase activity of the lower phase after aqueous two-phase extraction was 4246.96 nmol / min / mg, which significantly improved the enzyme activity.

[0036] Example 2 This embodiment investigates the effect of different PEG molecular weights on lipase purification, including the following steps: (1) Preparation of crude enzyme solution: Take fresh millet, crush it through a 50-mesh sieve, add 0.05 mol / L phosphate buffer (pH 8.0) at a material-to-liquid ratio of 1:5, stir and extract at 4℃ for 2 hours, centrifuge at 8000 r / min for 20 min, and collect the supernatant as crude enzyme solution.

[0037] (2) Aqueous two-phase extraction: Weigh a certain amount of PEG2000, PEG4000, PEG6000 and ammonium sulfate respectively, add deionized water to dissolve them, and construct an aqueous two-phase system so that the final mass fraction of PEG in the system is 15% and the mass fraction of ammonium sulfate is 12%.

[0038] (3) Phase separation and recovery: Add the crude enzyme solution to the above system (the volume ratio of crude enzyme solution to aqueous two-phase system is 1:4). Mix evenly at 10~30℃ and let stand for 30 min to separate the phases.

[0039] Table 1. Effect of different PEG molecular weights on lipase purification

[0040] Among them, the target phase yield (Y, %) is the percentage of enzyme activity recovered from the target phase (usually the enzyme-enriched phase) out of the total enzyme activity added to the system. The purification factor (PF) is the ratio of the specific activity of the enzyme in the target phase to the specific activity of the enzyme in the crude enzyme solution, reflecting the purification effect. The partition ratio (K) is the ratio of the enzyme activity in the upper phase to the enzyme activity in the lower phase.

[0041] The recovery rate and purification factor were the main factors verifying the purification effect. The results are shown in Table 1. When the molecular weight of PEG was between 4000 and 8000, the lipase recovery rate was high, and a good purification factor was maintained. When the molecular weight was >8000, the effect decreased. This is because as the molecular weight of PEG increases, the exclusion volume effect of the long polymer chain is enhanced, causing the protein to be expelled from the PEG-rich phase. At the same time, the high molecular weight significantly increases the viscosity of the system, resulting in a longer phase separation time and increased mass transfer resistance, which is not conducive to the effective extraction of enzymes. Considering the recovery rate, purification effect, and ease of operation, this invention preferably uses PEG of 4000-8000 as the phase-forming polymer.

[0042] Example 3 This embodiment investigates the effect of different inorganic salts on lipase purification, including the following steps: (1) Preparation of crude enzyme solution: Take fresh millet, crush it through a 50-mesh sieve, add 0.05 mol / L phosphate buffer (pH 8.0) at a material-to-liquid ratio of 1:5, stir and extract at 4℃ for 2 hours, centrifuge at 8000 r / min for 20 min, and collect the supernatant as crude enzyme solution.

[0043] (2) Aqueous two-phase extraction: Weigh a certain amount of PEG4000 and different inorganic salts (ammonium sulfate, potassium phosphate and sodium citrate), add deionized water to dissolve them, and construct an aqueous two-phase system so that the final mass fraction of PEG in the system is 15% and the mass fraction of inorganic salts is 12%.

[0044] (3) Phase separation and recovery: Add the crude enzyme solution to the above system (the volume ratio of crude enzyme solution to aqueous two-phase system is 1:4). Mix evenly at 10~30℃ and let stand for 30 min to separate the phases.

[0045] Table 2. Effects of different inorganic salts on lipase purification

[0046] The results are shown in Table 2. The type of inorganic salt had little effect on the recovery rate of lipase after extraction, but a significant impact on the purification factor. While the sodium carbonate system could also form an aqueous two-phase system, its strongly alkaline aqueous solution far exceeded the stable pH range of millet lipase, leading to irreversible denaturation of the enzyme protein and loss of activity. Although sodium citrate had a higher partition coefficient, the sulfate system was more effective at removing impurities. Therefore, considering the purity requirements, sulfate was the preferred choice.

[0047] Example 4 This example investigates the effect of different PEG concentrations on lipase purification, including the following steps: (1) Preparation of crude enzyme solution: Take fresh millet, crush it through a 50-mesh sieve, add 0.05 mol / L phosphate buffer (pH 8.0) at a material-to-liquid ratio of 1:5, stir and extract at 4℃ for 2 hours, centrifuge at 8000 r / min for 20 min, and collect the supernatant as crude enzyme solution.

[0048] (2) Aqueous two-phase extraction: Weigh a certain amount of PEG4000 and ammonium sulfate respectively, add deionized water to dissolve them, and construct an aqueous two-phase system so that the final mass fraction of PEG in the system is 12%~18% and the mass fraction of ammonium sulfate is 12%.

[0049] (3) Phase separation and recovery: Add the crude enzyme solution to the above system (the volume ratio of crude enzyme solution to aqueous two-phase system is 1:4). Mix evenly at 10~30℃ and let stand for 30 min to separate the phases.

[0050] Table 3. Effect of different PEG concentrations on lipase purification

[0051] The results are shown in Table 3. As the PEG concentration increases, the extraction recovery rate and purification factor show a trend of first increasing and then decreasing. The extraction effect is better when the concentration is 12%~14%.

[0052] Example 5 This embodiment investigates the effect of different inorganic salt concentrations on lipase purification, including the following steps: (1) Preparation of crude enzyme solution: Take fresh millet, crush it through a 50-mesh sieve, add 0.05 mol / L phosphate buffer (pH 8.0) at a material-to-liquid ratio of 1:5, stir and extract at 4℃ for 2 hours, centrifuge at 8000 r / min for 20 min, and collect the supernatant as crude enzyme solution.

[0053] (2) Aqueous two-phase extraction: Weigh a certain amount of PEG4000 and ammonium sulfate respectively, add deionized water to dissolve them, and construct an aqueous two-phase system so that the final mass fraction of PEG in the system is 14% and the mass fraction of ammonium sulfate is 10~20%.

[0054] (3) Phase separation and recovery: Add the crude enzyme solution to the above system (the volume ratio of crude enzyme solution to aqueous two-phase system is 1:4). Mix evenly at 10~30℃ and let stand for 30 min to separate the phases.

[0055] Table 4. Effects of different inorganic salt concentrations on lipase purification

[0056] The results are shown in Table 4. With increasing ammonium sulfate concentration, the recovery rate and purification fold of lipase increased significantly, then decreased slightly after reaching a concentration of approximately 16%. Therefore, an ammonium sulfate concentration of 16%–18% was selected.

[0057] Example 6 This embodiment investigates the effect of different extraction buffers on lipase purification, including the following steps: (1) Preparation of crude enzyme solution: Take fresh millet, crush it through a 50-mesh sieve, and add different buffer solutions at a material-to-liquid ratio of 1:5: 0.05 mol / L phosphate buffer (pH 7.0), 0.05 mol / L phosphate buffer (pH 8.0), and 0.05 mol / L Tris-HCl buffer (pH 8.5). Stir and extract at 4℃ for 2 hours, centrifuge at 8000 r / min for 20 min, and collect the supernatant as crude enzyme solution.

[0058] (2) Aqueous two-phase extraction: Weigh a certain amount of PEG4000 and ammonium sulfate respectively, add deionized water to dissolve them, and construct an aqueous two-phase system so that the final mass fraction of PEG in the system is 14% and the mass fraction of ammonium sulfate is 18%.

[0059] (3) Phase separation and recovery: Add the crude enzyme solution to the above system (the volume ratio of crude enzyme solution to aqueous two-phase system is 1:4). Mix evenly at 10~30℃ and let stand for 30 min to separate the phases.

[0060] The results showed that different buffer systems could effectively enrich lipases within the pH range of 7.0-8.5, and the specific activities of the obtained lipases did not differ significantly, indicating that the method of the present invention has good adaptability to buffer systems.

[0061] Example 7 In this embodiment, the enzyme solution after aqueous two-phase extraction is further purified by molecular sieve (gel chromatography) to obtain a high-purity enzyme preparation.

[0062] (1) Desalting treatment: The purified enzyme solution after aqueous two-phase extraction was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed overnight at 4°C against 20 mmol / L Tris-HCl buffer. The dialysate was changed 3 times during the process to remove most of the PEG and inorganic salts.

[0063] (2) Chromatographic separation: The dialyzed enzyme solution was filtered through a 0.22 μm filter membrane and loaded onto a Superdex 75 10 / 300GL gel chromatography column that had been pre-equilibrated with 20 mmol / L Tris-HCl buffer.

[0064] (3) Elution and collection: After loading the sample, rinse with equilibration buffer until there is no protein absorption peak in the eluent, then use buffer to perform isocratic elution and collect the eluent in separate tubes.

[0065] Results: The elution peak with the highest lipase activity was collected (see [link]). Figure 3 The enzyme solutions were combined. The specific activity of the finely purified lipase was determined to be 8675.54 nmol / min / mg.

[0066] See SDS-PAGE electrophoresis images after different purification steps. Figure 1 The numbers represent the protein distribution in the solution, from left to right: marker, crude enzyme solution (Example 1), enzyme solution after aqueous two-phase extraction (Example 1), and enzyme solution after chromatography (Example 7). The protein standard marker bands used in this invention are shown in the figure. Figure 2 By comparing the SDS-PAGE electrophoresis results, the molecular weight of different bands can be determined.

[0067] Comparative Example 1 To verify the superiority of the aqueous two-phase extraction process of this invention, a comparative experiment was conducted using the traditional ammonium sulfate fractionation precipitation method: (1) Extraction: Take the same millet powder as in Example 1 and prepare crude enzyme solution in the same way.

[0068] (2) Precipitation: Under ice bath conditions, solid ammonium sulfate was slowly added to the crude enzyme solution until the saturation reached 65%. After stirring for 30 minutes, it was left to stand overnight at 4°C.

[0069] (3) Post-treatment: Centrifuge at 10000 r / min for 20 min at 4℃, discard the supernatant, reconstitute the precipitate with a small amount of phosphate buffer (pH 8.0), and put it into a dialysis bag for dialysis for 24 hours to remove salt.

[0070] Table 5. Comparison of the effects of the two-phase aqueous method of the present invention and the traditional precipitation method.

[0071] The results are shown in Table 5. Compared with the traditional precipitation method, the method used in this invention significantly reduces the time, greatly improves the purification efficiency, and significantly increases the purification factor.

[0072] Comparative Example 2 This comparative example did not employ a direct chromatography purification method using an aqueous two-phase system: Take the same millet flour as in Example 1 and prepare crude enzyme solution using the same method. After obtaining the crude enzyme solution, directly load it onto a DEAE-Sepharose anion exchange column or molecular sieve for purification.

[0073] The results showed that the crude enzyme solution contained a large amount of pigments and colloidal substances, which led to a rapid decrease in the adsorption capacity of the chromatography medium and severe tailing of the elution peak, making it impossible to obtain a high concentration of enzyme solution. The lipase recovery rate was less than 20%, the specific activity was significantly lower than that of the aqueous two-phase pre-purification method, and the operating cost was significantly increased.

[0074] Comparative Example 3 This comparative example compares the distribution of target lipases in the upper and lower phases after aqueous two-phase extraction, including the following steps: (1) Preparation of crude enzyme solution: Take fresh millet, crush it through a 50-mesh sieve, add 0.05 mol / L phosphate buffer (pH 8.0) at a material-to-liquid ratio of 1:5, stir and extract at 4℃ for 2 hours, centrifuge at 8000 r / min for 20 min, and collect the supernatant as crude enzyme solution.

[0075] (2) Aqueous two-phase extraction: Weigh a certain amount of PEG4000 and ammonium sulfate respectively, add deionized water to dissolve them, and construct an aqueous two-phase system so that the final mass fraction of PEG in the system is 14% and the mass fraction of ammonium sulfate is 18%.

[0076] (3) Phase separation and recovery: Add the crude enzyme solution to the above system (the volume ratio of crude enzyme solution to aqueous two-phase system is 1:4). Mix evenly at 10~30℃ and let stand for 30 min to separate the phases.

[0077] Table 6. Distribution of target lipases in the upper and lower phases after aqueous two-phase extraction.

[0078] As shown in Table 6, the upper phase has a high protein concentration and low enzyme activity, while the lower phase has a low protein concentration and high enzyme activity. Furthermore, the examples demonstrate that the target lipase recovery rate is high in the lower phase, indicating that the purification effect of the lower phase after extraction is significant.

[0079] In summary, this invention addresses the technical challenges of complex millet raw material matrices and the difficulty in extracting and purifying lipases by proposing a method for extracting and purifying millet lipases based on a polyethylene glycol-inorganic salt aqueous two-phase system. This method, through the rational selection of PEG molecular weight, inorganic salt types, and their mass fractions, achieves the targeted distribution and efficient enrichment of millet lipases in the aqueous two-phase system, while significantly reducing interference from impurities such as proteins and pigments.

[0080] Compared with traditional ammonium sulfate fractionation precipitation and direct column chromatography purification methods, the process of this invention is simpler, operates under milder conditions, and significantly reduces processing time. Furthermore, the resulting lipase exhibits higher specific activity and purification fold, demonstrating good stability and reproducibility. In addition, the aqueous two-phase system used in this invention, with water as the main solvent, aligns with the development trends of green chemistry and food safety, and possesses excellent scale-up potential.

[0081] Therefore, the method of this invention is not only applicable to basic research and enzymatic property analysis of millet lipase, but also provides technical support for the regulation of millet storage stability, research on lipid rancidity mechanism and related food processing fields, and has high application value and promotion prospects.

[0082] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing millet lipase, characterized in that: include, Extraction of crude enzyme solution: After crushing the millet raw material, add buffer solution for extraction, and take the supernatant to obtain crude enzyme solution; Aqueous two-phase extraction: Aqueous two-phase system was constructed by mixing polyethylene glycol (PEG) and inorganic salts, crude enzyme solution was added, the mixture was allowed to stand and separate into layers, and the lower phase was removed; Post-processing: The collected extract was subjected to a lower phase to remove impurities, most of the salt and polyethylene glycol, to obtain millet lipase solution.

2. The method as described in claim 1, characterized in that: The buffer solution is a phosphate buffer or a Tris-HCl buffer with a pH of 7-9.

3. The method as described in claim 2, characterized in that: The mass-to-volume ratio of the pulverized millet raw material to the buffer solution is (1~2) g: 5 mL.

4. The method according to any one of claims 1 to 3, characterized in that: The polyethylene glycol has a molecular weight of 4000-8000.

5. The method as described in claim 4, characterized in that: The inorganic salt is selected from one or more of phosphates, sulfates, and citrates.

6. The method as described in claim 5, characterized in that: The inorganic salt is selected from ammonium sulfate.

7. The method as described in claim 1 or 6, characterized in that: The aqueous two-phase system contains polyethylene glycol (PEG) at a mass fraction of 12% to 14% and ammonium sulfate at a mass fraction of 16% to 18%.

8. The method as described in claim 7, characterized in that: The volume ratio of the crude enzyme solution to the aqueous two-phase system is 1:3~5.

9. The method as described in claim 1, characterized in that: The treatment of the millet lipase solution also includes, The lipase activity peak was collected by isocratic elution using molecular sieve chromatography to obtain a purified enzyme solution.

10. The method as described in claim 9, characterized in that: The process also includes a drying step, in which the refined enzyme solution is freeze-dried under vacuum to obtain millet lipase powder.

Images