Method for separating and purifying serrapeptase in fermentation liquor through two-step salting-out extraction

Abstract

The invention relates to the technical field of separation and purification, and discloses a method for extracting, separating and purifying serrapeptase in fermentation liquor through two-step salting-out. The serrapeptase in the fermentation liquor is separated and purified through a two-step salting-out extraction technology, specifically, the serrapeptase in the fermentation liquor is concentrated in a lower phase through one-step salting-out extraction, bacterial cell fragments are removed, then the serrapeptase is concentrated and purified through two-step salting-out extraction, and the serrapeptase is enriched in a middle phase. The method has good separation and purification effects, after two steps of salting-out extraction, the purification multiple of the serrapeptase is 3.5 times, the recovery rate is 90.7%, the electrophoresis purity of the obtained serrapeptase is close to 100% through SDS-PAGE gray analysis, and the serrapeptase freeze-dried powder can keep the enzyme activity of 88.6% after being stored for 180 days at the temperature of-80 DEG C.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification, and in particular to a method for separating and purifying serrapeptase in a fermentation broth by two-step salting-out extraction. Background Art

[0002] Serrapeptase is a proteolytic enzyme of the serine protease family, mainly produced by the Serratia marcescens strain E15. The enzyme has an affinity for non-living tissue proteins in the cocoon, which can dissolve them and help the silkworm to break out of the cocoon and become a butterfly, without affecting living tissue. The uniqueness of serrapeptase lies in its chemical structure that can inhibit binding with healthy tissue proteins, making it safer to use in the human body. Therefore, it has a variety of pharmacological effects, such as anti-inflammatory, wound healing, analgesic, and pathogen resistance. Serrapeptase has important research significance in the fields of biomedicine and industry due to its unique mechanism of action and wide application value.

[0003] There are two main bacteria that produce serrapeptase, namely Seratia marcescens and Bacillus subtilis. At present, serrapeptase used in the pharmaceutical industry is almost produced by fermentation of Seratia marcescens. Separation and purification is an important link, accounting for more than 60% of the total production cost.

[0004] Industrial separation methods for serrapeptase in fermentation broth include ammonium sulfate precipitation, acetone precipitation, ultrafiltration, gel filtration chromatography, ion exchange chromatography, etc., but there are some disadvantages. For example, the step-by-step precipitation of ammonium sulfate and the graded precipitation of organic solvents are cumbersome and time-consuming. The combination of multiple chromatography methods has strict requirements on operating conditions, and the enzyme is easily inactivated during the separation and purification process, which reduces the efficiency and recovery rate of the enzyme separation and purification.

[0005] The cost of separation and purification of serrapeptase downstream accounts for about 70% of its production cost. It is urgent to develop a cheap separation and purification method to reduce production costs. The existing serrapeptase purification method has many steps, long operation time, low enzyme activity yield, and expensive chromatography media. Summary of the invention

[0006] The efficiency and recovery rate of separating and purifying serrapeptase from Seratia marcescens fermentation broth are low. In order to solve the above problems, the present invention provides a two-step salting-out extraction method for separating and purifying serrapeptase in fermentation broth.

[0007] The specific technical scheme of the present invention is: On the one hand, the present invention provides a method for separating and purifying serrapeptase in a fermentation broth by two-step salting-out extraction, comprising the following steps: adding an organic solvent and an inorganic salt to the fermentation broth to perform a first step of salting-out mixing, standing, centrifuging to separate phases, obtaining a lower phase enriched with serrapeptase, taking the lower phase, adding an organic solvent and an inorganic salt to perform a second step of salting-out mixing, suspending evenly, standing, centrifuging to separate phases, obtaining an intermediate phase enriched with serrapeptase, and separating the intermediate phase; wherein: The organic solvent is one or more of tert-butyl alcohol, acetone, tert-butyl alcohol, n-butyl alcohol, and n-propyl alcohol; The inorganic salt is one or more of ammonium sulfate, sodium bicarbonate, magnesium sulfate, phosphate, and sodium citrate.

[0008] The present invention separates and purifies serrapeptase in fermentation broth by two-step salting-out extraction technology. Specifically, serrapeptase in fermentation broth is concentrated in the lower phase by one-step salting-out extraction, bacterial cell fragments are removed, and then serrapeptase is concentrated and purified by two-step extraction salting-out, so that serrapeptase is enriched in the middle phase. The method of the present invention has a good separation and purification effect. After two-step salting-out extraction, the purification multiple of serrapeptase is 3.5 times, and the recovery rate is 90.7%. The obtained serrapeptase has an electrophoretic purity of nearly 100% by SDS-PAGE grayscale analysis, and the lyophilized powder of serrapeptase can still maintain 88.6% of enzyme activity after being stored at -80°C for 180 days.

[0009] As a preferred embodiment of the above method, the organic solvent is tert-butyl alcohol, and the inorganic salt is ammonium sulfate.

[0010] The present invention finds that various C3 and C4 alcohols can be used as phase-forming organic solvents in SOE. Compared with other solvents, the recovery rate and purification multiple of the tert-butyl alcohol system are both the highest, and it is the most effective organic solvent. In addition, the present invention also finds that the extraction effect of ammonium sulfate is better than that of other salt systems. Tert-butyl alcohol is used as a phase-forming solvent of the extraction system, which can effectively remove small molecules such as lipids, phenols, pigments and enzyme inhibitors, and improves the amount of serrapeptase enriched in the lower phase during one-step extraction, and improves the amount of serrapeptase enriched in the middle phase during two-step extraction. The two-step extraction and enrichment are combined to greatly improve the purity of the separated serrapeptase.

[0011] As a preferred embodiment of the above method, in the first step of salting out mixing, the volume ratio of tert-butanol to fermentation broth is 1:(2.5~0.5).

[0012] As a preferred embodiment of the above method, in the first step of salting out mixing, the mass ratio of ammonium sulfate to the volume of the fermentation broth is (90g~150g):(1L).

[0013] As a preferred embodiment of the above method, in the first step of salting out mixing, the pH of the mixed system is adjusted to 5.0-10.0.

[0014] As a preferred embodiment of the above method, in the first step of salting out mixing, the temperature of the mixing system is adjusted to 4°C to 50°C.

[0015] As a preferred embodiment of the above method, in the second step of salting out and mixing, the volume ratio of the mass of ammonium sulfate to the lower phase is (180g~240g):(1L).

[0016] As a preferred embodiment of the above method, in the second step of salting out mixing, the pH of the mixed system is adjusted to 5.0-10.0.

[0017] As a preferred embodiment of the above method, the following steps are also included: The intermediate phase was dissolved with Tris-HCl buffer, transferred to a dialysis bag with a molecular weight cutoff of 14 kDa, and dialyzed under double distilled water for more than 12 h to desalinate. The alkaline protease concentrate obtained by dialysis and desalination was pre-frozen at -20°C to -10°C, and then transferred to -40°C to -30°C and freeze-dried under vacuum conditions of less than 50 Pa.

[0018] On the other hand, based on the above method, the present invention provides a method for use in separating and purifying serrapeptase in fermentation broth.

[0019] Compared with the prior art, the present invention has the following technical effects: The present invention separates and purifies serrapeptase in fermentation broth by two-step salting-out extraction technology. Specifically, serrapeptase in fermentation broth is concentrated in the lower phase by one-step salting-out extraction, bacterial cell fragments are removed, and then serrapeptase is concentrated and purified by two-step extraction salting-out, so that serrapeptase is enriched in the middle phase. The method of the present invention has a good separation and purification effect. After two-step salting-out extraction, the purification multiple of serrapeptase is 3.5 times, and the recovery rate is 90.7%. The obtained serrapeptase has an electrophoretic purity of nearly 100% by SDS-PAGE grayscale analysis, and the lyophilized powder of serrapeptase can still maintain 88.6% of enzyme activity after being stored at -80°C for 180 days.

[0020] The present invention separates and purifies serrapeptase in fermentation broth through a two-step salting-out extraction technology, thereby reducing separation costs. For example, the tert-butanol used in the method of the present invention can be reused, and even if it is recycled three times, the tert-butanol can still be used to extract high-purity serrapeptase, while the serrapeptase activity yield remains substantially unchanged. At the same time, the method of the present invention has a mild separation process, is simple to operate, and is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of the ratio of ammonium sulfate mass to fermentation broth volume on the first step of salting-out extraction of serrapeptase; FIG2 shows the effect of the volume ratio of tert-butyl alcohol to fermentation broth on the first step salting-out extraction distribution of serrapeptase; FIG3 shows the effect of pH on the first step of salting-out extraction distribution of serrapeptase; FIG4 shows the effect of temperature on the first step of salting-out extraction distribution of serrapeptase; FIG5 shows the effect of ammonium sulfate content and the phase volume ratio in the first step of salting-out extraction on the distribution of serrapeptase in the second step of salting-out extraction; FIG6 shows the effect of pH on the second step salting out extraction partition of serrapeptase; FIG7 shows the effect of the number of tert-butyl alcohol cycles on the extraction of serrapeptase; FIG8 is a diagram showing the results of SDS-PAGE analysis of serrapeptase after purification by two-step salting-out extraction. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0023] The serrapeptase fermentation broth used in the present invention is derived from the strain Serratia marcescens The fermentation broth of zjut-29, the strain covered is a public strain. Serratia marcescens The preparation method of zjut-29 fermentation liquid is as follows: the bacteria after slant activation culture are inoculated into 100 mL seed culture medium, and the seed liquid is obtained by culturing at 30°C and 200 rpm for 12 h. The seed liquid is transferred to 200 mL fermentation medium at an inoculation rate of 2% (v / v), and the serrapeptase fermentation liquid is obtained by culturing at 30°C and 200 rpm for 24 h.

[0024] In the following examples of the present invention, the pH of the system is adjusted by phosphate (dipotassium hydrogen phosphate and potassium dihydrogen phosphate).

[0025] Example 1 Screening of salting-out extraction system Serrapeptase fermentation broth (protein content 0.416 mg / mL, enzyme activity 621 U / mL) was used as the object of investigation. Different organic solvents (acetone, n-propanol, isopropanol, tert-butanol, n-butanol) and inorganic salts (ammonium sulfate, sodium bicarbonate, magnesium sulfate, sodium citrate, potassium dihydrogen phosphate) were paired and mixed to form a salting-out extraction system to investigate the distribution behavior of serrapeptase in the salting-out extraction system composed of different salts and organic solvents. The screening of the salting-out extraction system was carried out according to the following steps: 1. The pH of the screening system was set to 8.0, the organic solvent concentration was 1:2 (v / v) (volume ratio of organic solvent to fermentation broth), and the inorganic salt concentration was 12.0% (w / v) (ratio of inorganic salt mass (g) to fermentation broth volume (mL)). The operation steps are: (1) In a 50 mL centrifuge tube, add 20 mL of fermentation broth, then add inorganic salts, mix well on a vortex mixer, adjust the pH to 8.0 after the salt is completely dissolved, add organic solvent, vortex again for 3-5 min, mix thoroughly, let stand for phase separation, centrifuge at 3000 g for 10 min at 4°C, and observe three layers (upper organic phase (upper phase), middle interface precipitate (intermediate phase), and lower aqueous phase (lower phase)). Record the volume of the upper phase and collect the lower phase.

[0026] (2) Dissolve the precipitate at the intermediate interface in 50 mM Tris-HCl buffer (pH 8.0) at a ratio of 1:5 (v / v). Centrifuge at 10,000 g for 10 min and collect the supernatant, which is the intermediate phase supernatant.

[0027] (3) The lower phase collected in step (1) and the intermediate phase supernatant obtained in step (2) were dialyzed in distilled water at 4°C for 12 h. The volumes of the dialyzed lower phase and the intermediate phase supernatant were measured. The enzyme activity and protein concentration of the serrapeptase in the upper phase, intermediate phase, and lower phase in the salting-out extraction system were determined. The enzyme activity was determined using the Folin phenol reagent method, and the protein concentration was determined using the Coomassie Brilliant Blue G-250 method.

[0028] 2. The purification effect of serrapeptase was measured by the parameter purification multiple (P F ), recovery rate (R (%)) and bacterial removal rate (Rc (%)). The separation and purification results of the middle phase and the lower phase of serrapeptase are shown in Table 1, where the middle phase is represented by the middle interface phase and the lower phase is represented by the bottom aqueous phase. In Table 1, the salt mass to fermentation broth volume ratio is 12% (w / v), the organic solvent to fermentation broth volume ratio is 1:2 (v / v), the temperature is room temperature, and each system is controlled to pH 8.0 by adjusting the phosphate (dipotassium hydrogen phosphate and potassium dihydrogen phosphate) ratio; "ND" means that no phase is formed under the current conditions.

[0029] Table 1 Effects of different salting-out extraction systems on the purification multiples and enzyme activity yield of serrapeptase It can be seen from the test results of the separation and purification effect and Table 1 that when the mass of salt and the volume ratio of the fermentation broth are 12% (w / v), serrapeptase is mainly distributed in the lower phase. The extraction effect of ammonium sulfate and phosphate is better than that of other salt systems. Compared with other solvents, the recovery rate and purification multiple of the tert-butanol system are the highest, and it is the most effective organic solvent. Various C3 and C4 alcohols can be used as phase-forming organic solvents in SOE. Among them, tert-butanol has the strongest phase-forming ability and can effectively remove small molecules such as lipids, phenols, pigments and enzyme inhibitors. Due to its size and branched structure, tert-butanol is not easy to penetrate into the folded protein molecules, so it does not cause denaturation. In addition, for the salt concentration initially explored by salting-out extraction, the target enzyme was not better present in the lower phase. After preliminary screening in this embodiment, it was found that the two systems of ammonium sulfate-tert-butanol and phosphate-tert-butanol were suitable for directly separating and purifying serrapeptase from the fermentation broth, and can be further optimized later. Among them, the phosphate used in phosphate-tert-butanol is potassium dihydrogen phosphate.

[0030] In this example, the specific activity of the enzyme S A The calculation formula is as follows: ; In the formula, A0 (unit: U / mL) represents the measured unit enzyme activity, and C0 (unit: mg / mL) represents the unit protein concentration.

[0031] In this embodiment, the purification factor P of the system F The calculation formula is as follows: ; S in the formula Ap , S Ai Represent the purification specific activity and the initial specific activity, respectively.

[0032] In this embodiment, the recovery rate R (%) of the system is calculated as follows: ; In the formula, A p Indicates the activity of the purified enzyme, V p Indicates the volume of the purified enzyme solution, A i represents the initial enzyme activity, V i represents the initial enzyme solution volume.

[0033] In this embodiment, the calculation formula of the bacterial removal rate Rc (%) of the system is as follows: ; In the formula, A F Indicates the absorbance value of the bacterial phase where the purified enzyme is located, A f Indicates the absorbance value of bacteria in the fermentation broth, V F Represents the volume of the phase where the purified enzyme is located, V f Indicates the volume of the fermentation broth.

[0034] Example 2 Purification effect of salting-out extraction system after initial screening In this embodiment, the purification effect of the system after the initial screening of the salting-out extraction system is carried out according to the following steps: Under the conditions of salt mass to fermentation broth volume ratio of 12% (ratio of inorganic salt mass (g) to fermentation broth volume (mL)), tert-butanol to fermentation broth volume ratio of 1:2 (v / v), pH 8.0, and room temperature, the bacterial removal effect, enzyme activity yield, and purification multiple were investigated.

[0035] The operation method and determination method of the salting-out extraction in this example are the same as those in Example 1. The results of separation and purification of the serrapeptase in the lower phase are shown in Table 2.

[0036] Table 2 system Bacteria removal rate (%) Enzyme activity recovery rate (%) Purification multiple Ammonium sulfate-tert-butyl alcohol 99.8±2.2 92.7±1.2 1.83±0.01 Potassium dihydrogen phosphate-tert-butyl alcohol 99.6±1.5 93.8±0.4 1.73±0.03 As can be seen from Table 2, under this condition, the bacterial removal rate of the ammonium sulfate-tert-butyl alcohol system was 99.8%, the purification factor was 1.83, and the yield was 92.7%; the bacterial removal rate of the potassium dihydrogen phosphate-tert-butyl alcohol system was 99.6%, the purification factor was 1.78, and the enzyme activity yield was 93.5%. The purification effects of the two systems are not much different, and both deserve further optimization.

[0037] Example 3 Optimization of the first step of salting-out extraction of serrapeptase by ammonium sulfate-tert-butyl alcohol salting-out Since salting-out extraction is affected by many factors, it is necessary to optimize the ammonium sulfate-tert-butanol system. This embodiment adopts a single variable method, and different concentrations of tert-butanol are mixed with different concentrations of ammonium sulfate to form the first step of ammonium sulfate-tert-butanol salting-out extraction system, and the serrapeptase in the fermentation broth of different pH is extracted by salting-out at different temperatures. Among them, the volume ratio (v / v) of tert-butanol to fermentation broth is set to: 1:2.5, 1:2, 1:1.5, 1:1, 1:0.5; the volume ratio (w / v) of ammonium sulfate mass to fermentation broth is set to: 9%, 10.5%, 12%, 13.5%, 15%; the pH of the fermentation broth is adjusted to 1, 3, 5, 7.2, 9, 11 respectively; the temperature is adjusted to: 4°C, 25°C, 37°C, 40°C, 45°C, 50°C respectively.

[0038] The operation method of salting-out extraction in this example is the same as that in Example 1. The enzyme activity and protein concentration of the serrapeptase from the upper, middle and lower phase sample solutions of the salting-out extraction were determined. The purification multiple and recovery rate were calculated. The purification and recovery results of the serrapeptase in the lower phase are shown in Figure 1 , Figure 2 , Figure 3 , Figure 4 . Figure 1The effect of the ratio of ammonium sulfate mass to fermentation broth volume on the first step salting-out extraction distribution of serrapeptase, where the single variable is the ratio of ammonium sulfate mass to fermentation broth volume, the volume ratio of tert-butanol to fermentation broth is 1:2, the pH is 8.0, and the temperature is 40°C; Figure 2 The effect of the volume ratio of tert-butanol to fermentation broth on the first step salting-out extraction distribution of serrapeptase, where the single variable is the volume ratio of tert-butanol to fermentation broth, the volume ratio of ammonium sulfate mass to fermentation broth is 12%, the pH is 8.0, and the temperature is 40°C; Figure 3 The effect of pH on the first step salting-out extraction partition of serrapeptase, where the single variable is pH, the volume ratio of ammonium sulfate to fermentation broth is 12%, the volume ratio of tert-butyl alcohol to fermentation broth is 1:2, and the temperature is 40°C; Figure 4 The effect of temperature on the first step salting-out extraction partitioning of serrapeptase, where the single variable is temperature, the volume ratio of ammonium sulfate mass to fermentation broth is 12%, the volume ratio of tert-butanol to fermentation broth is 1:2, and the pH is 8.0.

[0039] From the results of this example (see Figure 1 , Figure 2 , Figure 3 , Figure 4 ) It can be seen that the optimal conditions for the first step of salting-out extraction are: the mass ratio of ammonium sulfate to the volume of the fermentation broth is 12% (w / v), the volume ratio of tert-butanol to the fermentation broth is 1:2 (v / v), pH 8.0, and the temperature is 40°C. At this time, the bacterial removal rate is more than 99.8%, the purification multiple of serrapeptase is 2.19 times, and the recovery rate is 97.5%.

[0040] Example 4 Optimization of the second step of salting-out extraction of serrapeptase by ammonium sulfate-tert-butyl alcohol salting-out extraction In order to concentrate and purify serrapeptase and enrich it in the middle phase. The lower phase without dialyzation extracted by the first step of salting out (according to the operating steps of Example 1, the salting out system obtained by screening in Example 3 "ammonium sulfate mass to fermentation broth volume ratio is 12% (w / v), tert-butanol to fermentation broth volume ratio is 1:2 (v / v), pH 8.0, temperature 40°C" was extracted) was used as the starting material for the second step of salting out extraction. Using the single variable method, this embodiment distributes serrapeptase to the middle phase by changing the volume ratio of ammonium sulfate mass to the lower phase of the first step of salting out extraction and the pH of the system. Among them, the ratio (w / v) of ammonium sulfate mass (g) to the volume (mL) of the lower phase of the first step of salting out extraction is set to: 18%, 19.5%, 21%, 22.5%, 24% respectively; the pH is adjusted to 1, 3, 5, 7.2, 9, 11 respectively.

[0041] The operation method of salting-out extraction in this example is the same as that in Example 1. The enzyme activity and protein concentration of serrapeptase from the upper, middle and lower phase sample solutions of salting-out extraction were determined. The purification multiple and recovery rate were calculated. Figure 5 , Figure 6 . Figure 5 The effect of ammonium sulfate content and the volume ratio of the lower phase of the first step salting-out extraction on the second step salting-out extraction distribution of serrapeptase, where the single variable is the volume ratio of ammonium sulfate mass to the undialyzed lower phase of the first step salting-out extraction, the volume ratio of tert-butanol to the undialyzed lower phase of the first step salting-out extraction is 1:2, the pH is 7.0, and the temperature is 40°C. Figure 6 The effect of pH on the partitioning of serrapeptase in the second step of salting-out extraction is shown in Figure 3, where the single variable is pH, the volume ratio of ammonium sulfate mass to the undialyzed lower phase of the first step of salting-out extraction is 22.5%, the volume ratio of tert-butyl alcohol to the undialyzed lower phase of the first step of salting-out extraction is 1:2, and the temperature is 40°C.

[0042] from Figure 5 , Figure 6 It can be seen that the optimal conditions for the second step of salting-out extraction are: the volume ratio of ammonium sulfate to the lower phase of the first step of salting-out extraction is 22.5% (w / v), the volume ratio of tert-butanol to the lower phase of the first step of salting-out extraction is 1:2 (v / v), pH 7.0, temperature 40°C, the purification multiple of serrapeptase is 1.61 times, and the recovery rate is 93.1%.

[0043] Example 5: Two-step salting-out extraction and separation and purification of serrapeptase in fermentation broth under optimal salting-out conditions According to the extraction operation of Example 1, the first step of salting-out extraction was performed using the salting-out system obtained by screening in Example 3, "ammonium sulfate mass to fermentation broth volume ratio of 12% (w / v), tert-butanol to fermentation broth volume ratio of 1:2 (v / v), pH 8.0, temperature 40° C." to extract serrapeptase in the fermentation broth. After the first step of salting-out extraction, the separation and purification results of serrapeptase in the lower phase are shown in Table 3.

[0044] According to the extraction operation of Example 1, the second step of salting out extraction was performed using the salting out system screened in Example 4, "the volume ratio of ammonium sulfate mass to the lower phase of the first step of salting out extraction was 22.5% (w / v), the volume ratio of tert-butyl alcohol to the lower phase of the first step of salting out extraction was 1:2 (v / v), pH 7.0, and temperature 40°C" to extract the serrapeptase in the lower phase of the first step of salting out extraction that was not dialyzed. After the second step of salting out extraction, the separation and purification results of the serrapeptase in the intermediate phase are shown in Table 3.

[0045] Table 3 Summary of the separation and purification of serrapeptase from fermentation broth using salting-out extraction After two-step salting-out extraction, the purification effect of serrapeptase is shown in Table 3. As can be seen from Table 3, at low salt concentration in the first step of salting-out extraction (SOE), the protease tends to be distributed in the lower phase of SOE, while removing impurities, bacteria and their fragments. In the second step of SOE, as the salt concentration increases, the protease tends to precipitate in the middle phase, concentrating and enriching the enzyme. Under the optimized conditions, after two-step salting-out extraction, the final recovery rate of the enzyme was 90.7%, and the purification multiple was 3.5 times.

[0046] Example 6 Effect of recycling of tert-butyl alcohol on serrapeptase extraction This example explores the effect of recycling and reusing tert-butanol on the extraction of serrapeptase. The recovery of tert-butanol was carried out by rotary evaporator. According to the boiling point of tert-butanol of 82.5°C, rotary evaporation was carried out at 45°C for 20 min, and the tert-butanol after rotary evaporation was collected. The three-phase separation system was reconstructed using the recovered tert-butanol to recover serrapeptase, and the effect on the purification of serrapeptase in the fermentation broth was tested to evaluate the extraction capacity of the recovered tert-butanol, and three parallel operations were performed.

[0047] Effect of tert-butyl alcohol recycling on salting out extraction Figure 7 shown.

[0048] from Figure 7 It can be seen that the tert-butanol recovered within three times can still extract high-purity serrapeptase, and its enzyme activity yield remains basically unchanged. When the salting-out extraction system is scaled up to industrial production, the recovery of tert-butanol can save a lot of costs and minimize the harm to the environment.

[0049] Example 7 Freeze-drying of serrapeptase after salting-out extraction and its storage stability The intermediate phase (prepared in Example 5) after the second step of salting out extraction was precipitated and dissolved with Tris-HCl buffer at pH 8.0, and the volume was measured and the enzyme activity of the concentrate was measured. The obtained concentrate was transferred to a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for more than 12 hours under double distilled water to desalt. The volume of the enzyme solution was measured and the activity of the enzyme was measured. The alkaline protease concentrate after dialyzation and desalination was transferred to a clean and dry culture dish and pre-frozen in a refrigerator at -20°C for 4 hours, and then transferred to -40°C and freeze-dried under a vacuum of less than 50 Pa for 24 hours. The purified and freeze-dried serrapeptase was placed in a refrigerator at -80°C for storage for 7, 30, 90 and 180 days, respectively, and samples were taken to measure the enzyme activity and specific activity.

[0050] Table 4 Storage stability of serrapeptase freeze-dried powder obtained by ammonium sulfate-tert-butyl alcohol salting-out extraction Time (day) Relative activity (%) 7 99.6±0.6 30 97.7±1.2 90 94.5±0.6 180 88.6±2.6 After the intermediate phase precipitate after the second step of salting out extraction was redissolved, the lyophilized powder product of serrapeptase was obtained after dialysis and freeze drying. The storage stability of serrapeptase lyophilized powder was investigated, and the results are shown in Table 4. As can be seen from Table 4, serrapeptase lyophilized powder can still maintain 88.6% enzyme activity after being stored at -80°C for 180 days.

[0051] Example 8 SDS-PAGE analysis of serrapeptase samples after salting out extraction The purity of serrapeptase was characterized by SDS-PAGE electrophoresis. The samples before and after purification were mixed with the loading buffer solution (the sample before purification was the fermentation broth, and the sample after purification was the lower phase after the first step of salting-out extraction prepared in Example 5, and the intermediate phase after the second step of salting-out extraction prepared in Example 5), and the formula of the loading buffer solution was shown in Table 5. Put it in 100 mL of boiling water and boil for 10 min, and the hydrogen bonds and -SS- in the protein sample can be completely broken. The secondary structure of the protein is completely released. 12% separation gel and 4% concentrated gel were prepared in the electrophoresis tank, and the formula is shown in Table 5. Add electrode buffer solution, adjust the voltage to 80 V, wait until the sample migrates to the junction of the concentrated gel and the separation gel, adjust the voltage to 120 V, and wait until the sample migrates to the bottom of the electrophoresis tank, and turn off the electrophoresis. After removing the gel and rinsing it with purified water, it was stained with Coomassie Brilliant Blue R-250 for 3 h, and then decolorized with a decolorizing solution (ratio of ethanol: acetic acid: water = 2:1:7). The molecular weight of the protein molecule was marked with a protein marker (15-130 kDa).

[0052] Table 5 Separation gel and concentration gel formula Reagents 12% separation gel (mL) 4% Concentrating Gel (mL) H2O 2.2 1.3 4×Tris / SDS separation gel buffer pH 8.8 1.25 0.5 40% Acr-Bis (29:1) 1.5 0.2 TEMED 0.005 0.002 10%APS 0.05 0.02 SDS-PAGE analysis of serrapeptase samples after salting out extraction Figure 8 As shown, lanes 1 and 2 are respectively the fermentation broth, the lower phase of the first step of salting-out extraction, and the middle phase of the second salting-out extraction. Figure 8 It can be seen that the molecular weight of the enzyme is about 52 kDa. Comparing the lower phase (Lane 2) of the first step of salting-out extraction with the fermentation broth (Lane 1), it can be found that most of the impurities are removed. Comparing the middle phase (Lane 3) of the second salting-out extraction with the lower phase (Lane 2) of the first step of salting-out extraction, it can be found that the second step of salting-out extraction further improves the effect of removing impurities, and its electrophoresis gray value can be close to 100%.

[0053] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for separating and purifying serrapeptase in a fermentation broth by two-step salting-out extraction, characterized in that: The following steps are involved: Adding an organic solvent and an inorganic salt to the fermentation broth to perform a first step of salting out and mixing, standing, centrifuging to separate phases, obtaining a lower phase enriched with serrapeptase, taking the lower phase, adding an organic solvent and an inorganic salt to perform a second step of salting out and mixing, suspending evenly, standing, centrifuging to separate phases, obtaining an intermediate phase enriched with serrapeptase, and separating the intermediate phase; wherein: The organic solvent is one or more of tert-butyl alcohol, acetone, tert-butyl alcohol, n-butyl alcohol, and n-propyl alcohol; The inorganic salt is one or more of ammonium sulfate, sodium bicarbonate, magnesium sulfate, phosphate, and sodium citrate.

2. A two-step salting-out extraction separation and purification method for serrapeptase in fermentation broth as claimed in claim 1, characterized in that: The organic solvent is tert-butyl alcohol, and the inorganic salt is ammonium sulfate.

3. A two-step salting-out extraction separation and purification method for serrapeptase in fermentation broth as claimed in claim 1 or 2, characterized in that: In the first step of salting out mixing, the volume ratio of tert-butyl alcohol to fermentation broth is 1:(2.5~0.5).

4. A two-step salting-out extraction separation and purification method for serrapeptase in fermentation broth as claimed in claim 1 or 2, characterized in that: In the first step of salting out mixing, the mass ratio of ammonium sulfate to the volume of fermentation broth is (90g~150g):(1L).

5. A two-step salting-out extraction separation and purification method for serrapeptase in fermentation broth as claimed in claim 1 or 2, characterized in that: In the first step of salting out mixing, the pH of the mixed system is adjusted to 5.0-10.

0.

6. A two-step salting-out extraction separation and purification method for serrapeptase in fermentation broth as claimed in claim 1 or 2, characterized in that: In the first step of salting out mixing, the temperature of the mixed system is adjusted to 4°C~50°C.

7. A two-step salting-out extraction separation and purification method for serrapeptase in fermentation broth as claimed in claim 1 or 2, characterized in that: In the second step of salting out and mixing, the volume ratio of the mass of ammonium sulfate to the lower phase is (180 g ~ 240 g): (1 L).

8. A two-step salting-out extraction separation and purification method for serrapeptase in fermentation broth as claimed in claim 1 or 2, characterized in that: In the second step of salting out mixing, the pH of the mixed system is adjusted to 5.0-10.

0.

9. A two-step salting-out extraction separation and purification method for serrapeptase in fermentation broth as claimed in claim 1 or 2, characterized in that: The following steps are also included: The intermediate phase was dissolved with Tris-HCl buffer, transferred to a dialysis bag with a molecular weight cutoff of 14 kDa, and dialyzed for desalination under double distilled water. The alkaline protease concentrate obtained by dialysis and desalination was pre-frozen at -20°C to -10°C, and then transferred to -40°C to -30°C and freeze-dried under vacuum conditions of less than 50 Pa.

10. Use of the method according to any one of claims 1 to 9 in separating and purifying serrapeptase in a fermentation broth.

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