Method for separating and purifying serrapeptase based on two-step salting-out extraction system of tert-butyl alcohol and inorganic salt

Abstract

The invention relates to the field of separation and purification, and discloses a method for separating and purifying serrapeptase based on a two-step salting-out extraction system of tert-butyl alcohol and inorganic salt. 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 the lower phase through salting-out extraction of tert-butyl alcohol and phosphate in the first step, macromolecular substances such as bacterial cell fragments are removed, then extraction salting-out of tert-butyl alcohol and sodium chloride in the second step is carried out, and the serrapeptase in the fermentation liquor is separated and purified. And concentrating and purifying the serrapeptase, so that the serrapeptase is enriched in the middle phase. The method has good separation and purification effects, after two steps of salting-out extraction, the purification multiple of the serrapeptase is 3.2 times, the recovery rate is 93.0%, 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 89.1% after being stored for 180 days at the temperature of-80 DEG C.

Description

Technical Field

[0001] The invention relates to the field of separation and purification, and in particular to a method for separating and purifying serrapeptase based on a two-step salting-out extraction system of tert-butyl alcohol and an inorganic salt. 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 phosphate precipitation, acetone precipitation, ultrafiltration, gel filtration chromatography, ion exchange chromatography, etc., but there are some disadvantages. For example, the step-by-step precipitation of phosphate 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] For example, a method for separating and purifying a crude enzyme solution containing serrapeptase is disclosed in a patent with application publication number CN105969691A, wherein the crude enzyme solution containing serrapeptase obtained by fermenting Serratia marcescens LL-413 strain is subjected to ammonium sulfate (NH4SO4) precipitation, dialysis desalination and freeze drying to obtain serrapeptase. However, the method disclosed in the patent has the problems of low separation and purification efficiency and recovery rate, and at the same time, the purity of the separated serrapeptase is low.

[0006] 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

[0007] The efficiency and recovery rate of separating and purifying serrapeptase from the fermentation broth of Seratia marcescens are low. In order to solve the above problems, the present invention provides a method for separating and purifying serrapeptase based on a two-step salting-out extraction system of tert-butanol and inorganic salt.

[0008] The specific technical scheme of the present invention is: The invention provides a method for separating and purifying serrapeptase based on a two-step salting-out extraction system of tert-butanol and inorganic salts. The method comprises the following steps: adding tert-butanol and phosphate to a fermentation broth containing serrapeptase 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 tert-butanol and sodium chloride to perform a second step of salting-out mixing, suspending uniformly, standing, centrifuging to separate phases, obtaining an intermediate phase enriched with serrapeptase, and separating the intermediate phase.

[0009] The present invention separates and purifies serrapeptase in fermentation broth by two-step salting-out extraction technology. Specifically, the serrapeptase in fermentation broth is concentrated in the lower phase by salting-out extraction of tert-butanol and phosphate in the first step, and most of the macromolecular impurities such as bacterial cell fragments are removed. Then, most of the small molecular impurities are removed by extraction and salting-out of tert-butanol and sodium chloride in the second step, and the serrapeptase is concentrated and purified, so that the 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 3.2 times, and the recovery rate is 93.0%. The obtained serrapeptase has an electrophoretic purity of nearly 100% by SDS-PAGE grayscale analysis, and the lyophilized powder of serrapeptase can still maintain 89.1% enzyme activity after being stored at -80°C for 180 days.

[0010] As a preferred embodiment of the above method, the phosphate is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium phosphate.

[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:(0.5~3).

[0012] As a preferred embodiment of the above method, in the first step of salting out mixing, the ratio of the mass of phosphate to the volume of the fermentation broth is (60g~100g):(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 mixing, the volume ratio of the mass of sodium chloride to the lower phase is (15g~75g):(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, in the second step of salting out mixing, the temperature of the mixed system is adjusted to 30°C to 55°C.

[0018] As a preferred embodiment of the above method, the method further comprises the following steps: 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.

[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, the serrapeptase in fermentation broth is concentrated in the lower phase by salting-out extraction of tert-butanol and phosphate in the first step, and most of the macromolecular impurities such as bacterial cell fragments are removed. Then, most of the small molecular impurities are removed by extraction and salting-out of tert-butanol and sodium chloride in the second step, and the serrapeptase is concentrated and purified, so that the 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 3.2 times, and the recovery rate is 93.0%. The obtained serrapeptase has an electrophoretic purity of nearly 100% by SDS-PAGE grayscale analysis, and the lyophilized powder of serrapeptase can still maintain 89.1% enzyme activity after being stored at -80°C for 180 days.

[0020] The invention separates and purifies serrapeptase in fermentation broth through two-step salting-out extraction technology, thereby reducing separation cost; the tert-butanol used in the method of the invention can be reused, and even if it is recycled three times, the tert-butanol can still extract high-purity serrapeptase, and the serrapeptase activity yield remains basically unchanged, which meets the requirements of green production; meanwhile, the method of the invention has a mild separation process, is simple to operate, and is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the effect of the ratio of phosphate mass to fermentation broth volume on the first step of salting-out extraction distribution of serrapeptase; FIG2 shows the effect of the volume ratio of tert-butyl alcohol to fermentation broth on the first step of 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 the mass of NaCl and the volume ratio of the lower phase on the second step salting-out extraction distribution of serrapeptase; FIG6 shows the effect of the volume ratio of tert-butyl alcohol to the lower phase on the second step salting-out extraction distribution of serrapeptase; FIG7 shows the effect of pH on the second step salting out extraction partition of serrapeptase; FIG8 shows the effect of temperature on the second step salting out extraction partition of serrapeptase; FIG9 shows the effect of the number of cycles of tert-butyl alcohol on the salting-out extraction effect of serrapeptase; FIG. 10 is a diagram showing the results of SDS-PAGE analysis of serrapeptase purified by SOE. 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 embodiment of the present invention is derived from the fermentation broth of the strain Serratia marcescens zjut-29, and the strain is a public strain. The preparation method of the fermentation broth of Serratia marcescens zjut-29 is as follows: the bacteria after slant activation culture are inoculated into 100 mL of seed culture medium, and the seed liquid is obtained by culturing at 30°C and 200 rpm for 12 h. The seed liquid is transplanted into 200 mL of fermentation medium at an inoculation amount of 2% (v / v), and the serrapeptase fermentation broth 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 Optimization of the first step of salting-out extraction of serrapeptase by phosphate-tert-butanol salting-out Since salting-out extraction is affected by many factors, it is necessary to optimize the phosphate-tert-butanol system. Using the single variable method, this embodiment uses different concentrations of tert-butanol mixed with different concentrations of phosphate potassium dihydrogen phosphate to form the first step of the phosphate-tert-butanol salting-out extraction system, and salting-out extracts serrapeptase in fermentation broths of different pH at different temperatures. Among them, the volume ratio (v / v) of tert-butanol to fermentation broth is set to: 1:3, 1:2, 1:1.5, 1:1, 1:0.67, 1:0.5; the volume ratio (w / v) of phosphate mass to fermentation broth (the ratio of phosphate mass (g) to the volume (mL) of fermentation broth) is set to: 6%, 7%, 8%, 9%, 10%; the pH of the fermentation broth is adjusted to 5, 6, 7, 8, 9, 10, respectively; the temperature is adjusted to: 4°C, 25°C, 37°C, 40°C, 45°C, 50°C, respectively. Salting-out extraction optimization is carried out according to the following steps: 1. The operation steps are: (1) In a 50 mL centrifuge tube, add 20 mL of fermentation broth, then add inorganic salt potassium dihydrogen phosphate, mix well on a vortex mixer, adjust the pH to 8.0 after the salt is completely dissolved, add tert-butyl alcohol, 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 serrapeptase from the upper phase, intermediate phase, and lower phase in the salting-out extraction system were determined, and it was found that most of the serrapeptase was enriched in the lower phase. 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 results of the separation and purification of serrapeptase in the lower phase are shown in Figure 1 , Figure 2 , Figure 3 , Figure 4 ,in: Figure 1The effect of the ratio of phosphate mass to fermentation broth volume on the first step salting-out extraction partition of serrapeptase, where the single variable is the ratio of phosphate 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 partition of serrapeptase, where the single variable is the volume ratio of tert-butanol to fermentation broth, the mass ratio of phosphate to fermentation broth volume is 8% (w / v), pH is 8.0, and temperature is 40°C; Figure 3 The effect of pH ratio on the first step salting-out extraction partitioning of serrapeptase, where the single variable is pH, the ratio of phosphate mass to fermentation broth volume is 8% (w / v), the volume ratio of tert-butanol 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 ratio of phosphate mass to fermentation broth volume is 8% (w / v), the ratio of tert-butanol to fermentation broth volume is 1:2, and the pH is 8.0.

[0029] from 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 phosphate to the volume of the fermentation broth is 8% (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 above 99.6%, the purification multiple of serrapeptase is 2.12 times, and the recovery rate is 96.5%.

[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 Optimization of the second step of salting-out extraction of serrapeptase In order to concentrate and purify serrapeptase and enrich it in the middle phase, this embodiment uses NaCl instead of insoluble phosphate for salting-out extraction. The lower phase of the first step of salting-out extraction without dialyzing is used as the starting material for the second step of salting-out extraction. Using a single variable method, this embodiment distributes serrapeptase in the middle phase by changing the volume ratio of NaCl mass to the lower phase of the first step of salting-out extraction, the volume ratio of tert-butyl alcohol to the lower phase of the first step of salting-out extraction, the system pH and the system temperature. Among them, the mass ratio of NaCl to the lower phase volume of the first step salting out extraction (w / v) (the ratio of the mass of NaCl (g) to the volume (mL) of the lower phase of the first step salting out extraction) was set to 1.5%, 3%, 4.5%, 6%, 7.5% (w / v), respectively; the volume ratio of tert-butanol to the lower phase of the first step salting out extraction was set to 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1 (v / v), respectively; the pH was adjusted to 1, 3, 5, 7.2, 9, 11, respectively; the temperature was adjusted to 30, 35, 35, 40, 45, 50, 55 °C, respectively.

[0035] 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 derived from the salting-out extraction mesophase were determined. The purification multiple and recovery rate were calculated. Figure 5 , Figure 6 , Figure 7 , Figure 8 . Figure 5 The effect of the mass of NaCl and the volume ratio of the lower phase on the second step salting-out extraction distribution of serrapeptase, where the single variable is the mass of NaCl and the volume ratio of the lower phase of the first step salting-out extraction, the volume ratio of tert-butanol to the fermentation broth is 1:2, the pH is 8.0, and the temperature is 40°C; Figure 6 The effect of the volume ratio of tert-butanol to the lower phase on the second step salting-out extraction partition of serrapeptase, where the single variable is the volume ratio of tert-butanol to the lower phase of the first step salting-out extraction, the volume ratio of NaCl mass to the lower phase of the first step salting-out extraction is 3%, pH is 8.0, and the temperature is 40℃; Figure 7The effect of pH on the partition of the second step salting-out extraction of serrapeptase, where the single variable is pH, the volume ratio of NaCl mass to the lower phase of the first step salting-out extraction is 3%, the volume ratio of tert-butyl alcohol to the lower phase of the first step salting-out extraction is 1:2, and the temperature is 40°C; Figure 8 The effect of temperature on the second step salting out extraction partitioning of serrapeptase, where the single variable is temperature, the volume ratio of NaCl mass to the lower phase of the first step salting out extraction is 3%, the volume ratio of tert-butanol to the lower phase of the first step salting out extraction is 1:2, and the pH is 8.0.

[0036] from Figure 5 , Figure 6 , Figure 7 , Figure 8 It can be seen that the optimal conditions for the second step of salting-out extraction are: the volume ratio of sodium chloride mass to the lower phase of the first step of salting-out extraction is 3% (w / v) (the actual salt content in the system is 8% phosphate + 3% sodium chloride), the volume ratio of tert-butyl alcohol to the lower phase of the first step of salting-out extraction is 1:2 (v / v), pH 8.0, and temperature 40°C. Under the optimal salting-out extraction conditions, the purification multiple of serrapeptase was 1.51 times, and the recovery rate was 96.4%.

[0037] Example 3: Two-step salting-out extraction 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 with the salting-out system screened in Example 1, "phosphate mass to fermentation broth volume ratio of 8% (percentage of potassium dihydrogen phosphate mass (g) to fermentation broth volume (mL)), 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 1.

[0038] 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 2, "the volume ratio of sodium chloride mass to the lower phase of the first step of salting out extraction is 3% (the percentage of sodium chloride mass (g) to the volume (mL) of the lower phase), the volume ratio of tert-butanol to the lower phase of the first step of salting out extraction is 1:2 (v / v), pH 8.0, 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 1.

[0039] Table 1 Summary of the separation and purification of serrapeptase from fermentation broth using salting-out extraction sample Total enzyme activity (U) Total protein (mg) Specific activity (U / mg) Purification multiple Enzyme activity yield (%) Fermentation Broth 12420±190 8.32±0.14 1493±20 1 100 First SOE (water phase) 111723±68 3.59±0.02 3270±12 2.12±0.12 98.5±1.32 Second SOE (intermediate phase) 10020±64 2.21±0.06 5232±18 3.20±0.05 93±0.26 After two-step salting-out extraction (SOE), the purification effect of serrapeptase is shown in Table 1. As can be seen from Table 1, at the low salt concentration of the first step SOE, the protease tends to be distributed in the lower phase of SOE, while removing impurities, bacteria and their fragments. In the second step 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 93.0%, and the purification multiple was 3.2 times.

[0040] Example 4 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.

[0041] Effect of tert-butyl alcohol recycling on salting out extraction Fig. 9 shown.

[0042] from Fig. 9 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.

[0043] Example 5 Freeze-drying of serrapeptase after salting-out extraction and its storage stability The intermediate phase (prepared in Example 3) 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 h 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 h, and then transferred to -40°C and a vacuum degree of less than 50 Pa for freeze-drying for 24 h. 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.

[0044] Table 2 Storage stability of serrapeptase freeze-dried powder obtained by ammonium sulfate-tert-butyl alcohol salting-out extraction Time (day) Relative activity (%) 7 99.4±1.2 30 96.1±0.5 90 93.4±0.7 180 89.1±1.7 After the intermediate phase precipitate was redissolved after the second step of salting out extraction, 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 2. As can be seen from Table 2, serrapeptase lyophilized powder can still maintain 89.1% enzyme activity after being stored at -80°C for 180 days.

[0045] Example 6 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 3, and the intermediate phase after the second step of salting-out extraction prepared in Example 3), and the formula of the loading buffer solution was shown in Table 3. 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 3. 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).

[0046] Table 3 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 Fig.10 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. Fig.10 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%.

[0047] 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.

[0048] 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 based on a two-step salting-out extraction system of tert-butyl alcohol and an inorganic salt, characterized in that: The following steps are involved: The fermentation broth containing serrapeptase is added with tert-butanol and phosphate to perform the first step of salting out and mixing, the mixture is allowed to stand, and the mixture is centrifuged to separate phases to obtain a lower phase enriched with serrapeptase, and the lower phase is taken out and tert-butanol and sodium chloride are added to perform the second step of salting out and mixing, the mixture is evenly suspended, the mixture is allowed to stand, and the mixture is centrifuged to separate phases to obtain an intermediate phase enriched with serrapeptase, and the intermediate phase is separated.

2. The method according to claim 1, characterized in that: The phosphate is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium phosphate.

3. The method according to claim 1 or 2, characterized in that: In the first step of salting out mixing, the volume ratio of tert-butanol to fermentation broth is 1:(0.5~3).

4. The method according to claim 1 or 2, characterized in that: In the first step of salting out mixing, the mass ratio of phosphate to volume of fermentation broth is (60g~100g):(1L).

5. The method according to 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. The method according to 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. The method according to claim 1 or 2, characterized in that: In the second step of salting out mixing, the ratio of the mass of sodium chloride to the volume of the lower phase is (15g~75g):(1L).

8. The method according to 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. The method according to claim 1 or 2, characterized in that: In the second step of salting out mixing, the temperature of the mixed system is adjusted to 30°C~55°C.

10. The method according to 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.

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