Separation and purification method of bromelain hydrolase and high protein hydrolysis active peptide fragment mixture

Abstract

The invention discloses a separation and purification method of a bromelain hydrolase and high protein hydrolysis active peptide fragment mixture, and belongs to the technical field of biology. The method comprises the following steps: firstly, pretreating bromelain to obtain a bromelain aqueous solution without insoluble impurities, then loading a supernatant to a cation exchange column for gradient elution, with a phase A being an equilibrium buffer solution composed of 40-60 mM of Tris-HCl, 3-8% of glycerol and 0.5-5 mM of DTT, and a phase B being an elution buffer solution composed of 40-60 mM of Tris-HCl, 400-600 mM of NaCl, 3-8% of glycerol and 0.5-5 mM of DTT, the volume ratio of the elution buffer solution being increased from 0 to 20%, the volume ratio of the elution buffer solution being increased from 0 to 20%, the volume ratio of the elution buffer solution being increased from 0 to 20%, and the volume ratio of the elution buffer solution being increased from 0 to 20%; the volume ratio of the elution buffer solution is increased from 20% to 100%, elution is performed by 5-10 times of the column volume, then the volume ratio of the elution buffer solution is increased from 20% to 100%, and elution is performed by 3-6 times of the column volume, so that bromelain and a component with a peptide fragment mixture as a main component are obtained through separation and purification. The method provides a new purification process for producing the bromelain hydrolase, and the peptide fragment mixture obtained by the method has higher activity and a relatively wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for separating and purifying a mixture of bromelain hydrolase and highly active protein hydrolysates. Background Technology

[0002] Bromelain is a general term for enzymes extracted from plants of the Bromeliaceae family. It includes proteolytic enzymes, phosphatases, peroxidases, cellulases, glycosidases, and some non-protein substances. Among them, proteolytic enzymes belong to the cysteine ​​hydrolases.

[0003] In the medical field, bromelain possesses various biological activities, including anti-inflammatory, immunomodulatory, and teeth whitening effects. Its anti-inflammatory activity makes it clinically applicable in the treatment of inflammations such as osteoarthritis and sinusitis. Although the mechanism of bromelain's anti-inflammatory action is not yet fully understood and requires further research, current studies suggest that its anti-inflammatory effect is mainly related to its proteolytic enzyme activity. Therefore, obtaining high-purity, high-activity bromelain is essential.

[0004] Bromelain hydrolase is mainly prepared through two methods: heterologous expression in *E. coli* and natural isolation and purification. *E. coli* production of recombinant proteins has advantages such as clear genetic background, high transformation efficiency, rapid growth and reproduction, and low cost, enabling large-scale production and expression of the target protein. However, because bromelain hydrolase easily forms inclusion bodies during heterologous expression in *E. coli*, purification often requires urea denaturation to achieve solubilization, followed by refolding to restore its original activity. This process suffers from drawbacks such as reduced protein activity and yield after refolding, and cumbersome procedures, failing to meet the needs of industrial production. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of Escherichia coli in producing bromelain, the present invention aims to provide a method for separating and purifying bromelain and a mixture of high-protein hydrolysing peptides. This method can not only successfully separate a high-purity bromelain from bromelain, but also obtain a component with a peptide mixture as the main component. The peptide mixture obtained by this method has higher activity than bromelain and bromelain aqueous solution, and has good application prospects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for separating and purifying a mixture of bromelain hydrolase and highly active protein hydrolysing peptides, comprising the following steps: (1) Dissolve bromelain powder in buffer solution, centrifuge to obtain supernatant, and obtain bromelain aqueous solution; (2) Dilute the bromelain aqueous solution obtained in step (1), load it onto a cation exchange column, and perform gradient elution with elution buffer containing NaCl to collect the mixture of bromelain hydrolase and high protein hydrolysis active peptides respectively. (3) The mixture of bromelain hydrolase and high protein hydrolyzable peptide obtained in step (2) is concentrated to obtain the final product.

[0007] Based on the above technical solution, further, the buffer in step (1) is PBS buffer or Tris-HCl buffer; preferably Tris-HCl buffer, with a buffer concentration of 40~60 mM; wherein it contains glycerol with a final concentration of 3~8% and DTT with a final concentration of 0.5~5 mM.

[0008] Based on the above technical solution, the supernatant in step (1) is further subjected to ultrafiltration concentration treatment, and the molecular weight cutoff of ultrafiltration is 3000~20000 Da.

[0009] Based on the above technical solution, further, the centrifugation conditions in step (1) are centrifugation at 5000~15000 rpm for 10~30 min at 0~4℃.

[0010] Based on the above technical solution, further, the dilution in step (2) specifically involves diluting the bromelain aqueous solution to 3~6 mg / mL, and then filtering it with a 0.22 μm microporous membrane.

[0011] Based on the above technical solution, further, in step (2), the cation exchange column uses highly cross-linked 5-10% agarose microspheres as the medium, and the column volume is 5-10 mL.

[0012] Based on the above technical solution, further, the cation exchange column in step (2) is an SP 6FF column or a CM6FF column, preferably an SP 6FF column.

[0013] Based on the above technical solution, further, the gradient elution in step (2) is a linear gradient elution. Phase A is an equilibration buffer with a composition of 40~60 mM Tris-HCl, pH=8.0, 3~8% glycerol, and 0.5~5 mM DTT. Phase B is an elution buffer with a composition of 40~60 mM Tris-HCl, pH=8, 400~600 mM NaCl, 3~8% glycerol, and 0.5~5 mM DTT. The flow rate is 0.5~1.5 mL / min. First, the volume percentage of the elution buffer in phase B is increased from 0 to 20%, eluting 5~10 times the column volume. Then, the volume percentage of the elution buffer in phase B is increased from 20% to 100%, eluting 3~6 times the column volume.

[0014] The present invention also provides a mixture of bromelain hydrolase and high protein hydrolysing active peptides obtained by the above-described separation and purification method.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a linear gradient elution method based on cation exchange chromatography to separate and purify bromelain, yielding two components: component I, stem bromelain, and component II, a peptide mixture as the main component. When activity was measured using casein as a substrate, the peptide mixture showed higher activity than stem bromelain. This invention provides a foundation for improving the separation and purification of stem bromelain and unexpectedly reveals that the peptide mixture exhibits higher activity. Furthermore, the process of this invention is simple, requires minimal equipment investment, and has well-defined process parameters, making it easy to operate and suitable for large-scale production. Attached Figure Description

[0016] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0017] Figure 1 This is a schematic diagram of the bromelain separation and purification process of the present invention.

[0018] Figure 2 The image shows the results of ion exchange chromatography purification of the bromelain in Example 2.

[0019] Figure 3 The image shows the SDS-PAGE images of the two components obtained from the separation and purification in Example 3.

[0020] Figure 4 This is a graph showing the MADLI identification results of component II in Example 4. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0022] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the art.

[0023] The bromelain raw material used in this example was produced by Guangxi Nanning Pangbo Biotechnology Co., Ltd.

[0024] Example 1: Pretreatment of bromelain Dissolve 1.2 g of bromelain in 30 mL of equilibration buffer (50 mM Tris-HCl pH=8.0, 5% glycerol, 1 mM DTT), centrifuge at 12000 rpm for 20-30 min, then take the supernatant and centrifuge at 4500 rpm for 10-20 min in a 10000 Da ultrafiltration concentration tube to further remove insoluble impurities. Collect the supernatant to obtain an aqueous solution of bromelain.

[0025] Example 2: Isolation and purification of bromelain See the separation and purification process. Figure 1 The cation exchange column was pre-equilibrated with at least 5 column volumes of equilibration buffer, followed by rinsing the loading loop with at least 2 column volumes of equilibration buffer. The supernatant (bromelain aqueous solution) was diluted to 4 mg / mL with equilibration buffer, filtered through a 0.22 μm microporous membrane, loaded onto a syringe, and the separation and purification program was started. The cation exchange chromatography system used was an AKTA Pure 25, and the ion exchange column was an SP 6FF column (5 mL). The column was eluted with 1 column volume of equilibration buffer. Phase A was then prepared as equilibration buffer (50 mM Tris-HCl pH=8.0, 5% glycerol, 1 mM DTT), and phase B as elution buffer (50 mM Tris-HCl (pH=8), 500 mM NaCl, 5% glycerol, 1 mM DTT) for linear gradient elution. Initially, the elution buffer concentration was increased from 0% to 20%, eluting for 6 column volumes. Then, the elution buffer concentration was increased from 20% to 100%, eluting for 4 column volumes, at a flow rate of 1.0 mL / min. After the program was complete, the column was eluted with at least 5 column volumes of elution buffer until a stable baseline was reached in the UV absorption.

[0026] Upon testing, the purified bromelain aqueous solution yielded two components (component I and component II), with significant differences in their content (see...). Figure 2 The purified components were collected and concentrated by ultrafiltration to increase the concentration of each component. After being flash-frozen in liquid nitrogen, they were stored at -80°C or used for subsequent testing.

[0027] Example 3: SDS-PAGE identification of purified components The two purified components were mixed with 2×loading buffer at a volume ratio of 1:1. 3 μL of protein marker and 10 μL of sample were added sequentially to the sample wells of the protein electrophoresis gel. The electrophoresis apparatus was first run at 120 V for 20 min, then changed to 200 V until the bromophenol blue reached the bottom of the separating gel. The protein gel was stained with Coomassie Brilliant Blue R250 staining solution, heated and shaken for 2 min, and then placed on a shaker and stained for 10 min. The gel was then destained with destaining solution, and the destaining solution was changed repeatedly and shaken until the protein bands were clear.

[0028] The results are as follows Figure 3 As shown, by Figure 3 It can be seen that sample I has a single band at 25 kD, while sample II has a large number of bands below 15 kD in addition to the band at 25 kD, indicating that small molecule proteins account for a higher proportion in peak II.

[0029] Example 4: Qualitative determination of purified components The purified two components underwent pre-mass spectrometry sample processing: First, 100 μg of sample (concentration previously measured using the Coomassie Brilliant Blue method) was added to a final concentration of 10 mM using DTT solution, and the mixture was treated at 56 °C for 1 h. Then, iodoacetamide (IAA) solution was added to a final concentration of 20 mM, and the mixture was reacted in the dark for 30-40 min. Subsequently, 2 mg / mL trypsin solution was added to the above solution at a sample-to-trypsin mass ratio of 1:20, and the mixture was enzymatically digested at 37 °C for 16-18 h. Acidification was then performed by adding pure trifluoroacetic acid (TFA) solution at a volume ratio of 1:100, followed by repeated pipetting and sonication to remove air bubbles, and centrifugation at 12,000 rpm for 10 min. The sample was then desalted. First, 200 μL of 1% TFA was added to equilibrate the desalting column, and this operation was repeated once. Next, the acidified sample was added to the desalting column, and after complete drying, it was rinsed with 200 μL of 1% TFA, the effluent was discarded, and this operation was repeated once. Finally, it was eluted with 200 μL of an elution buffer containing 80% acetonitrile and 0.1% TFA aqueous solution, and the eluted sample was collected. The collected desalted sample was lyophilized at -100℃. The lyophilized protein sample was redissolved with 20-30 μL of 0.1% formic acid (FA) solution. After adding the formic acid solution, the sample was first tapped a few times and then sonicated until the protein powder was completely dissolved. The sample was then centrifuged at 12,000 rpm for 20-30 min, and the supernatant was collected for mass spectrometry identification. The mass spectrometer was an Orbitrap Explories 480 with a mass range of 40-6000 m / z and a resolution of 480,000 FWHM (m / z=200). Data acquired by mass spectrometry were searched in the Ananas_comosus_fasta database.

[0030] The mass spectrometry identification results are shown in Table 1. The matching results in the Ananas_comosus_fasta database indicate that both belong to the same bromelain hydrolase stem bromelain (P14518). However, since sample II also contains small molecular weight proteins in SDS-PAGE, it is speculated that it may be a mixture of peptide fragments of bromelain hydrolase.

[0031] Table 1. Information related to protein mass spectrometry identification

[0032] Component II was identified by MALDI, and the results are as follows: Figure 4 As shown in the figure, a corresponding peak appears at a molecular weight of around 10 kD, indicating that sample II contains stem bromelain fragments with a molecular weight of around 10 kD. This confirms that the low molecular weight protein component in component II is a mixture of peptides, and based on the peptide matching results, the possible cleavage sites are inferred to be between NNESSMMY.

[0033] Example 5: Activity determination of purified components Bromelain hydrolyzes casein to produce tyrosine, which has a maximum absorption peak at 275 nm. Therefore, the activity of each component can be determined by the casein method. The activities of each sample were determined using stem bromelain (component I), a peptide mixture (component II), and an aqueous solution of bromelain, respectively. The specific methods are described below: Dilute the sample to the same concentration (0.065 mg / ml) with an appropriate amount of buffer (50 mM Tris-HCl, pH=8), accurately measure 100 μL, incubate in a 37℃ water bath for 10 min, quickly add 500 μL of casein solution incubated at 37℃, react for 10 min, then add 500 μL of trichloroacetic acid solution to stop the reaction, incubate for 10 min, centrifuge at 12000 rpm for 10 min at 4℃, and retain the supernatant.

[0034] Take another 100 μL of sample solution and incubate at 37℃ for 10 min. Quickly add 500 μL of trichloroacetic acid solution incubated at 37℃, react for 10 min, then add 500 μL of casein solution, incubate for 10 min, centrifuge at 12000 rpm for 10 min at 4℃, use the filtrate as a blank, and measure the absorbance at 275 nm using spectrophotometry. Perform three parallel experiments, denoted as A1, A2, and A3. Take the average value of the three experiments, denoted as A1. .

[0035] Weigh 5 mg of tyrosine standard, dilute to 100 mL with 0.1 mol / L hydrochloric acid solution, use water as a blank, and measure the absorbance at 275 nm. Record this as As.

[0036] One unit of activity (U) is defined as the amount of enzyme required per minute to hydrolyze casein to produce 1 μg of tyrosine during the reaction. The specific activity of the sample is calculated using the following formula: Specific activity U / mg =

[0037] In the formula, 1.1: total volume of the reaction system (mL); 10: Reaction time (min); 50: The number of μg of tyrosine contained per mL.

[0038] The activity assay results are shown in Table 2. As can be seen from the table, the peptide mixture has a greater activity than stem bromelain, indicating that the peptide mixture plays a major role in the activity of bromelain, while stem bromelain contributes relatively little to the activity.

[0039] Table 2. Activity of each sample determined by the casein method

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for separating and purifying a mixture of bromelain hydrolase and highly active protein hydrolysing peptides, characterized in that, Includes the following steps: (1) Dissolve bromelain powder in buffer solution, centrifuge to obtain supernatant, and obtain bromelain aqueous solution; (2) Dilute the bromelain aqueous solution obtained in step (1), load it onto a cation exchange column, and perform gradient elution with elution buffer containing NaCl to collect the mixture of bromelain hydrolase and high protein hydrolysis active peptides respectively. (3) The mixture of bromelain hydrolase and high protein hydrolyzable peptide obtained in step (2) is concentrated to obtain the final product.

2. The separation and purification method according to claim 1, characterized in that, The buffer solution in step (1) is PBS buffer or Tris-HCl buffer; preferably Tris-HCl buffer, with a buffer concentration of 40~60 mM; containing glycerol at a final concentration of 3~8% and DTT at a final concentration of 0.5~5 mM.

3. The separation and purification method according to claim 1, characterized in that, The supernatant described in step (1) is concentrated by ultrafiltration, with a molecular weight cutoff of 3000~20000 Da.

4. The separation and purification method according to claim 1, characterized in that, The centrifugation conditions in step (1) are centrifugation at 5000~15000 rpm for 10~30 min at 0~4℃.

5. The separation and purification method according to claim 1, characterized in that, The dilution in step (2) specifically involves diluting the bromelain aqueous solution to 3~6 mg / mL and then filtering it through a 0.22 μm microporous membrane.

6. The separation and purification method according to claim 1, characterized in that, In step (2), the cation exchange column uses highly cross-linked 5-10% agarose microspheres as the medium, and the column volume is 5-10 mL.

7. The separation and purification method according to claim 6, characterized in that, The cation exchange column in step (2) is an SP6FF column or a CM 6FF column, preferably an SP 6FF column.

8. The separation and purification method according to claim 1, characterized in that, The gradient elution described in step (2) is a linear gradient elution. Phase A is the equilibration buffer, consisting of 40-60 mM Tris-HCl, 3-8% glycerol, and 0.5-5 mM DTT. Phase B is the elution buffer, consisting of 40-60 mM Tris-HCl, 400-600 mM NaCl, 3-8% glycerol, and 0.5-5 mM DTT. The flow rate is 0.5-1.5 mL / min. First, the volume percentage of the phase B elution buffer is increased from 0 to 20%, eluting 5-10 column volumes. Then, the volume percentage of the phase B elution buffer is increased from 20% to 100%, eluting 3-6 column volumes.

9. The mixture of bromelain hydrolase and high-protein hydrolyzable peptide fragments obtained by the separation and purification method according to any one of claims 1-8.

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