A method for the isolation and purification of tryptophan side-chain oxidase

Abstract

The present invention discloses a method for the isolation and purification of tryptophan side-chain oxidase, belonging to the field of biotechnology. The method of the present invention comprises the following steps: inoculating Pseudomonas ATCC 29574 into 2% BBL liquid medium and culturing until the bacteria reach the stationary phase, and centrifuging to collect the bacterial cells; taking the bacterial cells, adding a bacterial cell lysate, and ultrasonically disrupting to completely disrupt the bacterial cells, and centrifuging to take the supernatant, namely separating the crude enzyme solution; transferring the crude enzyme solution to an ultrafiltration tube for centrifugation, and collecting the enzyme solution in the filter of the ultrafiltration tube; performing affinity chromatography on the enzyme solution through an affinity chromatography column filled with amino agarose coupled with 3-indoleacrylic acid to obtain the purified tryptophan side-chain oxidase. By means of the method of affinity chromatography, a specific ligand is coupled to the chromatography column, so that the tryptophan side-chain oxidase in the fermentation broth specifically binds to the ligand, significantly improving the purity of the tryptophan side-chain oxidase.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to the separation and purification of proteins, and specifically relates to a method for separating and purifying tryptophan side-chain oxidase. Background Art

[0002] Tryptophan side-chain oxidase (TSO) is an enzyme that can act on the α-carbon atom on the tryptophan side chain and dehydrogenate it. It consists of three isozymes with relative molecular masses of 60 kDa, 44 kDa, and 42 kDa respectively. It can oxidize tryptophan in polypeptides, proteins, and some tryptophan analogues. Tryptophan side-chain oxidase is mainly isolated from a Pseudomonas fluorescens ATCC29574, but there is currently no systematic and efficient method for its separation and purification.

[0003] With the development of protein separation and purification technologies, the purification methods of natural proteins have evolved from traditional large-dose, low-purity salting-out separation to small-dose, high-purity automated column chromatography separation. The AKTA protein purifier is a fully automated instrument equipped with a mixer, a pump system, a conductivity detector, a UV detector, and a pH detector. It has the characteristics of convenience, high efficiency, and high sensitivity. By replacing the chromatography columns with different packings and different specifications, different processes in the protein purification process can be satisfied. Therefore, it is widely used in the rapid detection, separation, and purification of biological samples, effectively purifying and separating specific biological small molecules from various biological samples (such as plants, animals, and microorganisms), and is suitable for small-scale production in laboratories and the like.

[0004] Currently, the whole-genome sequencing of Pseudomonas fluorescens ATCC 29574 has not been completed, and it is impossible to construct recombinant colonies through the gene expressing tryptophan side-chain oxidase. It is impossible to capture it using an antibody with a tag. For a natural protein, enzyme solution can be obtained through traditional gel filtration chromatography. However, since only screening is carried out based on the relative molecular mass of the protein, the obtained sample is all proteins within a certain relative molecular mass range, with poor specificity, low sensitivity, time-consuming and laborious due to multiple chromatographies, and ultimately the sample purity is also low. Affinity chromatography utilizes the interaction between proteins and certain ligands for specific binding to separate non-specifically adsorbed substances, and then by changing the buffer conditions, the proteins specifically adsorbed on the ligand are dissociated to form a second chromatographic peak. In this way, the target protein and impurities are separated. The proteins obtained by this method have high specificity and high sample purity, and are suitable for samples with high purity requirements. By connecting different ligands, it can be applied to the separation and purification of different substances. Summary of the Invention

[0005] The object of the present invention is to overcome the disadvantages and deficiencies existing in the prior art, and to provide a method for separating and purifying tryptophan side-chain oxidase. Through the present invention, a protein solution with a relatively high purity can be obtained, which meets the requirements for small-scale laboratory production, laboratory cell experiments, and animal experiments.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A method for separating and purifying tryptophan side-chain oxidase, comprising the following steps:

[0008] (1) Inoculate Pseudomonas fluorescens ATCC 29574 into 2% BBL liquid medium and culture until the bacteria reach the stationary phase, then centrifuge to collect the bacterial cells.

[0009] (2) Take the bacterial cells, add the bacterial cell lysate, and ultrasonically disrupt the cells until they are completely disrupted, then centrifuge and take the supernatant to obtain the crude enzyme solution.

[0010] Among them, the preferred formulation of the bacterial cell lysate is: 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton-X, adjust the pH to 8.5 - 9.0. Add phenylmethylsulfonyl fluoride (PMSF) before use to make its concentration 1 μg / mL.

[0011] (3) Transfer the crude enzyme solution obtained in step (2) to an ultrafiltration tube and centrifuge, and collect the enzyme solution in the filter of the ultrafiltration tube.

[0012] (4) The enzyme solution obtained in step (3) is subjected to affinity chromatography through an affinity chromatography column filled with amino agarose coupled with 3-indole acrylic acid to obtain purified tryptophan side-chain oxidase.

[0013] In some embodiments, step (1) includes the resuscitation, enrichment, and fermentation of Pseudomonas fluorescens ATCC 29574, and centrifugal separation to collect the bacterial cells. Specifically, it can be: inoculate the frozen Pseudomonas fluorescens ATCC 29574 into a nutrient broth medium and culture at 30 °C and 150 rpm for 24 h. Then add the inoculated bacterial liquid to 2% BBL liquid medium at a ratio of 1:20 and mix and culture at 30 °C and 200 rpm for 36 h. After the bacteria complete logarithmic growth and reach the stationary growth stage, centrifuge at 4 °C and 8000×g for 1 h to collect the precipitate.

[0014] In some embodiments, in step (2), the preferred formulation of the bacterial cell lysate is: 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton-X, adjust the pH to 8.5 - 9.0. Add PMSF before use to make its concentration 1 μg / mL.

[0015] In some embodiments, in step (2), the ultrasonic disruption is carried out in an ice-water bath, and the conditions for ultrasonic disruption are as follows: 140 W, working for 10 s, intermittent for 10 s, and the working time is 30 - 40 min; the conditions for centrifugation are: centrifuging at 4 °C and 10000×g for 30 min.

[0016] In some embodiments, step (3) is as follows: Transfer the crude enzyme solution obtained in step (2) to an Amicon Ultra-15 10K ultrafiltration tube (before use, the ultrafiltration tube should be washed with ultrapure water and pre-cooled at 4 °C). If the crude enzyme solution is less than 12 mL, add 0.002 M acetate buffer solution with pH 5.5 to make up to the 12 mL scale line. Place the ultrafiltration tube in a centrifuge with the filter membrane panel facing up, and centrifuge at 4 °C and 4000×g for 40 min. The acceleration and deceleration during centrifugation should not be too large. After centrifugation, pour off the lower filtrate and collect the enzyme solution in the filter.

[0017] In some embodiments, in step (4), the 3-indoleacrylic acid-coupled amino agarose is prepared by a method including the following steps:

[0018] 1) Dissolve 3-indoleacrylic acid, 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate, and N-hydroxysuccinimide in anhydrous dimethylformamide, and ensure that the addition amounts of carbodimethylamine and N-hydroxysuccinimide are in excess. Shake the mixed solution for reaction to generate a pale yellow ester substance. The preferred shaking reaction time is 24 - 48 h.

[0019] 2) Equilibrate the amino agarose with 0.1 M phosphate buffer solution with pH 8.5, and then add the ester substance obtained in step 1) to the equilibrated amino agarose at a ratio of 1:20, and shake the reaction to obtain 3-indoleacrylic acid-coupled amino agarose. The preferred shaking reaction time is 1 h.

[0020] In some embodiments, step (4) is carried out using an AKTA protein purifier, specifically: Equilibrate the affinity chromatography column with 0.002 M acetate buffer solution with pH 5.5 until the effluent A 280 is less than 50 mAU; after equilibration, push the enzyme solution in the sample loop to the affinity chromatography column with 0.002 M acetate buffer solution with pH 5.5; then elute with 0.002 M, 0.05 M, and 0.2 M acetate buffer solutions with pH 5.5 until A 280 is less than 50 mAU, and collect the eluates of the acetate buffer solutions at each gradient concentration.

[0021] The present invention overcomes the problem of impure enzyme solution extracted by traditional methods using fermentation broth and molecular sieves. By means of affinity chromatography, a specific ligand is coupled to the chromatography column, enabling the tryptophan side-chain oxidase in the fermentation broth to specifically bind to the ligand, significantly improving the purity of the product. The separation and purification are carried out using an AKTA protein purifier, which has a high degree of automation, simple steps, high separation efficiency, good reproducibility, and can be widely applied to laboratory preparation. The present invention has the following advantages:

[0022] (1) The present invention selects 3-indoleacrylic acid as the coupling ligand, which has better effects than other indole-ring-containing coupling ligands (such as L-tryptophan, N-acetyl-L-tryptophan, etc.), and can effectively improve the binding rate of tryptophan side-chain oxidase to the affinity chromatography column.

[0023] (2) The present invention uses the method of affinity chromatography to separate and purify tryptophan side-chain oxidase. Compared with the gel filtration method, it can screen and separate the impurity proteins with a molecular weight similar to that of tryptophan side-chain oxidase, and obtain a tryptophan side-chain oxidase solution with a higher purity.

[0024] (3) The present invention selects 2% BBL liquid medium as the fermentation broth, which can not only ensure the protein content of the crude enzyme solution of tryptophan side-chain oxidase, but also reduce the reaction consumption cost, and to a certain extent, facilitate the judgment of the fragmentation end point. Description of the Drawings

[0025] Figure 1 : SDS-PAGE detection of the enzyme solution purified by the 3-indoleacrylic acid-coupled amino agarose affinity chromatography column; Left 1: Protein marker; Left 2: 0.2M acetate eluent; Left 3: 0.05M acetate eluent.

[0026] Figure 2 : SDS-PAGE detection of the enzyme solution purified by the N-acetyl-L-tryptophan-coupled amino agarose affinity chromatography column; Left 1: Protein marker; Left 2: 0.2M acetate eluent; Left 3: 0.05M acetate eluent. Detailed Embodiments

[0027] The following further describes the present invention in detail with reference to the embodiments. It should be noted that the technical features described in the following embodiments or the combination of technical features should not be considered in isolation, and they can be combined with each other to achieve better technical effects.

[0028] Example 1: Preparation of the crude enzyme solution

[0029] (1) Inoculate the frozen Pseudomonas fluorescens ATCC 29574 into the sterilized Nutrient Broth medium and culture it at 30 °C and 150 rpm for 24 h.

[0030] (2) Add the inoculated bacterial liquid obtained in step (1) to the sterilized 2% BBL liquid medium (the BBL liquid medium is purchased from Qingdao Haibo Biotechnology, and the 2% BBL liquid medium is BBL liquid medium: distilled water = 1:49 (v / v)) at a ratio of 1:20 (v / v), and mix and culture at 30 °C and 200 rpm for 36 h. The bacteria complete logarithmic growth and reach a stable growth stage, and the bacterial content in the culture solution is about 20×10 10 / mL.

[0031] In this step, BBL liquid media with other volume fractions are also used. When culturing bacteria with other volume fractions of the medium, there are problems such as difficulty in ultrasonic disruption, difficulty in judging the end point of disruption, or insufficient bacterial concentration.

[0032] (3) Transfer the fermentation broth obtained in step (2) to a centrifuge tube, centrifuge at 4 °C and 8000×g for 1 h, carefully pour off the supernatant, and retain the lower bacterial cell precipitate. The storage temperature of the bacterial cell precipitate is -80 °C.

[0033] (4) Taking 2 g of wet-weight bacteria as an example. Take 2 g of wet-weight bacteria, add 10 mL of bacterial cell lysate, and under ice-water bath conditions, ultrasonically disrupt the bacteria. The ultrasonic disruption conditions are: 140 W, working for 10 s, intermittent for 10 s, and the working time is 30 - 40 min. If the bacterial suspension becomes clear and transparent after ultrasonic treatment, it means that the bacteria are completely disrupted. The formula of the bacterial cell lysate is: 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton-X, adjust the pH to 8.5 - 9.0; add phenylmethylsulfonyl fluoride (PMSF) working solution before use to make its concentration 1 μg / mL.

[0034] (5) Centrifuge the liquid after ultrasonic disruption at 4 °C and 10000×g for 30 min, and take the supernatant, which is the crude enzyme solution.

[0035] (6) Transfer the crude enzyme solution obtained in step (5) to an Amicon Ultra-15 10K ultrafiltration tube (before use, the ultrafiltration tube should be washed with ultrapure water and pre-cooled at 4 °C). If the crude enzyme solution is less than 12 mL, add 0.002 M acetate (sodium acetate - hydrochloric acid) buffer with pH 5.5 to make up to the 12 mL scale line. Place the ultrafiltration tube in a centrifuge with the filter membrane panel facing up, and centrifuge at 4 °C and 4000×g for 40 min. The acceleration and deceleration during centrifugation should not be too large. After centrifugation, pour off the lower filtrate and collect the enzyme solution in the filter.

[0036] Example 2: Coupling of the ligand of the tryptophan side-chain oxidase affinity chromatography column

[0037] (1) Dissolve 4 g of 3-indole acrylic acid, 12 g of 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate, and 4 g of N-hydroxysuccinimide in 100 mL of anhydrous dimethylformamide. Shake the mixture for reaction for 24 - 48 h to generate a pale yellow ester substance.

[0038] (2) Take an appropriate amount of amino agarose filler in a centrifuge tube, and add an equal volume of 0.1 M phosphate buffer solution with pH 8.5 for equilibration. Subsequently, add the ester substance obtained in step (1) to the equilibrated amino agarose according to the mass ratio of ester substance to amino agarose filler of 1:20, and react with shaking at room temperature for 1 h to obtain amino agarose filler conjugated with 3-indole acrylic acid.

[0039] (3) Pack the amino agarose filler conjugated with 3-indole acrylic acid obtained in step (2) into a chromatography column according to the chromatography column packing method. Then open the outlet at the lower end of the chromatography column to drain the conjugation mixture, and wash it with 30 column volumes of 0.1 M phosphate buffer solution with pH 6.9. If it is not used for a long time, the amino agarose affinity chromatography column conjugated with 3-indole acrylic acid is stored at 4 °C with 0.02% sodium azide.

[0040] Meanwhile, prepare an amino agarose affinity chromatography column conjugated with N-acetyl-L-tryptophan or L-tryptophan as a control, and replace 4 g of 3-indole acrylic acid in step (1) above with 5 g of N-acetyl-L-tryptophan or 4 g of L-tryptophan, and the other operations are the same as above.

[0041] Example 3: Separation and extraction of tryptophan side chain oxidase by affinity chromatography

[0042] The protein purifier used in this example is the NGC Quest 10 Chromatography System, and this method is also applicable to other AKTA protein purifier equipment.

[0043] Load the crude enzyme solution of Example 1 onto the protein purifier, with a loading volume of 1 mL. Use the affinity chromatography column prepared in Example 2, and equilibrate the affinity chromatography column with 30 column volumes of 0.002 M acetate buffer solution with pH 5.5 until the effluent A 280 is less than 50 mAU, and the flow rate is 2 mL / min. After equilibration, push the enzyme solution in the sample loop to the affinity chromatography column with 4 mL of 0.002 M acetate buffer solution with pH 5.5. Subsequently, elute with 20 column volumes of acetate buffer solutions with concentrations of 0.002 M, 0.05 M, and 0.2 M and pH 5.5 until A 280 is less than 50 mAU, and collect the eluates of acetate buffer solutions at each gradient concentration.

[0044] Subsequently, wash the column with 5 column volumes of 0.2 M acetate buffer at pH 5.5 and ultrapure water. If not used for a long time, store it with 20% ethanol solution.

[0045] The collected eluate was added to different Amicon Ultra-15 10K ultrafiltration tubes respectively, and centrifuged at 4 °C and 4000×g for 30 - 40 min according to the operation in step (6) of Example 1 to obtain the tryptophan side-chain oxidase enzyme solution.

[0046] Example 4: SDS-PAGE determination of the purified TSO enzyme solution

[0047] (1) Prepare 30 mL of 12% separating gel according to the following table, and mix well by repeated vibration. After mixing, pour the gel along one side of the glass plate to about 0.5 cm below the lower edge of the comb teeth. Subsequently, add anhydrous ethanol to cover the upper part of the separating gel. Let it stand for 45 min until it completely solidifies. Then pour out the anhydrous ethanol and suck out the remaining moisture with filter paper. Prepare 15 mL of stacking gel, insert the comb and let it stand for 30 min until it completely solidifies.

[0048]

[0049] (2) Take 36 μL of protein sample (the tryptophan side-chain oxidase enzyme solution obtained in Example 3), add 10 μL of 5× protein loading buffer and 4 μL of PMSF, and boil for 5 min. Connect the electrophoresis tank to the electrophoresis instrument, add 1× electrophoresis buffer, and carefully pull out the comb. Use a pipette to aspirate 20 μL of the boiled protein sample and add it dropwise to each well in turn. First, run at a constant voltage of 80 V for 30 min, then change the voltage to 120 V and run for about 2 h until the protein marker band runs to the bottom of the gel, and stop the electrophoresis.

[0050] After the electrophoresis is completed, scrape off the stacking gel and retain the separating gel. Put the separating gel into a staining box, add Coomassie R-250 staining solution, and shake and stain for 10 min. Subsequently, recover the staining solution, add decolorizing solution and decolorize overnight. After the bands are clear, take a photo, and the results are shown in Figure 1 and Figure 2 .

[0051] Comparison Figure 1 Figure 2 It can be seen that the protein band purified by the 3-indoleacrylic acid-coupled amino agarose affinity chromatography column is clear and concentrated at about 46 KD. However, there is an obvious tailing phenomenon near the target protein eluted by the 0.2 M acetate eluate of the protein band purified by the N-acetyl-L-tryptophan-coupled amino agarose affinity chromatography column, and the protein concentration and purity obtained are inferior to those purified by the 3-indoleacrylic acid-coupled amino agarose affinity chromatography column. The target protein was not purified by the L-tryptophan-coupled amino agarose affinity chromatography column.

[0052] Analyze using Image J Figure 1 The gray value of the band around 46 kD (the second from the left) was measured, and through calculation and analysis, the purity of the target protein was approximately 67.2%.

[0053] Example 5: Determination of the enzyme activity of the purified TSO enzyme solution by spectrophotometry

[0054] The activity of tryptophan side-chain oxidase (TSO enzyme) eluted with 0.2 M acetate buffer in Example 3 was determined based on the spectrophotometry method in GB / T 15400-2018 with appropriate modifications.

[0055] (1) Preparation of experimental group samples: Use a pipette to add 100 μL of L-tryptophan standard series solutions with a concentration of 300 μg / mL into EP tubes respectively. Measure the enzyme solution concentration using an ultra-micro spectrophotometer. Take an appropriate amount of enzyme solution to make the enzyme content in the solution 5 μg. Number the tubes and mix well by vortex oscillation. Subsequently, place the EP tubes in a constant temperature incubator and incubate at 37°C overnight.

[0056] (2) Preparation of blank group samples: Add 100 μL of L-tryptophan standard series solutions with different concentration gradients respectively. At the same time, add deionized water with the same volume as the enzyme solution added in step (1) to each group. Number the tubes and mix well by vortex oscillation. Place the EP tubes in a constant temperature incubator and incubate at 37°C overnight.

[0057] (3) After incubation, add 100 μL of 10% potassium hydroxide solution to each EP tube and mix well by oscillation. Add 500 μL of p-dimethylaminobenzaldehyde solution and mix well by oscillation. Let it stand at room temperature for 30 min. Subsequently, add 20 μL of 0.2% sodium nitrite solution to each EP tube, shake well, and let it stand at room temperature for 25 min.

[0058] (4) Observe the color of the solutions in the EP tubes of different groups. The blank group is blue, and the experimental group is colorless, indicating that the tryptophan has been metabolized by the TSO enzyme. This result shows that the separation and purification method of the present invention can separate and purify TSO enzyme with high purity.

Claims

1. A method for the isolation and purification of tryptophan side chain oxidase, characterized in that: It includes the following steps: (1) Inoculate Pseudomonas fluorescens ( Pseudomonas fluorescens ) ATCC 29574 into 2% BBL liquid medium and culture until the bacteria reach the stationary phase, then centrifuge to collect the bacterial cells; (2) Take the bacterial cells, add the bacterial cell lysate, and ultrasonically disrupt until the bacterial cells are completely disrupted. Centrifuge and take the supernatant to obtain the crude enzyme solution. (3) Transfer the crude enzyme solution obtained in step (2) to an ultrafiltration tube for centrifugation, and collect the enzyme solution in the filter of the ultrafiltration tube. (4) The enzyme solution obtained in step (3) is subjected to affinity chromatography through an affinity chromatography column filled with 3-indoleacrylic acid-coupled amino agarose to obtain purified tryptophan side-chain oxidase.

2. The separation and purification method of a tryptophan side chain oxidase according to claim 1, characterized in that: Step (1) is as follows: Inoculate the frozen Pseudomonas fluorescens ATCC 29574 into a nutrient broth medium and culture it at 30 °C and 150 rpm for 24 h; then add the inoculated bacterial liquid to a 2% BBL liquid medium at a ratio of 1:20 and mix and culture it at 30 °C and 200 rpm for 36 h; centrifuge at 4 °C and 8000×g for 1 h and collect the precipitate.

3. The separation and purification method of a tryptophan side-chain oxidase according to claim 1, characterized in that: The formula of the bacterial cell lysate described in step (2) is: 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton-X, adjust the pH to 8.5 - 9.0; add phenylmethylsulfonyl fluoride before use to make its concentration 1 μg / mL.

4. The separation and purification method of a tryptophan side chain oxidase according to claim 1, characterized in that: In step (2), the ultrasonic disruption is carried out in an ice-water bath, and the ultrasonic disruption conditions are: 140 W, working for 10 s, intermittent for 10 s, and the working time is 30 - 40 min; the centrifugation conditions are: centrifuge at 4 °C and 10000×g for 30 min.

5. The separation and purification method of a tryptophan side-chain oxidase according to claim 1, wherein: Step (3) is as follows: Transfer the crude enzyme solution obtained in step (2) to an Amicon Ultra-15 10K ultrafiltration tube. If the crude enzyme solution is less than 12 mL, add 0.002 M acetate buffer with a pH of 5.5 to make up to the 12 mL scale line; place the ultrafiltration tube in a centrifuge with the filter membrane panel facing up, centrifuge at 4 °C and 4000×g for 40 min; after centrifugation, pour off the lower filtrate and collect the enzyme solution in the filter.

6. The method for separation and purification of a tryptophan side chain oxidase according to claim 1, characterized in that: In step (4), the 3-indoleacrylic acid-coupled amino agarose is prepared by a method including the following steps: 1) Dissolve 3-indoleacrylic acid, 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate, and N-hydroxysuccinimide in anhydrous dimethylformamide, shake and react the mixture to generate a pale yellow ester substance. 2) Equilibrate the amino agarose with 0.1 M phosphate buffer with a pH of 8.5, and then add the ester substance obtained in step 1) to the equilibrated amino agarose at a ratio of 1:20 and shake and react to obtain 3-indoleacrylic acid-coupled amino agarose.

7. The method for separation and purification of a tryptophan side-chain oxidase according to claim 1, wherein: Step (4) is carried out using an AKTA protein purifier. The affinity chromatography column is equilibrated with 0.002 M acetate buffer at pH 5.5 until the effluent A 280 is less than 50 mAU; after equilibration, the enzyme solution in the sample loop is pushed onto the affinity chromatography column with 0.002 M acetate buffer at pH 5.5; subsequently, it is eluted with 0.002 M, 0.05 M, and 0.2 M acetate buffer at pH 5.5 until A 280 is less than 50 mAU, and the eluates of the acetate buffer at each gradient concentration are collected.

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