Separation and purification method of phthalate degrading enzyme

Abstract

The invention discloses a separation and purification method of a phthalate degrading enzyme, which comprises the following steps: extracting an intracellular enzyme solution of a phthalate degrading bacterium as a crude enzyme solution, selecting a protein liquid chromatography system, and sequentially carrying out DEAE ion exchange, HAP adsorption chromatography purification, Phenyl-Sepharose hydrophobic chromatography purification and Superdex-200 gel filtration to obtain the phthalate degrading enzyme. And separating and purifying the crude enzyme of the phthalate degrading bacteria to obtain the degrading enzyme. The method is high in feasibility and good in separation and purification effect, and the obtained degrading enzyme can efficiently degrade PAEs, so that a foundation is laid for the attempt of constructing more effective microbial degradation engineering strains in the later period.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a method for separating and purifying phthalate-degrading enzyme. Background Art

[0002] Phthalate esters (PAEs) are one of the main organic pollutants in groundwater. The use of microorganisms to remediate PAEs-polluted groundwater has broad application prospects.

[0003] When microorganisms degrade PAEs, specific key enzymes play a role in each step of the metabolic pathway. Therefore, the separation and purification of PAEs metabolic enzymes must be carried out on the basis of a clear metabolic pathway and a full understanding of the target protein. Only by selectively choosing the best separation and purification technology can it be completed, and then the optimal reaction conditions and functional characteristics can be mastered, and it is possible to successfully apply it to the development and application of biological enzyme preparations. The separation and purification of enzymes are the basis for studying the structure, chemical composition and biological functions of enzymes, and are an indispensable part of protein research. The separation and purification of enzymes usually rely on their molecular size, charge properties, properties, solubility and transfer binding sites. In order to obtain pure enzymes, various methods are often combined. Chromatography is the most commonly used method for protein purification. It can not only prepare a large amount of purified protein, but also maintain the biological activity of the protein. For unknown PAEs metabolic enzymes, they can be separated and purified according to their physical and chemical properties combined with conventional enzyme extraction and purification methods, mainly including ion exchange chromatography, gel filtration chromatography, affinity chromatography and some other chromatography techniques.

[0004] Previous studies found that the strain Sphingobium yanoikuyae SHJ can achieve efficient degradation of PAEs in simulated shallow groundwater. Through whole-genome gene sequence sequencing, the genes related to PAEs degradation were found, and the mechanism of PAEs degradation by Sphingobium yanoikuyae SHJ was understood and supplemented at the genomic level. And the PAEs-degrading enzyme system was screened by isobaric tags for relative and absolute quantification (iTRAQ) protein quantification analysis technology. However, its qualitative and functional analysis both rely on existing databases. Some information such as the functional characteristics of key PAEs-degrading enzymes cannot be found in existing databases, or the matching degree with the existing data in the existing databases is not high. In addition, although the enzyme proteins matched in the database have similar functions, it is impossible to determine their key roles in the PAEs degradation pathway. Summary of the Invention

[0005] In order to overcome the above-mentioned disadvantages of the prior art, the object of the present invention is to provide a method for separating and purifying phthalate-degrading enzymes, which can separate and purify the degrading enzymes that play a key role in the PAEs degradation pathway from the crude enzyme solution of degrading bacteria without relying on a database, and solve the problem that the functional information is restricted by the database.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for separating and purifying phthalate-degrading enzymes, which extracts the intracellular enzyme solution of phthalate-degrading bacteria as the crude enzyme solution, selects a protein liquid chromatography system, and sequentially passes through DEAE ion exchange, HAP adsorption chromatography purification, Phenyl-Sepharose hydrophobic chromatography purification and Superdex-200 gel filtration to separate and purify the degrading enzymes from the crude enzyme solution of phthalate-degrading bacteria.

[0008] In one embodiment, the phthalate-degrading bacteria is Sphingobium yanoikuyae SHJ, and the protein liquid chromatography system is an AKTA Purifier fast protein liquid chromatography system.

[0009] In one embodiment, the method for extracting the crude enzyme solution of phthalate-degrading bacteria is as follows:

[0010] Inoculate the bacterial suspension of phthalate-degrading bacteria into an inorganic salt liquid medium containing DEP, and incubate it at a constant temperature under anaerobic conditions to obtain a culture solution. Centrifuge to collect the bacterial cells, wash the bacterial cells with a buffer solution and then suspend them, ultrasonically disrupt them in an ice bath, centrifuge the disrupted cell solution, and collect the supernatant to obtain the intracellular enzyme solution.

[0011] In one embodiment, the bacterial suspension of phthalate-degrading bacteria is obtained by the following method:

[0012] Inoculate the phthalate-degrading bacteria into a peptone-yeast extract medium containing DEP, culture it until the logarithmic growth phase, centrifuge to collect the bacterial cells, wash them with a buffer solution and then resuspend them in sterile normal saline.

[0013] In one embodiment, in the inorganic salt liquid medium and the peptone-yeast extract medium, the content of DEP is 50 mg·L -1 ; the buffer solution for washing the bacterial cells when obtaining the bacterial suspension is a phosphate buffer with a pH of 7.4; the buffer solution for washing the bacterial cells obtained from the culture solution is Tris-HCl with a pH of 7.5, and before performing DEAE ion exchange, Tris-HCl with a concentration of 1 M and a pH of 7.8 is added to the intracellular enzyme to make the final concentration of Tris-HCl 20 mM.

[0014] In one embodiment, the DEAE ion exchange is carried out as follows:

[0015] Equilibrate the column volume of the DEAE ion exchange chromatography column with Buffer A, and then load the sample. The sample to be loaded is the intracellular enzyme solution degassed and filled with nitrogen under ice bath conditions; after loading, elute with a gradient of Buffer A and Buffer B, and collect one bottle every fixed volume after the column. Keep the temperature under ice bath throughout the process;

[0016] The HAP adsorption chromatography purification is carried out as follows:

[0017] Equilibrate the column volume of the HAP adsorption chromatography column with Buffer A, and then load the sample. The sample to be loaded is the active fraction collected after the DEAE ion exchange column; after loading, elute with a gradient of Buffer A and Buffer C, and collect one bottle every fixed volume after the column. Keep the temperature under ice bath throughout the process;

[0018] The Phenyl-Sepharose hydrophobic chromatography purification is carried out as follows:

[0019] Equilibrate the column volume of the Phenyl-Sepharose hydrophobic chromatography column with Buffer D, and then load the sample. The sample to be loaded is the active fraction collected after the HAP adsorption chromatography column; after loading, elute with a gradient of Buffer D and Buffer A, and collect one bottle every fixed volume after the column. Keep the temperature under ice bath throughout the process.

[0020] The Superdex-200 gel filtration is carried out as follows:

[0021] Concentrate the active sample collected after the Phenyl-Sepharose hydrophobic chromatography column with a Concentration Cell, degas and fill it with nitrogen for standby as the Superdex-200 gel filtration sample; equilibrate the column volume of the Superdex-200 gel filtration column with Buffer E, and then load the sample. After loading, elute with Buffer E isocratically, and collect one bottle every fixed volume after the column. Keep the temperature under ice bath throughout the process.

[0022] In one embodiment, the Buffer A is 50 mM Tris-HCl, pH 7.8; the Buffer B is 50 mM Tris-HCl containing NaCl, pH 7.8, and the NaCl concentration is 2 M; the Buffer C is 50 mM Tris-HCl containing KH 2 PO 4 with a concentration of 50 mM Tris-HCl, pH 7.8, and the concentration of KH 2 PO 4 is 0.5 M; the Buffer D is containing (NH4 ) 2 PO 4 at a concentration of 50 mM Tris-HCl, pH 7.8, KH 2 PO 4 at a concentration of 0.8 M; said Buffer E is 50 mM Tris-HCl containing KCl, pH 7.8, with a KCl concentration of 100 mM.

[0023] In one embodiment, the gradient elution for DEAE ion exchange is as follows: 0 - 1 CV, 100% Buffer A; 1 - 3 CV, 0 - 50% Buffer B; 3 - 4 CV, 50% Buffer B; 4 - 5 CV, 100% Buffer B;

[0024] The gradient elution for HAP adsorption chromatography purification is as follows: 0 - 1 CV, 100% Buffer A; 1 - 6 CV, 0 - 100% Buffer C; 6 - 7 CV, 100% Buffer C;

[0025] The gradient elution for Phenyl-Sepharose hydrophobic chromatography purification is as follows: 0 - 1 CV, 100% Buffer D; 1 - 6 CV, 100 - 0% Buffer D; 6 - 7 CV, 0% Buffer D;

[0026] The isocratic elution is as follows: 0 - 1 CV, 100% Buffer Buffer E.

[0027] In one embodiment, during the DEAE ion exchange process, the liquid temperatures before and after the column and the column temperature are maintained at 0 °C, and during the HAP adsorption chromatography purification, Phenyl-Sepharose hydrophobic chromatography purification, and Superdex-200 gel filtration processes, the liquid temperatures before and after the column and the column temperature are maintained at 4 °C.

[0028] In one embodiment, SDS-polyacrylamide gel electrophoresis technology is used for detection, and four purified enzyme bands are isolated and purified from the crude enzyme solution of phthalate-degrading bacteria, namely Band I, Band II, Band III, and Band IV; among them, the final purification fold of Band I is 1.21, the apparent molecular weight is 58.50 kD, and it has transferase activity, dioxygenase activity, and transporter protein function; the final purification fold of Band II is 1.21, the apparent molecular weight is 35.75 kD, and it has isomerase activity, hydrolase activity, and transferase activity; the final purification fold of Band III is 1.19, the apparent molecular weight is 68.94 kD, and it is a carbon-carbon lyase that catalyzes the cleavage of C-C bonds; the final purification fold of Band IV is 1.19, the apparent molecular weight is 62.76 kD, and it has transferase activity.

[0029] In one embodiment, the coding gene sequence of the degradation enzyme obtained by separation and purification can be aligned with the whole genome sequence of the strain, and the corresponding gene sequence is obtained by cloning technology to obtain a highly efficient engineering bacterium with the ability to degrade PAEs.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. Based on the research on the determination of PAEs degradation enzyme activity and its enzymatic properties, etc., the present invention establishes an enzyme separation and purification system to realize the separation and purification of the PAEs degradation crude enzyme liquid mixture of Sphingobium yanoikuyae SHJ. Finally, four purified enzyme bands (bands I, II, III, and IV) are successfully purified, and protein identification and function prediction are carried out on them. Among them, the final purification fold of band I is 1.21, the apparent molecular weight is 58.50 kD, and it has transferase activity, dioxygenase activity, and transporter protein function; the final purification fold of band II is 1.21, the apparent molecular weight is 35.75 kD, and it has isomerase activity, hydrolase activity, and transferase activity; the final purification fold of band III is 1.19, the apparent molecular weight is 68.94 kD, and it is a carbon-carbon lyase that catalyzes the cleavage of C-C bonds; the final purification fold of band IV is 1.19, the apparent molecular weight is 62.76 kD, and it has transferase activity.

[0032] 2. The PAEs degradation enzyme isolated and purified by the present invention can efficiently degrade PAEs, which will lay a foundation for the subsequent attempt to construct a more effective microbial degradation engineering strain.

[0033] 3. The present invention adopts the AKTA Purifier fast protein liquid chromatography system. With the supporting chromatographic column and method template, it can realize the rapid separation and purification of protein mixtures through various purification techniques such as ion exchange, gel filtration, hydrophobic chromatography, reverse chromatography, and affinity chromatography. It is also equipped with a fraction collector with peak collection function, and can also be controlled by software operation, having the advantages of high resolution, convenient enzyme recognition, and mild operation conditions.

[0034] 4. The present invention proposes a method for isolating PAEs degradation enzyme from the PAEs-degrading microorganism Sphingobium yanoikuyae SHJ. The method is reasonably designed, highly feasible, and has good separation and purification effects. Description of the Drawings

[0035] Figure 1 It is a diagram for measuring the purity and apparent molecular weight of the purified protein during the PAEs degradation process in the embodiment of the present invention, where:

[0036] Lane 1: Low molecular weight protein standard. From top to bottom, they are β-galactosidase (116,000), bovine serum albumin protease (66,200), ovalbumin (45,000), lactate dehydrogenase (35,000), β-lactoglobulin (18,400), restriction endonuclease bsp98 type 1 (25,000), lysozyme (14,400);

[0037] Lane 2: Crude enzyme solution;

[0038] Lane 3: Purified protein I collected from the DEAE column;

[0039] Lane 4: Purified protein I collected from the HAP column;

[0040] Lane 5: Purified protein I collected from the Phenyl-sepharose-A column;

[0041] Lane 6: Purified protein I collected from the Superdex 200-B column;

[0042] Lane 7: Purified protein II collected from the Superdex 200-B column

[0043] Figure 2 It is in the determination diagram of the purity and apparent molecular weight of the purified protein during the PAEs degradation process in the embodiment of the present invention:

[0044] Lane 1: Low molecular weight protein standard. From top to bottom, they are β-galactosidase (116,000), bovine serum albumin protease (66,200), ovalbumin (45,000), lactate dehydrogenase (35,000), β-lactoglobulin (18,400), restriction endonuclease bsp98 type 1 (25,000), lysozyme (14,400);

[0045] Lane 2: Crude enzyme solution;

[0046] Lane 3: Purified protein II collected from the DEAE column;

[0047] Lane 4: Purified protein II collected from the HAP column;

[0048] Lane 5: Purified protein II collected from the Phenyl-sepharose-B column;

[0049] Lane 6: Purified protein III collected from the Superdex 200-D column;

[0050] Lane 7: Purified protein IV collected from the Superdex 200-D column Detailed implementation mode

[0051] The implementation mode of the present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0052] In order to deeply understand the mechanism of action of PAEs-degrading bacterium Sphingobium yanoikuyae SHJ in degrading PAEs, and to attempt to construct a more effective microbial degradation engineering bacterium in the future, the present invention proposes a method for isolating PAEs-degrading enzymes from the PAEs-degrading bacterium Sphingobium yanoikuyae SHJ with reasonable design and strong feasibility. Based on the previous studies on protease activity determination and its enzymatic properties, the present invention establishes an enzyme separation and purification system, selects an AKTA Purifier fast protein liquid chromatography system (AKTA-FPLC, GE Healthcare, USA), and separates and purifies the protein mixture through four chromatography techniques: diethylaminoethyl cellulose (DEAE) ion exchange, hydroxyapatite (HAP) adsorption chromatography purification, phenyl-agarose hydrophobic chromatography purification, and Superdex-200 gel filtration in sequence.

[0053] The specific implementation process of the method for isolating and purifying phthalate-degrading enzymes of the present invention is as follows. The main steps include:

[0054] (1) Extraction of crude PAEs-degrading enzyme solution: Inoculate the PAEs-degrading bacterium Sphingobium yanoikuyae SHJ into a nutrient broth peptone medium (NB) containing 50 mg·L -1 DEP, culture at 30 °C and 150 r·min -1 until the logarithmic growth phase (24 h), centrifuge at 4000 xg for 10 min, collect the bacterial cells, wash them 3 times with sterilized phosphate buffer (PBS, pH 7.4), and then resuspend them in sterile normal saline (0.90%) to obtain a bacterial suspension. Inoculate the bacterial suspension into an inorganic salt liquid medium (MSM) containing 50 mg·L -1 DEP, and adjust its OD600 nm to 0.3. Incubate at a constant temperature under anaerobic conditions (4000 mL brown bottle, sealed without headspace, 17 °C, static, dark) for 96 h, and then collect the culture solution. Centrifuge the collected culture solution at 8000 xg at 4 °C for 10 min, collect the bacterial cells, and the supernatant is the extracellular enzyme; wash the bacterial cells three times with 10 mM Tris-HCl (pH 7.5) buffer, then suspend the bacterial cells with the buffer (V:W = 1:3 mL / g), ultrasonically disrupt them in an ice bath, ultrasonicate for 3 s each time, with an interval of 3 s, for a total of 20 min, and centrifuge the disrupted cell solution at 13000 xg at 4 °C for 30 min, and collect the supernatant to obtain the intracellular enzyme solution.

[0055] In this step, by using a solution containing 50 mg·L-1 Enrich and culture the degrading bacteria with beef extract peptone medium containing DEP. Add 50 mg·L -1 of DEP to the beef extract peptone medium to enable it to adapt to the pollutant environment in advance, which has the effect of domestication. Incubate at 30 °C and 150 r·min -1 until the logarithmic growth phase (24 h), at which time the bacterial activity is the highest.

[0056] In this step, after washing 3 times with sterilized phosphate buffer (PBS, pH 7.4), resuspend the enriched strains in sterile normal saline (0.90%). By inoculating the bacterial suspension into the inorganic salt liquid medium (MSM) containing 50 mg·L -1 of DEP respectively, using diethyl phthalate (DEP), one of the high-concentration detected substances of PAEs in shallow groundwater sediments, as the research object, inoculate a large number of enriched strains into the MSM containing 50 mg·L -1 of DEP and culture for 96 h to synthesize related degrading enzyme systems in vivo. By measuring the progress curve of the enzymatic reaction, it is determined that the appropriate reaction time for measuring the enzyme activity of the extracellular enzyme of the crude DEP biodegradation enzyme solution is 180 min, while the appropriate reaction time for measuring the enzyme activity of the intracellular enzyme is 30 min; from the enzyme properties, it is known that the specific activity of the extracellular enzyme is the highest when the enzyme concentration is 0.065 μg·μL -1 and that of the intracellular enzyme reaches the highest when the enzyme concentration is 0.01 μg·μL -1 ; the enzymatic reaction rate of both the extracellular enzyme and the intracellular enzyme is the largest when the carbon source DEP concentration is 100 mg·L -1 ; the extracellular enzyme has the highest activity at a system pH of 7.2, while the intracellular enzyme has the highest activity at pH 6.5; the extracellular enzyme has the highest activity at about 45 °C, while the intracellular enzyme has the highest activity at 25 °C. The reason for culturing for 96 h is that through the study of the DEP degradation pathway, it is found that the obtained metabolites and the key time points of the degradation pathway are 24 h and 96 h, and no other products are generated after 96 h.

[0057] (2) DEAE ion exchange chromatography purification: Add 1 M Tris-HCl (pH 7.8) to the intracellular enzyme to make its final concentration 20 mM Tris-HCl, and then degas and fill with nitrogen under ice bath conditions for standby. Before use, equilibrate the DEAE ion exchange chromatography column with Buffer A (50 mM Tris-HCl, pH 7.8) for 2 CV (column volume), rinse pump B, rinse pump A, and then load the sample with pump B (diluted 50% with Buffer A, gradient-target: 50% B), and the flow rate is 5 mL·min -1, the liquid before and after the column is kept at low temperature (ice bath), and the column is kept at low temperature (0 °C) with a circulating water pump; after loading the sample, pump B is rinsed, pump A is rinsed, and then gradient elution is performed with Buffer A and Buffer B (50 mM Tris-HCl containing 2 M NaCl, pH 7.8) (0-1 CV, 100% A; 1-3 CV, 0-50% B; 3-4 CV, 50% B; 4-5 CV, 100% B), and the flow rate is 5 mL·min -1 , one bottle is collected every 25 mL after the column, and the liquid before and after the column and the column are also kept at low temperature.

[0058] Ion exchange column chromatography is a method for separating different components based on the difference in the net charge of proteins under certain pH conditions. The stationary phase of the anion exchange chromatography column is positively charged and combines with negatively charged proteins, mainly used for the separation of protein samples with relatively low isoelectric points. The selection of the ionic strength and pH value of the starting buffer should fully consider the pH value of the target protein to effectively separate the impurity proteins. The reason for choosing Tris-HCl (pH 7.8) in this experiment is mainly to consider the source environment of the target protein and the continuity of the subsequent elution steps. The elution results show that when the crude enzyme extract passes through the DEAE chromatography column, the NaCl concentration in the elution solution is about 0.65 mol·L -1 when a single active elution peak is obtained.

[0059] After detecting the protein concentration and enzyme activity of all collection tubes, the peak time of the protein concentration peak and the enzyme activity peak corresponding to the active elution peak in the collection tubes basically coincide. Refer to Figure 1 and Figure 2 , the SDS-PAGE gel electrophoresis purity detection shows that compared with the crude enzyme solution of the intracellular enzyme, a large amount of impurity proteins have been removed and the enzyme sample volume has been significantly reduced after the DEAE ion exchange chromatography column, and the protein band is clearer, indicating that the crude enzyme solution has been purified and separated and concentrated.

[0060] (3) HAP adsorption chromatography purification: The HAP adsorption chromatography column is equilibrated with Buffer A (50 mM Tris-HCl, pH 7.8) for 2 CV, pump B is rinsed, pump A is rinsed, and then sample is loaded with pump B (gradient-target: 100% B), and the flow rate is 1.5 mL·min -1 , the liquid before and after the column is kept at low temperature (ice bath), and the column is kept at low temperature (4 °C) with a circulating water pump; the sample to be loaded is the active part collected after the DEAE ion exchange column. After loading the sample, pump B is rinsed, pump A is rinsed, and then Buffer A and Buffer C (containing 0.5 M KH 2 PO 4Gradient elution (0 - 1 CV, 100% A; 1 - 6 CV, 0 - 100% C; 6 - 7 CV, 100% C) with 50 mM Tris - HCl, pH 7.8 at a flow rate of 1.5 mL·min -1 , and collect one bottle every 15 mL after the column. Keep the temperature low before the column, after the column, and for the column itself as well.

[0061] HAP hydroxyapatite ceramic particles are a special ion - exchange agent. Its main component is calcium phosphate, containing positively charged calcium ions and negatively charged phosphate ions in the molecule. Phosphate ions can bind to positively charged proteins by ionic bonds and can be eluted by a gradient of sodium chloride or phosphate solution. Calcium ions bind to the free carboxyl groups of negatively charged proteins in a metal - chelation manner, which is insensitive to sodium chloride and can be eluted by a phosphate - solution gradient.

[0062] Collect the eluate of the active peak after the DEAE column and pass it through the HAP adsorption chromatography column. The elution results show that when the concentration of KH 2 PO 4 is 0.05 mol·L -1 and 0.35 mol·L -1 , two active elution peaks (I and II) are obtained after the HAP column respectively. Through the detection of protein concentration and enzyme activity of all collected tubes, the peak values of protein concentration and enzyme activity corresponding to the two active elution peaks show that the proteins in each collected tube have enzyme activity. Referring to Figure 1 and Figure 2 , SDS - PAGE gel electrophoresis purity detection shows that compared with the purified enzyme solution after the DEAE ion - exchange chromatography column, the impurity proteins in the purified enzyme solution after the HAP adsorption chromatography column have been largely removed, and each main protein band is clearer, indicating that the crude enzyme solution has been further purified and separated.

[0063] (4) Phenyl - Sepharose hydrophobic chromatography purification: Equilibrate the Phenyl - Sepharose hydrophobic chromatography column with Buffer D (containing 0.8 M (NH 4 ) 2 PO 4 in 50 mM Tris - HCl, pH 7.8) for 2 CV, rinse pump A, rinse pump B, and then load the sample with pump B (gradient - target: 100% B) at a flow rate of 2 mL·min -1, the liquid before and after the column is kept at low temperature (ice bath), and the column is kept at low temperature (4 °C) by a circulating water pump; the sample to be loaded is the active part collected after the HAP adsorption chromatography column. After loading the sample, pump A is rinsed, pump B is rinsed, and then gradient elution is performed with Buffer D and Buffer A (50 mM Tris-HCl, pH 7.8) (0-1 CV, 100% D; 1-6 CV, 100-0% D; 6-7 CV, 0% D), and the flow rate is 2 mL·min -1 , one bottle is collected every 20 mL after the column, and the liquid before and after the column and the column are also kept at low temperature.

[0064] 40% - 50% of the protein surface is non-polar. These regions bind to hydrophobic adsorbents at high or medium salt concentrations, and the binding ability varies with the salt concentration. The hydrophobic structure comes from the hydration structure of protein molecules. High-concentration salt solutions can disrupt the hydration structure of biomolecules, exposing the hydrophobic groups inside the protein to the outer surface of the molecule, thus binding to the hydrophobic medium. That is to say, proteins bind to the hydrophobic chromatography column with high-concentration salt solutions and are eluted with low-concentration salt solutions, while hydrophobic chromatography is the opposite of ion exchange. Adsorption with high salt and elution with low salt. The eluted sample is directly diluted and loaded onto other chromatography columns, which can be used as a bridge connecting chromatographic steps to replace salting-out precipitation.

[0065] The elution solution of the two active peaks I and II after the HAP column (denoted as HAP I and HAP II) is collected in this elution step and passed through the Phenyl-Sepharose hydrophobic chromatography column (PS-A and PS-B) respectively. The elution results show that in the elution solution (NH 4 ) 2 PO 4 concentrations are 0.8 mol·L -1 and 0 mol·L -1 respectively, two elution peaks (PS-A I, PS-A II, PS-B I, PS-B II) are obtained after the Phenyl-Sepharose column. After detecting the protein concentration and enzyme activity of all collected tubes, the protein concentration peak and enzyme activity peak corresponding to each active elution peak show that the proteins in each collected tube have enzyme activity. Referring to Figure 1 and Figure 2 , SDS-PAGE gel electrophoresis purity detection shows that compared with the purified enzyme solution after the HAP adsorption chromatography column, most of the impurity proteins in the purified enzyme solution after the Phenyl-Sepharose hydrophobic chromatography column have been removed, each main protein band is clearer, and the target band is concentrated around 30 - 70 kDa, indicating that the crude enzyme solution has been further purified and separated, but the target protein still does not form a single band.

[0066] (5) Superdex-200 Gel Filtration: The active sample collected after Phenyl-Sepharose hydrophobic chromatography column was concentrated to 1.5 mL using a Concentration Cell (molecular weight cut-off 30,000 Da), degassed and filled with nitrogen (in an ice bath), and reserved as the subsequent Superdex-200 gel filtration sample. The Superdex-200 gel filtration column was equilibrated with Buffer E (50 mM Tris-HCl containing 100 mM KCl, pH 7.8) for 2 CV, the pump B was rinsed, the pump A was rinsed, and then the sample was loaded using pump B (gradient-target: 100% B) at a flow rate of 1 mL·min -1 , and the liquid before and after the column was kept at low temperature (ice bath), and the column was kept at low temperature (4 °C) using a circulating water pump; after loading the sample, the pump B was rinsed, the pump A was rinsed, and then isocratic elution was performed with Buffer E for 1 CV (0 - 1 CV, 100% E) at a flow rate of 2 mL·min -1 , and one bottle was collected every 20 mL after the column, and the liquid before and after the column and the column were also kept at low temperature.

[0067] Gel filtration is also known as molecular sieve and molecular exclusion chromatography. This gel column uses a porous gel material as a carrier. When a protein solution is supported by this material, the blocking effects on proteins of different molecular sizes are different. Large protein molecules are eluted first, and small molecules are eluted later, thus achieving the purpose of separation and purification. Gel filtration chromatography is generally used for the last step of purification. The operating conditions are relatively mild and do not require organic solvents. The buffer composition has little effect on gel filtration chromatography, as long as the buffer composition does not affect the shape and biological activity of the sample. The selected buffer conditions should be suitable for the stability and activity of the protein, such as 25 - 150 mM KCl. Superdex-200 gel filtration requires the specimen to be highly concentrated, and the sample loading volume can only be between 1% and 2% of the column volume. The collected liquid of the four active peaks obtained after the Phenyl-Sepharose column (denoted as PS-AI, PS-AII, PS-B I, PS-B II) was respectively concentrated to 1.5 mL using a Concentration Cell (molecular weight cut-off 30,000 Da), and then subjected to Superdex-200 gel filtration (Superdex-A, Superdex-B, Superdex-C, and Superdex-D). The elution results showed that when the KCl concentration in the elution solution was between 0.1 mol·L -1 , multiple single elution peaks were obtained respectively after the Superdex-200 gel column.

[0068] After detecting the protein concentration and enzyme activity of all collection tubes, it can be seen from the protein concentration peak and enzyme activity peak of the collection tubes corresponding to each active elution peak that the protein in the collection tubes corresponding to each elution peak is inconsistent with its enzyme activity. It is still necessary to further separate the enzyme solution of the collection tubes corresponding to each single active elution peak by SDS-PAGE and check the purity of the protein. Refer to Figure 1 and Figure 2 In bands 6 and 7, SDS-PAGE gel electrophoresis purity detection shows that compared with the purified enzyme solution after Phenyl-Sepharose hydrophobic chromatography column, the impurity proteins in the purified enzyme solution after Superdex-200 gel filtration column have been completely removed, and the bands of each target protein are clearer, and the target protein forms a single band, indicating that the PAEs degrading enzyme has been separated and purified from the crude enzyme extract through four chromatography techniques: DEAE ion exchange, HAP adsorption chromatography purification, Phenyl-Sepharose hydrophobic chromatography purification and Superdex-200 gel filtration.

[0069] (6) Determination of the purity and molecular weight of the PAEs degrading enzyme: The purity of the PAEs degrading enzyme solution and the apparent molecular weight of the purified enzyme were detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Take the active enzyme solution collected after Superdex-200 gel filtration purification and concentrate it to 0.1 - 1 mL (so that the final protein concentration after concentration is about 1 mg·mL -1 ) using an ultrafiltration concentration device (Amicon Stirred Ultrafiltration Cells, molecular weight cut-off 30,000 Da, Millpore, USA), and perform SDS-PAGE electrophoresis under reducing conditions. If the PAGE electrophoresis shows an obvious single band, it indicates that the separation and purification result of the active protein is good and reaches its electrophoretic purity. By comparing with the molecular weights of low molecular weight standard proteins, the relationship between the protein molecular weight and the relative mobility was obtained, and based on this, the molecular weight of the DEP degrading enzyme was calculated.

[0070] Finally, four purified enzyme bands were successfully purified in the present invention (i.e., Figure 1 、 Figure 2The bands I, II, III, and IV) were isolated and purified, and protein identification and function prediction were performed. Among them, the final purification fold of band I was 1.21, and the apparent molecular weight was 58.50 kD. It had transferase activity, dioxygenase activity, and transporter protein function; the final purification fold of band II was 1.21, and the apparent molecular weight was 35.75 kD. It had isomerase activity, hydrolase activity, and transferase activity; the final purification fold of band III was 1.19, and the apparent molecular weight was 68.94 kD. It was a carbon-carbon lyase that catalyzed the cleavage of C-C bonds; the final purification fold of band IV was 1.19, and the apparent molecular weight was 62.76 kD. It had transferase activity.

[0071] The enzymes isolated and purified from the highly efficient degrading bacteria can be identified by mass spectrometry, and their coding gene sequences are compared with the known whole genome sequences of the strains. Finally, it is determined that the coding gene sequences of these pure degrading enzymes are also in the whole genome sequence of the strain Sphingobium yanoikuyae SHJ, indicating that the isolation and purification of PAEs degrading enzymes are successful, and their degradation functions are encoded and expressed by the genes of the strain Sphingobium yanoikuyae SHJ. Therefore, the corresponding (several of which reached 100% in identity and coverage in the experiment, and several were about 80%, so "corresponding" here can be considered as consistent in alignment) gene sequences can be obtained by cloning technology to obtain highly efficient engineering bacteria with the ability to degrade PAEs. This will lay a foundation for the construction of more effective microbial degradation engineering strains in the later stage, provide a good gene source for effectively using genetic engineering means to clone PAEs degrading enzyme genes, and ultimately achieve the goal of cheaply producing PAEs degrading engineering bacteria.

Claims

1. A method for separating and purifying a phthalate-degrading enzyme, characterized in that: The intracellular enzyme liquid of phthalate-degrading bacteria was extracted as crude enzyme liquid, and a protein liquid chromatography system was selected to separate and purify the degradation enzyme from the crude enzyme liquid of phthalate-degrading bacteria through DEAE ion exchange, HAP adsorption chromatography purification, Phenyl-Sepharose hydrophobic chromatography purification and Superdex-200 gel filtration in sequence.

2. The method for separating and purifying phthalate degrading enzyme according to claim 1, characterized in that: The phthalate-degrading bacteria is Sphingobium yanoikuyae SHJ, and the protein liquid chromatography system is an AKTA Purifier fast protein liquid chromatography system.

3. The method for separating and purifying phthalate degrading enzyme according to claim 1 or 2, characterized in that: The method for extracting the crude enzyme solution of phthalate-degrading bacteria is as follows: The bacterial suspension of phthalate-degrading bacteria is inoculated into an inorganic salt liquid culture medium containing DEP, cultured at a constant temperature under oxygen-limited conditions to obtain a culture solution, the bacterial bodies are collected by centrifugation, the bacterial bodies are washed with a buffer solution and then suspended, ultrasonically disrupted in an ice bath, the disrupted cell fluid is centrifuged, and the supernatant is collected to obtain the intracellular enzyme solution.

4. The method for separating and purifying phthalate degrading enzyme according to claim 3, characterized in that: The bacterial suspension of the phthalate-degrading bacteria is obtained by the following method: The phthalate-degrading bacteria were inoculated into a beef extract peptone medium containing DEP, cultured to the logarithmic growth phase, and the bacteria were collected by centrifugation, washed with a buffer solution, and then resuspended in a sterile physiological saline.

5. The method for separating and purifying phthalate degrading enzyme according to claim 4, characterized in that: The inorganic salt liquid culture medium and the beef extract peptone culture medium both contain 50 mg·L DEP. -1 ; The buffer for washing the bacteria when obtaining the bacterial suspension is a phosphate buffer, pH 7.4; the buffer for washing the bacteria obtained from the culture medium is Tris-HCl, pH 7.5, and before performing DEAE ion exchange, Tris-HCl with a concentration of 1 M and pH 7.8 is added to the intracellular enzyme to make the final concentration of Tris-HCl 20 mM.

6. The method for separating and purifying phthalate degrading enzyme according to claim 5, characterized in that: The DEAE ion exchange process is as follows: The column volume of the DEAE ion exchange chromatography column was balanced with Buffer A, and then the sample was loaded, and the loaded sample was the intracellular enzyme solution degassed and nitrogen-filled under ice bath conditions; after loading, Buffer A and Buffer B were used for gradient elution, and one bottle was collected for each fixed volume after the column, and the temperature was maintained by ice bath during the whole process; The HAP adsorption chromatography purification process is as follows: Use Buffer A to balance the column volume of the HAP adsorption chromatography column, and then load the sample, which is the active part collected after the DEAE ion exchange column; after loading, use Buffer A and Buffer C for gradient elution, and collect one bottle for each fixed volume after the column. Use an ice bath to maintain the temperature during the whole process; The Phenyl-Sepharose hydrophobic chromatography purification process is as follows: Use Buffer D to balance the column volume of the Phenyl-Sepharose hydrophobic chromatography column, and then load the sample, which is the active part collected after the HAP adsorption chromatography column; After loading, Buffer D and Buffer A were used for gradient elution. A fixed volume was collected in one bottle after the column. The temperature was maintained in an ice bath during the whole process. The Superdex-200 gel filtration process is as follows: The active sample collected after the Phenyl-Sepharose hydrophobic chromatography column was concentrated with a Concentration Cell, degassed and nitrogen-filled for later use as a Superdex-200 gel filtration sample; the column volume of the Superdex-200 gel filtration column was balanced with Buffer E, and then the sample was loaded. After loading, eluted isocratically with Buffer E, and one bottle was collected for each fixed volume after the column. The temperature was maintained in an ice bath during the whole process.

7. The method for separating and purifying phthalate degrading enzyme according to claim 6, characterized in that: The Buffer A is 50 mM Tris-HCl, pH 7.8; the Buffer B is 50 mM Tris-HCl, pH 7.8, containing NaCl, and the NaCl concentration is 2 M; the Buffer C is 50 mM Tris-HCl, pH 7.8, containing KH2PO4, and the KH2PO4 concentration is 0.5 M; the Buffer D is 50 mM Tris-HCl, pH 7.8, containing (NH4)2PO4, and the KH2PO4 concentration is 0.8 M; the Buffer E is 50 mM Tris-HCl, pH 7.8, containing KCl, and the KCl concentration is 100 mM.

8. The method for separating and purifying phthalate degrading enzyme according to claim 5, characterized in that: The gradient elution of the DEAE ion exchange is: 0-1CV, 100% Buffer A; 1-3CV, 0-50% Buffer B; 3-4CV, 50% Buffer B; 4-5CV, 100% Buffer B; The gradient elution of the HAP adsorption chromatography purification is: 0-1CV, 100% Buffer A; 1-6CV, 0-100% Buffer C; 6-7CV, 100% Buffer C; The gradient elution of the Phenyl-Sepharose hydrophobic chromatography purification is: 0-1CV, 100% Buffer D; 1-6CV, 100-0% Buffer D; 6-7CV, 0% Buffer D; The isocratic elution is: 0-1CV, 100% Buffer Buffer E.

9. The method for separating and purifying phthalate degrading enzyme according to claim 1 or 2, characterized in that: Using SDS-polyacrylamide gel electrophoresis technology, four purified enzyme bands were separated and purified from the crude enzyme solution of phthalate-degrading bacteria, namely band I, band II, band III and band IV; among them, the final purification factor of band I was 1.21, and the apparent molecular weight was 58.50kD, which had transferase activity, dioxygenase activity and transporter function; the final purification factor of band II was 1.21, and the apparent molecular weight was 35.75kD, which had isomerase activity, hydrolase activity and transferase activity; the final purification factor of band III was 1.19, and the apparent molecular weight was 68.94kD, which was a carbon-carbon lyase that catalyzed the breakage of CC bonds; the final purification factor of band IV was 1.19, and the apparent molecular weight was 62.76kD, which had transferase activity.

10. The method for separating and purifying phthalate degrading enzyme according to claim 1, characterized in that: The coding gene sequence of the isolated and purified degradation enzyme was compared with the whole genome sequence of the strain, and the corresponding gene sequence was cloned to obtain an efficient engineered bacterium capable of degrading PAEs.

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