Method for improving stability of dehalogenase through synanthrin covalent cross-linking immobilization technology
Through inulin covalent cross-linking immobilization technology, the problem of low thermal stability of dehalogenase is solved, significantly improving its thermal, pH and organic solvent stability, and extending the service life of the enzyme.
Patent Information
- Application Number
- CN202411947707.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-03-18
AI Technical Summary
The thermal stability of dehalogenase is low and it is difficult to maintain activity in high temperature environments.
Through inulin covalent cross-linking immobilization technology, inulin is covalently cross-linked with dehalogenase to form an immobilized enzyme and improve its thermal stability.
It significantly improves the thermal stability, pH stability and organic solvent stability of dehalogenase, and extends the service life of the enzyme.
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Figure CN119639727A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of improving the stability of dehalogenase, and particularly relates to a method for improving the stability of dehalogenase through inulin covalent cross-linking immobilization technology. Background Art
[0002] In order to meet the development needs of green disinfection, the degradation of various typical chemical agents by biocatalysis is considered to be "one of the most ideal disinfectants". Lyophilized powder of alkyl halide dehalogenase (haloalkane dehalogenases, HLD, EC 3.8.1.5) can be used as an enzyme source for the disinfection of H-type agents. The dehalogenase (DhaA) in this type of enzyme has the effect of catalyzing the hydrolysis and detoxification of mustard gas (HD), and is currently one of the best biological enzymes for catalytic degradation of HD. Due to its unique structure, biological enzymes are easily inactivated by factors such as temperature, ionic strength, solvents, pH value and even humidity. Therefore, in order to make biological enzymes be actually applied in battlefield environments, it is necessary to improve the environmental tolerance of biological enzymes.
[0003] Immobilized enzymes have a wider application space compared to free enzymes due to their good stability, high reuse rate, and easy separation from the reaction system. Inulin (Inu) is a natural fructose polymer with low price and comes from plant tubers. Inulin molecules contain multiple hydroxyl groups. After covalent cross-linking with enzymes, water molecules bound to hydroxyl groups can form a hydration layer around the enzyme molecules, thereby improving the structural stability of the enzyme and its tolerance to adverse environments. Summary of the invention
[0004] 1. Technical issues to be resolved
[0005] The invention provides a method for improving the stability of dehalogenase by using inulin covalent cross-linking immobilization technology, so as to solve the technical problem of low thermal stability of dehalogenase.
[0006] (II) Technical solution
[0007] In order to solve the above technical problems, the present invention proposes a method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology, the method comprising the following steps:
[0008] S1. Construction of strain expressing recombinant dehalogenase
[0009] The recombinant plasmid of the dehalogenase is transformed into competent cells and cultured overnight to obtain a recombinant strain; a single colony is picked in a culture medium and cultured overnight; the colony is transferred to a culture medium and cultured until OD600=0.6-0.8; IPTG is added for induction overnight; the bacterial cells are collected by centrifugation to obtain a recombinant dehalogenase;
[0010] S2. Purification of recombinant dehalogenase
[0011] The recombinant strain single colony is cultured in a culture medium overnight; the colony is transferred to a culture medium and cultured until OD600 = 0.6-0.8; IPTG is added for induction overnight, the bacteria are collected, the bacteria are lysed by ultrasonic disruption, and the supernatant is collected by high-speed centrifugation; the target protein in the supernatant is purified to obtain a purified recombinant dehalogenase;
[0012] S3. Covalent cross-linking and immobilization of dehalogenase
[0013] Inulin is dissolved in an acetic acid-sodium acetate buffer solution, and after being fully dissolved, a sodium periodate solution is added, and the reaction is carried out at room temperature in the dark; the sodium periodate oxidizes the ortho-hydroxyl group of the inulin to an aldehyde group, and then a small amount of ethylene glycol is added to terminate the oxidation reaction; the reaction solution is dialyzed to remove excess sodium periodate and ethylene glycol; then, the buffer solution is replaced with a phosphate buffer solution; the purified recombinant dehalogenase obtained in step S2 and sodium cyanoborohydride are added, the mixture is fully mixed, and the reaction is carried out overnight; the aldehyde group of the inulin reacts with the amino group of the dehalogenase under the reduction of the sodium cyanoborohydride to obtain an inulin-modified dehalogenase;
[0014] The inulin-modified dehalogenase reaction mixture is dialyzed to remove excess sodium cyanoborohydride; the reaction mixture is separated and purified; the reaction mixture containing the inulin-modified dehalogenase is eluted, the main elution peak is collected, and freeze-dried with a freeze dryer to complete the covalent cross-linking immobilization of the dehalogenase.
[0015] Further, in step S1, the recombinant plasmid of the dehalogenase is transformed into competent cells, and after coating, the plate is inverted and placed in a 37°C oven for overnight culture to obtain a recombinant strain; a single colony is picked in a liquid LB medium, and cultured overnight at 150 rpm in a 37°C shaker; the next day, it is transferred to a liquid LB medium at a ratio of 1% (v / v), and cultured at 200 rpm in a 37°C shaker until OD600 = 0.6-0.8; IPTG with a final concentration of 1 mM is added for induction overnight; the bacteria are collected by centrifugation to obtain a recombinant dehalogenase.
[0016] Further, in step S2, a single colony of the recombinant strain is placed in a liquid LB medium and cultured overnight at 150 rpm in a shaker at 37°C; the next day, it is transferred to a liquid LB medium at a ratio of 1% (v / v), and cultured at 200 rpm in a shaker at 37°C until OD600 = 0.6-0.8; IPTG with a final concentration of 1 mM is added for induction overnight, the bacteria are collected, the bacteria are lysed by ultrasonic disruption, and the supernatant is collected by high-speed centrifugation; the target protein in the supernatant is purified to obtain a purified recombinant dehalogenase.
[0017] Further, IPTG was added to a final concentration of 1 mM and the temperature for overnight induction was 18°C.
[0018] Furthermore, in step S2, nickel column affinity chromatography is used to purify the target protein in the supernatant.
[0019] Further, in step S3, 200 mg of inulin is weighed and dissolved in 20 mM acetic acid-sodium acetate buffer. After sufficient dissolution, 10 mM sodium periodate solution is added and reacted at room temperature in the dark for 20 minutes; the ortho-hydroxyl group of inulin is oxidized to an aldehyde group by sodium periodate, and then a small amount of ethylene glycol is added to terminate the oxidation reaction; the reaction solution is dialyzed with a dialysis bag with a molecular weight cutoff of 2 kDa to remove excess sodium periodate and ethylene glycol; then, the buffer is replaced with 20 mM phosphate buffer; 25 mg of the purified recombinant dehalogenase obtained in step S2 and 12.5 mg of sodium cyanoborohydride are added, mixed thoroughly, and reacted at 4°C overnight; the aldehyde group of inulin reacts with the amino group of the dehalogenase under the reduction of sodium cyanoborohydride to obtain an inulin-modified dehalogenase.
[0020] Furthermore, in step S3, after adding 10 mM sodium periodate solution, the final concentrations of inulin and sodium periodate are 2.5 mg / ml and 5 mM, respectively.
[0021] Further, in step S3, the inulin-modified dehalogenase reaction mixture is fully dialyzed using a dialysis bag with a molecular weight cutoff of 2 kDa to remove excess sodium cyanoborohydride; the above reaction solution is separated and purified; the reaction mixture containing the inulin-modified dehalogenase is eluted and the main elution peak is collected, and freeze-dried using a freeze dryer to complete the covalent cross-linking immobilization of the dehalogenase.
[0022] Furthermore, in step S3, the reaction solution is separated and purified using a Superdex 200 gel filtration column.
[0023] Furthermore, in step S3, the equilibrium buffer used for separation and purification is 20 mM phosphate buffer, the flow rate is 2 mL / min, and the detection wavelength is 280 nm.
[0024] (III) Beneficial effects
[0025] The present invention provides a method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology, including constructing a strain expressing recombinant dehalogenase, purifying the recombinant dehalogenase, and covalent cross-linking immobilization of the dehalogenase. The present invention adopts a chemical modification method to covalently cross-link inulin with alkyl dehalogenase to prepare immobilized alkyl dehalogenase, which can significantly improve the thermal stability of the dehalogenase, the stability under different pH conditions and in organic solvents. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1SDS-PAGE analysis of the DhaA expression product of the recombinant bacteria (lane 1: protein marker, lane 2: before DhaA induction, lane 3: after DhaA induction);
[0027] Figure 2 The results of anion exchange chromatography purification of dehalogenase were analyzed by SDS-PAGE;
[0028] Figure 3 The preparation principle of inulin covalently modified DhaA;
[0029] Figure 4 Purify Inu-DhaA by gel filtration column;
[0030] Figure 5 To identify the purified Inu-DhaA by SDS-PAGE electrophoresis;
[0031] Figure 6 is the relative activity of inulin-modified DhaA;
[0032] Figure 7 The 56°C thermal stability of Inu-DhaA;
[0033] Figure 8 Circular dichroism spectra of the sample before and after heating;
[0034] Fig. 9 The intrinsic fluorescence spectra of the samples before and after heating;
[0035] Fig.10 Long-term thermal stability of Inu-DhaA;
[0036] Fig.11 is the pH stability of Inu-DhaA;
[0037] Fig.12 is the circular dichroism spectra before and after treatment at pH 4.0;
[0038] Fig.13 is the intrinsic fluorescence spectrum before and after treatment at pH 4.0;
[0039] Fig.14 is the organic solvent stability of Inu-DhaA;
[0040] Fig.15 This is the intrinsic fluorescence spectrum after treatment with organic solvents. DETAILED DESCRIPTION
[0041] In order to make the purpose, content and advantages of the present invention more clear, the specific implementation methods of the present invention are further described in detail below in conjunction with the drawings and examples.
[0042] This example proposes a method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology, which specifically includes the following steps:
[0043] S1. Construction of strain expressing recombinant dehalogenase
[0044] The recombinant plasmid pET28a(+)-DhaA of dehalogenase (DhaA) was transformed into BL21(DE3) competent cells, and after plating, the cells were inverted and cultured in a 37°C oven overnight to obtain the recombinant strain BL21(DE3) / pET28a(+)-DhaA; single colonies were picked in a clean workbench and placed in 5 mL of liquid LB culture medium, and cultured in a 37°C shaker at a speed of 150 rpm overnight; the next day, the colonies were transferred to 100 mL of liquid LB culture medium at a ratio of 1% (v / v), and cultured in a 37°C shaker at a speed of 200 rpm until OD600 was 0.6-0.8; IPTG with a final concentration of 1 mM was added for low temperature (18°C) induction overnight; and the bacterial cells were collected by centrifugation to obtain the recombinant dehalogenase.
[0045] The protein bands of the bacteria before and after induction were identified by SDS-PAGE. Figure 1 As shown in the figure, it can be seen that after IPTG induction, a new protein band appeared in lane 3 at a molecular weight of about 30 kDa compared with lane 2, indicating that the recombinant dehalogenase was successfully constructed and induced to express.
[0046] S2. Purification of recombinant dehalogenase
[0047] A single colony of BL21(DE3) / pET28a(+)-DhaA was placed in 5 mL of liquid LB culture medium, and cultured overnight at 150 rpm in a 37°C shaker; the next day, the colony was transferred to 100 mL of liquid LB culture medium at a ratio of 1% (v / v), and cultured at 200 rpm in a 37°C shaker until OD600=0.6-0.8; IPTG with a final concentration of 1 mM was added for induction overnight at low temperature (18°C), the bacteria were collected, the bacteria were lysed by ultrasonic disruption, and the supernatant was collected by high-speed centrifugation. Since the vector carries a His tag when constructing the recombinant plasmid, it can bind to nickel ions by affinity, so the target protein in the supernatant is purified by nickel column affinity chromatography to obtain a purified recombinant dehalogenase.
[0048] The equilibration buffer used for purification was 20 mM Tris-HCl buffer (bufferA, pH 8.0) containing 20 mM imidazole and 0.5 M NaCl, and the elution buffer was 20 mM Tris-HCl buffer (bufferB, pH 8.0) containing 0.5 M imidazole and 0.5 M NaCl.
[0049] The results of SDS-PAGE electrophoresis are as follows Figure 2 The purified target protein was replaced with a 20 mM phosphate buffer (pH 7.4) and stored at -20°C for later use.
[0050] S3. Covalent cross-linking and immobilization of dehalogenase
[0051] Weigh 200 mg of inulin and dissolve it in 20 mM acetic acid-sodium acetate buffer (pH 5.8). After it is fully dissolved, add 10 mM sodium periodate solution. The final concentrations of inulin and sodium periodate are 2.5 mg / ml and 5 mM, respectively. Incubate at room temperature in the dark for 20 minutes. Figure 3 As shown, sodium periodate oxidizes the ortho-hydroxyl group of inulin to an aldehyde group, and then a small amount of ethylene glycol is added to terminate the oxidation reaction. The reaction solution is fully dialyzed with a dialysis bag with a molecular weight cutoff of 2 kDa to remove excess sodium periodate and ethylene glycol. Subsequently, the buffer is replaced with 20 mM phosphate buffer (pH 7.2). 25 mg of the purified recombinant dehalogenase obtained in step S2 and 12.5 mg of sodium cyanoborohydride (NaCNBH 3 ), mixed thoroughly, and reacted at 4°C overnight. The aldehyde group of inulin reacted with the amino group of DhaA under the reduction of sodium cyanoborohydride to obtain inulin-modified dehalogenase (Inu-DhaA).
[0052] The inulin-modified dehalogenase reaction mixture was fully dialyzed using a dialysis bag with a molecular weight cutoff of 2 kDa to remove excess sodium cyanoborohydride. The dialyzate was 20 mM phosphate buffer (pH 7.2). The reaction solution was separated and purified using a Superdex 200 gel filtration column (2.6 cm 60 cm). The equilibrium buffer used for purification was 20 mM phosphate buffer (pH 7.2), the flow rate was 2 mL / min, and the detection wavelength was 280 nm. Figure 4 As shown, the reaction mixture containing Inu-DhaA showed two well-separated elution peaks after elution, of which the main elution peak corresponded to Inu-DhaA. The elution peak was collected, concentrated and placed in a -20°C refrigerator. In addition, the elution peak position of unmodified DhaA was 93.2 mL, while the elution peak position of Inu-DhaA was 88.5 mL. Compared with unmodified DhaA, the elution peak of Inu-DhaA drifted to the left, indicating that inulin covalent modification can significantly increase the hydration volume of DhaA.
[0053] The elution peak corresponding to Inu-DhaA was collected and identified by SDS-PAGE electrophoresis. Figure 5As shown, DhaA presents one main electrophoresis band (lane 1), corresponding to a molecular weight of about 35 kDa. After inulin covalently binds to DhaA, the electrophoresis band corresponding to DhaA almost completely disappears, and a fuzzy and diffuse electrophoresis band appears above the band (lane 2). After purification by Superdex 200 gel filtration column, the dispersion of these bands becomes narrower, and the bands corresponding to DhaA and the bands with a molecular weight greater than 90 kDa completely disappear. This indicates that after separation and purification, a small amount of unmodified DhaA and Inu-DhaA with a high degree of binding are removed, further improving the purity of Inu-DhaA. The elution peak collected above is concentrated and freeze-dried with a freeze dryer to complete the covalent cross-linking immobilization of the dehalogenase.
[0054] The catalytic activity of covalently cross-linked immobilized dehalogenase was shown in Figure 6 As shown, compared with the unmodified alkyl dehalogenase (set as 100%), the relative enzyme activity of the purified Inu-DhaA still remains at about 88.0±4.1%. This shows that after inulin covalently cross-links DhaA, it may affect its activity by affecting the structure of DhaA. However, inulin covalent modification can maintain the biological activity of DhaA to a large extent, which indicates that inulin covalent modification has little effect on the active center of DhaA.
[0055] Thermal stability test of covalently cross-linked immobilized dehalogenase:
[0056] Take appropriate amount of liquid DhaA and Inu-DhaA lyophilized powder and heat them in a 56°C water bath. After 1, 2, 4, 6 and 8 hours of water bath heating, take out some samples and measure the biological activity of the heat-treated samples. Set the biological activity of each sample before heating treatment as 100%, and calculate the relative biological activity of the samples according to the following formula:
[0057]
[0058] The results are as follows Figure 7 As shown. The biological activity of unimmobilized DhaA decreased rapidly after heating at 56°C, with 35.47% and 24.38% of activity remaining in the first and second hours respectively, and its biological activity was almost completely lost after the fourth hour; while Inu-DhaA still had 83.34% of activity after 1 hour of heating treatment, and 50.81%-60.07% of activity in the fourth to eighth hours, which was much higher than the activity of DhaA. This shows that the thermal stability of DhaA is greatly improved after inulin immobilization.
[0059] The changes in the secondary and tertiary structures of DhaA before and after immobilization and heat treatment were analyzed using a circular dichroism spectrometer and a fluorescence spectrophotometer. Figure 8As shown. After DhaA was cross-linked and immobilized with inulin, the percentage of α-helix and β-fold of DhaA decreased. After heating, the α-helix and β-fold content of DhaA and Inu-DhaA decreased, but the decrease of DhaA was greater. This shows that DhaA immobilized with inulin cross-linking can resist the damage of DhaA secondary structure caused by heating.
[0060] like Fig. 9 As shown in the figure, from the results of endogenous fluorescence spectrum analysis, it can be seen that the maximum absorption wavelengths of the fluorescence spectra of DhaA and Inu-DhaA are both at 333nm, but the fluorescence spectrum intensity of Inu-DhaA is slightly lower than that of DhaA. This shows that inulin modification has a slight effect on the tertiary structure of DhaA. After the sample was heated, the maximum absorption wavelengths of the fluorescence spectra of DhaA and Inu-DhaA were red-shifted. Among them, the maximum absorption wavelength of the fluorescence spectrum of DhaA was red-shifted from 333nm to 339nm, while the maximum absorption wavelength of the fluorescence spectrum of Inu-DhaA was red-shifted from 333nm to 336nm, and the degree of red-shift was lower than that of DhaA. This shows that inulin cross-linking and immobilization improves the thermal stability of the tertiary structure of DhaA.
[0061] Long-term stability test of covalently cross-linked immobilized dehalogenase under high temperature conditions (37°C and 54°C):
[0062] The Inu-DhaA freeze-dried powder was placed in ovens at 37°C and 54°C, and the biological activity of Inu-DhaA was measured at regular intervals, with unmodified DhaA as a control. Fig.10 As shown in the figure, the enzyme activity of unmodified DhaA dropped sharply after being placed at 37℃ for 1 day, and its enzyme activity was almost completely lost on the 4th day; the enzyme activity of unmodified DhaA was almost completely lost after being placed at 54℃ for 1 day. In contrast, the enzyme activity of Inu-DhaA was 95% after being placed at 37℃ for 1 day, with only a slight decrease; its enzyme activity could still be maintained at about 92.3% on the 4th day, and then the enzyme activity had a slow upward trend, and its enzyme activity was still maintained at about 88% on the 14th day, and then the activity slowly decreased, and it was still 85.6% active on the 90th day. The enzyme activity of Inu-DhaA was 90% after being placed at 54℃ for 1 day, with only a slight decrease; its enzyme activity could still be maintained at about 81% on the 4th day, and then its enzyme activity slowly decreased until it was still about 46% on the 90th day. This shows that inulin covalent cross-linking DhaA can greatly improve the thermal stability of DhaA, and the longer the storage time, the more obvious the advantage of maintaining enzyme activity of this method.
[0063] pH stability test of covalently cross-linked immobilized dehalogenase:
[0064] The stability of Inu-DhaA was tested under different pH conditions (3.0-9.0). Fig.11 As shown in the figure, as the pH value of the solution decreases, the activity retention rate of DhaA decreases sharply. In contrast, in the pH range of 3.0-6.0, the activity retention rate of Inu-DhaA is significantly higher than that of DhaA. Among them, at a pH of 5.0, the activity retention rate of Inu-DhaA is about 70.23%, while the activity retention rate of DhaA is only about 23.77%; between pH 3.0-4.0, the activity of DhaA is almost completely lost, but Inu-DhaA still has an activity retention rate of 20.6%-52.49%. Therefore, inulin covalent cross-linking can greatly improve the pH stability of DhaA.
[0065] The secondary structure changes of DhaA before and after immobilization and before and after treatment at pH 4.0 for 1 hour were analyzed using a circular dichroism spectrometer and an intrinsic fluorescence spectrophotometer. Fig.12 As shown, after treatment at pH 4.0, the negative characteristic peak of the circular dichroism spectrum of DhaA near 216-218nm decreased, while the negative characteristic peak of Inu-DhaA in this wavelength range increased, indicating that after treatment at pH 4.0, the β-fold content of DhaA decreased, while the β-fold content of Inu-DhaA increased, and it is possible that the β-fold content can better increase the stability of DhaA.
[0066] from Fig.13 It can be seen from the endogenous fluorescence spectrum that after treatment at pH 4.0, the maximum absorption peak wavelength of the fluorescence spectrum of DhaA has undergone a significant red shift, and the fluorescence intensity has been greatly reduced. Among them, the maximum absorption wavelength of the fluorescence spectrum of DhaA has red-shifted from 333nm to 338nm. The degree of red shift of Inu-DhaA is much lower than that of DhaA, and the decrease in fluorescence intensity is smaller. Among them, the maximum absorption wavelength of the fluorescence spectrum of Inu-DhaA has red-shifted from 334nm to 336nm. This shows that the pH stability of DhaA can be improved after inulin cross-linking and immobilizing DhaA.
[0067] Organic solvent stability test of covalently cross-linked immobilized dehalogenase:
[0068] The stability of Inu-DhaA was tested in the presence of the organic solvent dimethyl sulfoxide (DMSO). Fig.14 As shown in the figure, after being treated with 40% (v / v) DMSO solution, the activity retention rate of DhaA was only 15.1%. In contrast, the activity retention rate of inu-DhaA was 52.45%, which was significantly higher than that of DhaA. The results showed that after inulin covalently cross-linked DhaA, the hydration layer formed by inulin around the DhaA molecule could resist the attack of DMSO, thereby improving the organic solvent stability of DhaA.
[0069] The tertiary structure changes of DhaA before and after immobilization and after 1 hour of treatment with 40% DMSO were analyzed using an endogenous fluorescence spectrophotometer. Fig.15 As shown in the figure, it can be seen that after treatment with 40% DMSO, the maximum absorption wavelength of the endogenous fluorescence spectrum of DhaA has undergone a significant red shift. Among them, the maximum absorption wavelength of the fluorescence spectrum of DhaA has red-shifted from 333nm to 341nm. The degree of red shift of Inu-DhaA is much lower than that of DhaA, and the decrease in fluorescence intensity is smaller. Among them, the maximum absorption wavelength of the fluorescence spectrum of Inu-DhaA has red-shifted from 333nm to 338nm. This shows that after inulin cross-linking and immobilizing DhaA, it can moderately resist the destruction of the secondary structure of DhaA by organic solvents, thereby improving its stability.
[0070] The present invention prepares Inu-DhaA based on inulin covalent cross-linking immobilization. Compared with DhaA, the activity of Inu-DhaA remains about 88%, indicating that inulin covalent cross-linking immobilization can substantially maintain the biological activity of DhaA; based on inulin covalent cross-linking immobilization, the thermal stability, pH stability and organic solvent stability of DhaA can be greatly improved, and the damage to the secondary structure and tertiary structure of DhaA in environments such as high temperature, low pH and organic solvents can be significantly alleviated.
[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology, characterized in that: The method comprises the following steps: S1. Construction of strain expressing recombinant dehalogenase The recombinant plasmid of the dehalogenase is transformed into competent cells and cultured overnight to obtain a recombinant strain; a single colony is picked in a culture medium and cultured overnight; the colony is transferred to a culture medium and cultured until OD600=0.6-0.8; IPTG is added for induction overnight; the bacterial cells are collected by centrifugation to obtain a recombinant dehalogenase; S2. Purification of recombinant dehalogenase The recombinant strain single colony is cultured in a culture medium overnight; the colony is transferred to a culture medium and cultured until OD600 = 0.6-0.8; IPTG is added for induction overnight, the bacteria are collected, the bacteria are lysed by ultrasonic disruption, and the supernatant is collected by high-speed centrifugation; the target protein in the supernatant is purified to obtain a purified recombinant dehalogenase; S3. Covalent cross-linking and immobilization of dehalogenase Inulin is dissolved in an acetic acid-sodium acetate buffer solution, and after being fully dissolved, a sodium periodate solution is added, and the reaction is carried out at room temperature in the dark; the sodium periodate oxidizes the ortho-hydroxyl group of the inulin to an aldehyde group, and then a small amount of ethylene glycol is added to terminate the oxidation reaction; the reaction solution is dialyzed to remove excess sodium periodate and ethylene glycol; then, the buffer solution is replaced with a phosphate buffer solution; the purified recombinant dehalogenase obtained in step S2 and sodium cyanoborohydride are added, the mixture is fully mixed, and the reaction is carried out overnight; the aldehyde group of the inulin reacts with the amino group of the dehalogenase under the reduction of the sodium cyanoborohydride to obtain an inulin-modified dehalogenase; The inulin-modified dehalogenase reaction mixture is dialyzed to remove excess sodium cyanoborohydride; the reaction mixture is separated and purified; the reaction mixture containing the inulin-modified dehalogenase is eluted, the main elution peak is collected, and freeze-dried with a freeze dryer to complete the covalent cross-linking immobilization of the dehalogenase.
2. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 1, characterized in that: In step S1, the recombinant plasmid of the dehalogenase is transformed into competent cells, and after coating, the plate is inverted and placed in a 37°C oven for overnight culture to obtain a recombinant strain; a single colony is picked in a liquid LB culture medium, and cultured overnight at 150 rpm in a 37°C shaker; the next day, it is transferred to a liquid LB culture medium at a ratio of 1% (v / v), and cultured at 200 rpm in a 37°C shaker until OD600 = 0.6-0.8; IPTG with a final concentration of 1 mM is added for overnight induction; and the bacterial cells are collected by centrifugation to obtain a recombinant dehalogenase.
3. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 1, characterized in that: In step S2, a single colony of the recombinant strain is placed in a liquid LB medium and cultured overnight at 37°C in a shaker at 150 rpm; the next day, the colony is transferred to a liquid LB medium at a ratio of 1% (v / v), and cultured at 37°C in a shaker at 200 rpm until OD600 = 0.6-0.8; IPTG with a final concentration of 1 mM is added for induction overnight, the bacteria are collected, the bacteria are lysed by ultrasonic disruption, and the supernatant is collected by high-speed centrifugation; the target protein in the supernatant is purified to obtain a purified recombinant dehalogenase.
4. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 2 or 3, characterized in that: IPTG was added to a final concentration of 1 mM and the temperature for overnight induction was 18°C.
5. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 3, characterized in that: In step S2, the target protein in the supernatant is purified by nickel column affinity chromatography.
6. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 1, characterized in that: In step S3, 200 mg of inulin is weighed and dissolved in 20 mM acetic acid-sodium acetate buffer. After fully dissolved, 10 mM sodium periodate solution is added, and the mixture is reacted at room temperature in the dark for 20 minutes; the ortho-hydroxyl group of inulin is oxidized to an aldehyde group with sodium periodate, and then a small amount of ethylene glycol is added to terminate the oxidation reaction; the reaction solution is dialyzed with a dialysis bag with a molecular weight cutoff of 2 kDa to remove excess sodium periodate and ethylene glycol; then, the buffer is replaced with 20 mM phosphate buffer; 25 mg of the purified recombinant dehalogenase obtained in step S2 and 12.5 mg of sodium cyanoborohydride are added, the mixture is fully mixed, and the mixture is reacted at 4° C. overnight; the aldehyde group of inulin reacts with the amino group of the dehalogenase under the reduction of sodium cyanoborohydride to obtain an inulin-modified dehalogenase.
7. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 6, characterized in that: In step S3, after adding 10 mM sodium periodate solution, the final concentrations of inulin and sodium periodate are 2.5 mg / ml and 5 mM, respectively.
8. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 1, characterized in that: In step S3, the inulin-modified dehalogenase reaction mixture is fully dialyzed using a dialysis bag with a molecular weight cutoff of 2 kDa to remove excess sodium cyanoborohydride; the above reaction solution is separated and purified; the reaction mixture containing the inulin-modified dehalogenase is eluted and the main elution peak is collected, and freeze-dried using a freeze dryer to complete the covalent cross-linking immobilization of the dehalogenase.
9. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 8, characterized in that: In step S3, the reaction solution is separated and purified using a Superdex 200 gel filtration column.
10. The method for improving the stability of dehalogenase by inulin covalent cross-linking immobilization technology according to claim 9, characterized in that: In step S3, the equilibrium buffer used for separation and purification is 20 mM phosphate buffer, the flow rate is 2 mL / min, and the detection wavelength is 280 nm.