Method for improving stability of dehalogenase through nanotube and sodium alginate immobilization technology
Through the nanotube and sodium alginate immobilization technology, the stability of dehalogenase is improved, and the problems of insufficient stability of dehalogenase and toxic byproduct generation in the existing technology are solved, and a more efficient and environmentally friendly dehalogenase catalytic effect is achieved.
Patent Information
- Application Number
- CN202411946914.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-03-21
AI Technical Summary
The existing dehalogenase technology has the problem of inability to take into account multiple substrates, low cleaning efficiency, poor environmental tolerance, and toxic by-products will be produced during the catalysis process.
Through nanotube and sodium alginate immobilization technology, recombinant dehalogenase strains were constructed, dehalogenase was purified, and immobilized by covalent cross-linking of carbon nanotubes and sodium alginate to improve the stability of dehalogenase.
It significantly improves the thermal stability and pH stability of dehalogenase, enhances its stability to the external environment, and reduces the generation of toxic by-products.
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Figure CN119662628A_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 by using nanotube and sodium alginate immobilization technology. Background Art
[0002] Green disinfection has gradually become an important research direction in the field of military disinfection, and is also the main research focus of green biological disinfection materials. Lyophilized powder of alkyl halide dehalogenase (HLD, EC 3.8.1.5) can be used as an enzyme source for decontamination of H-type toxic agents. Dehalogenase is currently one of the best biological enzymes for catalytic degradation of mustard gas (HD). Although there are various enzyme-based disinfectants used for decontamination of chemical agents at home and abroad, there are common problems such as the inability of the same disinfectant to take into account multiple substrates, low decontamination efficiency, poor environmental tolerance, and the generation of toxic byproducts during the catalytic process.
[0003] Compared with free enzymes, immobilized dehalogenases have a wider application space due to their good stability, high reuse rate, and easy separation from the reaction system. Carbon nanotubes are commonly used carriers for immobilized dehalogenases. They have great potential in biomedicine and catalytic carrier applications due to their large specific surface area, ordered nanopore structure, good thermal properties, outstanding chemical stability and biocompatibility. Sodium alginate is also a commonly used natural polymer carrier for immobilized dehalogenases. The G block of its molecular chain easily binds to divalent metal ions (such as Ca 2+ , Ba 2+ ) cross-linked into a three-dimensional mesh structure gel, forming an "egg box" model. When the enzyme interacts with the sodium alginate gel, the enzyme molecules are fixed inside the gel, thereby improving the stability and reusability of the dehalogenase. Summary of the invention
[0004] 1. Technical issues to be resolved
[0005] The invention proposes a method for improving the stability of dehalogenase by using nanotube and sodium alginate immobilization technology, so as to solve the technical problem of improving the stability of dehalogenase to the external environment.
[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 nanotube and sodium alginate 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. Immobilization of dehalogenase
[0013] Add carbon nanotubes to water and perform ultrasound to obtain a suspension of carbon nanotubes; take the suspension and the purified recombinant dehalogenase solution obtained in step S2, shake and mix at room temperature to obtain carbon nanotube-immobilized dehalogenase; prepare a sodium alginate solution and dialyze it in water overnight; take the dialyzed sodium alginate solution and mix it with the purified recombinant dehalogenase solution obtained in step S2 to obtain sodium alginate-immobilized dehalogenase, mix it with the carbon nanotube-immobilized dehalogenase, and then add it dropwise into a calcium nitrate solution to form small balls to obtain carbon nanotube- and sodium alginate-immobilized dehalogenase.
[0014] 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.
[0015] 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.
[0016] Further, IPTG was added to a final concentration of 1 mM and the temperature for overnight induction was 18°C.
[0017] Furthermore, in step S2, nickel column affinity chromatography is used to purify the target protein in the supernatant.
[0018] Further, in step S3, 10 mg of carbon nanotubes are added to 40 mL of water and ultrasonicated to obtain a suspension of carbon nanotubes; 250 μL of the suspension and 150 μL of the purified recombinant dehalogenase solution obtained in step S2 are taken, and the mixture is shaken and mixed at room temperature for 3 hours to obtain a carbon nanotube-immobilized dehalogenase; a 4% sodium alginate solution is prepared and dialyzed in water overnight; 1 mL of the dialyzed sodium alginate solution is taken and mixed with 300 μL of the purified recombinant dehalogenase solution obtained in step S2 to obtain a sodium alginate-immobilized dehalogenase, which is mixed with the carbon nanotube-immobilized dehalogenase and then added dropwise to 5 mL of calcium nitrate solution to form small balls, which are placed at 4° C. for 2 hours to obtain a dehalogenase immobilized by carbon nanotubes and sodium alginate.
[0019] Furthermore, in step S3, ultrasonication is performed for 1 hour at 30% power, for 1 second, and then for 5 seconds.
[0020] Furthermore, in step S3, the concentration of the calcium nitrate solution is 2%.
[0021] (III) Beneficial effects
[0022] The present invention proposes a method for improving the stability of dehalogenase by using nanotube and sodium alginate immobilization technology, including constructing a strain expressing recombinant dehalogenase, purifying the recombinant dehalogenase, and covalently cross-linking and immobilizing the dehalogenase. The present invention uses carbon nanotubes and sodium alginate to improve the thermal stability of dehalogenase to the external environment and the stability under different pH conditions. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 SDS-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);
[0024] Figure 2 The results of anion exchange chromatography purification of dehalogenase were analyzed by SDS-PAGE;
[0025] Figure 3 is the thermal stability of the immobilized dehalogenase (heat treatment at 50°C);
[0026] Figure 4 pH stability of immobilized dehalogenase. DETAILED DESCRIPTION
[0027] 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.
[0028] This example proposes a method for improving the stability of dehalogenase by using nanotube and sodium alginate immobilization technology, which specifically comprises the following steps:
[0029] S1. Construction of strain expressing recombinant dehalogenase
[0030] 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.
[0031] 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.
[0032] S2. Purification of recombinant dehalogenase
[0033] 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.
[0034] 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.
[0035] 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.
[0036] S3. Immobilization of dehalogenase
[0037] Take 10 mg of carbon nanotubes (CNTs) and add them to 40 mL of water. Ultrasonicate for 1 h at 30% power, ultrasonicate for 1 s, and pause for 5 s to obtain a suspension of CNTs; take 250 μL of the suspension and 150 μL of the purified recombinant dehalogenase solution (1.689 mg / mL) obtained in step S2, shake and mix at room temperature for 3 h to obtain carbon nanotube-immobilized dehalogenase (CNTs-DhaA); prepare a 4% sodium alginate solution and dialyze it in water overnight; take 1 mL of the dialyzed sodium alginate solution and mix it with 300 μL of the purified recombinant dehalogenase solution obtained in step S2 to obtain sodium alginate-immobilized dehalogenase (SA-DhaA), mix it with CNTs-DhaA, and then add it drop by drop into 5 mL The pellets were formed in a 2% calcium nitrate solution and placed at 4°C for 2h to obtain carbon nanotube and sodium alginate immobilized dehalogenase (CNTs-SA-DhaA); the supernatant was taken to measure the protein concentration and the protein content in the pellets was calculated; the supernatant was filtered out, the water on the surface of the pellets was dried, the mass of the pellets was measured, and the protein concentration in the pellets was calculated.
[0038] The relative activity of the dehalogenase immobilized on carbon nanotubes and sodium alginate was tested. The results are shown in Table 1. When the dehalogenase was immobilized on carbon nanotubes alone, the catalytic activity of the original enzyme was almost completely maintained; however, when sodium alginate or carbon nanotubes and sodium alginate were used simultaneously, the catalytic activity of the original enzyme was greatly reduced.
[0039] Table 1 Relative activity of dehalogenase immobilized on carbon nanotubes-sodium alginate
[0040]
[0041] The thermal stability results of carbon nanotube-alginate immobilized dehalogenase are shown in Figure 3 As shown in the figure, after heating in a 50℃ water bath for 1 hour, the carbon nanotube-immobilized dehalogenase still had about 60% activity, but the other two immobilization methods and the original enzyme all had about 20% activity, indicating that carbon nanotube-immobilized dehalogenase can improve the thermal stability of dehalogenase. However, after 2 hours, CNTs-DhaA still had about 15% activity, while the other samples still had about 5% activity, indicating that the carbon nanotube-immobilized dehalogenase method is superior to the other two methods in terms of thermal stability.
[0042] The pH stability results of dehalogenase immobilized on carbon nanotubes-sodium alginate are as follows Figure 4 As shown in the figure, as the pH value decreases, the catalytic activity of all samples decreases; but between pH values of 3.0-5.0, the decrease in the activity of SA-DhaA and CNTs-SA-DhaA is significantly smaller than that of CNTs-DhaA and DhaA, indicating that sodium alginate may partially inhibit the effect of low pH on the activity of dehalogenase. The above analysis shows that carbon nanotubes can improve the thermal stability of dehalogenase in the short term. Sodium alginate can partially inhibit the effect of low pH on the activity of dehalogenase.
[0043] 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 nanotube and sodium alginate 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. Immobilization of dehalogenase Add carbon nanotubes to water and perform ultrasound to obtain a suspension of carbon nanotubes; take the suspension and the purified recombinant dehalogenase solution obtained in step S2, shake and mix at room temperature to obtain carbon nanotube-immobilized dehalogenase; prepare a sodium alginate solution and dialyze it in water overnight; take the dialyzed sodium alginate solution and mix it with the purified recombinant dehalogenase solution obtained in step S2 to obtain sodium alginate-immobilized dehalogenase, mix it with the carbon nanotube-immobilized dehalogenase, and then add it dropwise into a calcium nitrate solution to form small balls to obtain carbon nanotube- and sodium alginate-immobilized dehalogenase.
2. The method for improving the stability of dehalogenase by nanotube and sodium alginate 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 nanotube and sodium alginate 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 nanotube and sodium alginate immobilization technology as claimed in claim 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 nanotube and sodium alginate immobilization technology according to claim 1, 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 nanotube and sodium alginate immobilization technology according to claim 1, characterized in that: In step S3, 10 mg of carbon nanotubes are added to 40 mL of water and ultrasonicated to obtain a suspension of carbon nanotubes; 250 μL of the suspension and 150 μL of the purified recombinant dehalogenase solution obtained in step S2 are taken, and the mixture is shaken and mixed at room temperature for 3 hours to obtain a carbon nanotube-immobilized dehalogenase; a 4% sodium alginate solution is prepared and dialyzed in water overnight; 1 mL of the dialyzed sodium alginate solution is taken and mixed with 300 μL of the purified recombinant dehalogenase solution obtained in step S2 to obtain a sodium alginate-immobilized dehalogenase, which is mixed with the carbon nanotube-immobilized dehalogenase and then added dropwise to 5 mL of calcium nitrate solution to form small balls, which are placed at 4° C. for 2 hours to obtain a dehalogenase immobilized by carbon nanotubes and sodium alginate.
7. The method for improving the stability of dehalogenase by nanotube and sodium alginate immobilization technology according to claim 1, characterized in that: In step S3, ultrasonication is performed for 1 h at 30% power, for 1 s, and then for 5 s.
8. The method for improving the stability of dehalogenase by nanotube and sodium alginate immobilization technology according to claim 1, characterized in that: In step S3, the concentration of the calcium nitrate solution is 2%.