Method for improving stability of dehalogenase through biomineralization technology
Through biomineralization technology, mineralized peptides are co-expressed with dehalogenase and mineralized in vitro in situ, which solves the problem of insufficient stability of dehalogenase and significantly improves its thermal stability and environmental tolerance.
Patent Information
- Application Number
- CN202411947897.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-04-25
AI Technical Summary
The existing dehalogenases have problems such as insufficient stability, poor environmental tolerance and toxic by-products produced during catalytic degradation of Class H poisons.
Through biomineralization technology, a recombinant dehalogenase strain was constructed, and the mineralized peptides were co-expressed with dehalogenase through genetic engineering, and the biomineralization of dehalogenase was performed by in vitro in situ mineralization.
It significantly improves the thermal stability of dehalogenase, enhances its stability to the external environment, and reduces the generation of toxic by-products.
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Figure CN119876114A_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 a biomineralization technology. Background Art
[0002] In order to meet the development needs of green disinfection, biocatalytic degradation of H-type agents has gradually become an important research direction in the field of military disinfection. Lyophilized powder of alkyl halide dehalogenase (HLD, EC 3.8.1.5) can be used as an enzyme source for decontamination of H-type agents. Dehalogenase has the function of catalyzing hydrolysis and detoxification of mustard gas (HD), and is currently one of the best dehalogenases for catalytic degradation of HD. Although there are various enzyme-based disinfectants used for decontamination of chemical agents at home and abroad, there are common problems that the same disinfectant cannot take into account multiple substrates, has low decontamination efficiency, poor environmental tolerance, and produces toxic byproducts during the catalytic process. For this reason, the military at home and abroad has begun to conduct in-depth research on dehalogenase genetic engineering, hoping to improve the stability of dehalogenase from the perspective of genetic modification.
[0003] Biomineralization is a common phenomenon in nature, which can link organic matter such as proteins with inorganic matter. Studies have shown that peptides composed of certain amino acid sequences in some mineralizing proteins are the main players in guiding mineralization. Therefore, mineralizing peptides can be fused with target proteins through genetic engineering to give them good biomineralization ability, thereby improving the stability of dehalogenases. Summary of the invention
[0004] 1. Technical issues to be resolved
[0005] The invention proposes a method for improving the stability of dehalogenase by biomineralization 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 biomineralization technology, which comprises 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. Dehalogenase Biomineralization
[0013] By adopting the in vitro in situ mineralization method, CaCl2 solution and phosphate buffer solution are added to the purified recombinant dehalogenase obtained in step S2 to carry out dehalogenase biomineralization.
[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] Furthermore, in step S3, an in vitro in situ mineralization method is used to add CaCl2 solution and phosphate buffer to 0.2 mg of recombinant dehalogenase to carry out dehalogenase biomineralization.
[0019] Furthermore, in step S3, the mineralization time is 4 hours.
[0020] Furthermore, in step S3, the calcium ion concentration in the CaCl2 solution is 10 mM.
[0021] Furthermore, in step S3, the protein concentration is 0.1 mg / ml.
[0022] (III) Beneficial effects
[0023] The present invention proposes a method for improving the stability of dehalogenase by biomineralization technology, including constructing a strain expressing a recombinant dehalogenase, purifying the recombinant dehalogenase, and biomineralizing the dehalogenase. The present invention genetically modifies the dehalogenase through genetic engineering, co-expresses it with a polypeptide that can induce mineralization, obtains a recombinant dehalogenase with a mineralizing peptide, and can form a mineralized precipitate under mild conditions. The present invention utilizes an immobilization strategy to significantly improve the thermal stability of the dehalogenase. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 SDS-PAGE analysis of the expression product of recombinant bacteria DhaA-W6 (lane 1: protein marker, lane 2: before DhaA-W6 induction, lane 3: after DhaA-W6 induction);
[0025] Figure 2 Purification of DhaA-W6 by nickel column affinity chromatography;
[0026] Figure 3 The results of anion exchange chromatography purification of recombinant dehalogenase were analyzed by SDS-PAGE;
[0027] Figure 4 The effect of mineralization time on the mineralization rate of dehalogenase;
[0028] Figure 5 is the effect of calcium ion concentration on the mineralization rate of dehalogenase;
[0029] Figure 6 is the effect of protein concentration on the mineralization rate of dehalogenase;
[0030] Figure 7 The thermal stability of mineralizing dehalogenase (heat treatment at 50℃). DETAILED DESCRIPTION
[0031] 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.
[0032] This example proposes a method for improving the stability of dehalogenase by biomineralization technology, which specifically comprises the following steps:
[0033] S1. Construction of strain expressing recombinant dehalogenase
[0034] The recombinant plasmid pET28a(+)-DhaA-W6 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-W6; 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 = 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.
[0035] 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 DhaA-W6 was successfully constructed and induced to express.
[0036] S2. Purification of recombinant dehalogenase
[0037] A single colony of BL21(DE3) / pET28a(+)-DhaA-W6 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 crushing, and the crushed 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 crushed supernatant is purified by nickel column affinity chromatography to obtain a purified recombinant dehalogenase.
[0038] 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.
[0039] Purification chromatogram Figure 2 As shown, it can be seen that a single elution peak is obtained when the sample is eluted. The elution peak F1 in Figure a is collected separately, concentrated and then subjected to SDS-PAGE electrophoresis to analyze the molecular weight and protein purity of the sample.
[0040] The results of SDS-PAGE electrophoresis are as follows Figure 3 As shown in the figure, it can be seen that the molecular weight of the elution peak F1 is consistent with the molecular weight of the original dehalogenase DhaA, that is, the elution peak F1 is the target protein of the recombinant dehalogenase DhaA-W6. The target protein was purified in large quantities, the buffer was replaced in 20mM phosphate buffer (pH 7.4), and stored at -20°C for use.
[0041] S3. Dehalogenase Biomineralization
[0042] Using the in vitro in situ mineralization method, a certain concentration of CaCl2 solution and phosphate buffer were added to 0.2 mg of the purified recombinant dehalogenase obtained in step S2, and the effects of mineralization time, calcium ion concentration and protein concentration on the mineralization rate were investigated.
[0043] 0.2 mg of DhaA and DhaA-W6 were added to 5 mM calcium chloride solution and 3 mM sodium dihydrogen phosphate buffer, pH 7.4, at 4°C, centrifuged at 1, 2, 4, 6 and 8 hours, washed three times, and the protein concentration of the supernatant was measured to calculate the mineralization rate. Figure 4 It can be seen that with the change of time, the mineralization rate of the original dehalogenase DhaA has been lower than that of the recombinant dehalogenase; in the first two hours, the mineralization speed is fast, and in the fourth hour, the mineralization rate of DhaA-W6 reaches more than 95%, and the subsequent mineralization tends to be saturated. Therefore, the mineralization time is selected to be 4 hours.
[0044] The calculation formula of mineralization rate is:
[0045]
[0046] Where c0 is the original concentration of the protein, V0 is the original volume of the protein, c solution is the concentration of supernatant in each wash after mineralization, V solution is the total volume of the washing solution.
[0047] Effect of calcium ion concentration on the mineralization rate of recombinant dehalogenase:
[0048] 0.2 mg of DhaA-W6 was added to 1.25, 2.5, 5, 10, and 20 mM calcium chloride solution and 6 mM sodium dihydrogen phosphate buffer, pH 7.4, respectively, and placed at 4°C for 4 hours. After centrifugation, the supernatant protein concentration was measured three times, and the mineralization rate was calculated according to the above method. The results are shown in Figure 5 As shown in the figure, it can be seen that the mineralization rate of the recombinant dehalogenase increases with the increase of calcium ion concentration, and basically reaches a saturated state at a concentration of 10mM. Therefore, the calcium ion concentration is selected to be 10mM.
[0049] Effect of protein concentration on the mineralization rate of recombinant dehalogenase:
[0050] DhaA-W6 was added to 10 mM calcium chloride solution and 6 mM sodium dihydrogen phosphate buffer, pH 7.4, respectively, and placed at 4°C for 4 hours, then centrifuged and washed three times. The protein concentration of the supernatant was measured, and the mineralization rate was calculated according to the above method. Figure 6 As shown in the figure, it can be seen that the mineralization rate of the recombinant dehalogenase decreases with the increase of protein concentration, indicating that the lower the protein concentration, the easier it is to mineralize; however, too low a concentration will seriously affect the yield, so a protein concentration of 0.1 mg / ml was selected.
[0051] Thermal stability of recombinant dehalogenases for biomineralization:
[0052] Heat DhaA-W6 in a 50°C water bath and take appropriate samples at 1, 2, 3, 4, 5, and 6 hours to measure enzyme activity. Figure 7 It can be seen that as time goes by, the enzyme activity of Cap-DhaA-W6 decreases less after mineralization than before mineralization. This shows that the recombinant dehalogenase has a significant advantage in thermal stability.
[0053] 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 biomineralization 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. Dehalogenase Biomineralization By adopting the in vitro in situ mineralization method, CaCl2 solution and phosphate buffer solution are added to the purified recombinant dehalogenase obtained in step S2 to carry out dehalogenase biomineralization.
2. The method for improving the stability of dehalogenase by biomineralization 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 biomineralization 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 biomineralization technology according to 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 biomineralization 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 biomineralization technology according to claim 1, characterized in that: In step S3, an in vitro in situ mineralization method is used to add CaCl2 solution and phosphate buffer to 0.2 mg of recombinant dehalogenase to perform dehalogenase biomineralization.
7. The method for improving the stability of dehalogenase by biomineralization technology according to claim 1, characterized in that: In step S3, the mineralization time is 4 hours.
8. The method for improving the stability of dehalogenase by biomineralization technology according to claim 1, characterized in that: In step S3, the calcium ion concentration in the CaCl2 solution is 10 mM.
9. The method for improving the stability of dehalogenase by biomineralization technology according to claim 1, characterized in that: In step S3, the protein concentration was 0.1 mg / ml.