Application of human CYP2C9 recombinant enzyme in denitrification of nitrocellulose

Abstract

The present invention discloses an application of a human CYP2C9 recombinase in the denitration of nitrocellulose. The present invention applies the human CYP2C9 recombinase to the denitration of nitrocellulose, which can convert the nitrate ester group into nitrate and nitrite. After reacting with nitrocellulose for 7 days, the nitrogen content of the nitrocellulose with an original nitrogen content of 12.7% is reduced to 11.585%, and the denitration treatment time is significantly shortened compared with the traditional biological method. The present invention has low production cost, simple process, mild reaction conditions, and is environmentally friendly, and can achieve efficient denitration of nitrocellulose.

Description

Technical Field

[0001] The invention belongs to the field of biological denitrification and relates to the application of human CYP2C9 recombinase in nitrocellulose denitrification. Background Art

[0002] Nitrocellulose is the product of the esterification reaction between cellulose and nitric acid. The nitrate group produced is the characteristic group in the nitrocellulose macromolecular chain. Theoretically, the nitrogen content of nitrocellulose is close to 14.14%, but in reality, the maximum nitrogen content of nitrocellulose is 13.8%. Nitrocellulose is one of the most widely used cellulose esters in modern technology. Low-nitrogen nitrocellulose with a nitrogen content of less than 12% is widely used as an industrial product, including photographic film, ink, leather and various nitro paints, while high-nitrogen nitrocellulose with a nitrogen content of more than 12%, commonly known as nitrocellulose, is the main energy component of single-base, double-base and triple-base propellants, providing propulsion energy for weapons.

[0003] At present, the methods for denitrification of high-nitrogen nitrocellulose mainly include chemical methods and biological methods. The chemical method mainly uses alkaline substances to hydrolyze nitrocellulose. For example, Zhongan Tao uses alkaline denitrification to achieve nitrogen reduction of high-nitrogen nitrocellulose, which usually requires alkaline reagents, organic reagents, etc., and its post-treatment is prone to cause environmental pollution (ZHONGAN TAO, SHIYING LI, YAJUN DING, et al. The Preparation and Properties of Low-Nitrogen Nitrocellulose by Alkaline Denitration [J]. Central European Journal of Energetic Materials, 2020, 17 (4): 535-551.). The biological method mainly uses microorganisms to degrade nitrocellulose, such as the degradation of flaky nitrocellulose by microorganisms in a composting environment, the biodegradability of nitrocellulose by sewage sludge inocula during methane production, and the degradation of dissolved nitrocellulose by isolated cultures in shake flask experiments. Degradation usually requires a complex biological environment, the active strains are difficult to determine, and the reaction time is generally long, requiring at least 20 days or more.

[0004] Cytochrome P450 enzymes (CYP) are a superfamily of integral membrane conserved proteins present in animals, plants, microorganisms and humans. They are involved in many important reactions in drug synthesis, steroid and carcinogen metabolism, and protect organisms from toxic compounds. Human CYP contains at least 57 genes and 58 pseudogenes, which are divided into 18 families and 43 subfamilies, and are mainly present in the kidney, small intestine and liver tissues of the human body. CYP is centered on heme porphyrin iron and can metabolize a variety of xenobiotics. It is associated with a variety of reactions such as epoxidation, hydroxylation, dealkylation, deamination, desulfurization, dehalogenation, sulfoxide oxidation and N-oxide reduction. Among them, the human CYP2C9 enzyme can hydroxylate and metabolize many drugs of different properties, mainly acidic substrates. Currently, there are more than 100 drugs that are substrates of CYP2C9 enzyme, including phenytoin, warfarin, coumarin acetate, tolbutamide, glibenclamide, glimepiride, diclofenac, ibuprofen, mefenamic acid, losartan, irbesartan, torsemide, etc. Summary of the invention

[0005] The purpose of the present invention is to provide an application of human CYP2C9 recombinant enzyme in nitrocellulose denitrification. The present invention screens out a highly efficient and specific enzyme, human CYP2C9 recombinant enzyme, for nitrocellulose denitrification. During the denitrification of nitrocellulose by the CYP2C9 enzyme, the nitrate ester group can be reduced to nitrite, thereby achieving green, harmless and efficient denitrification of nitrocellulose, which can not only make up for the disadvantages of chemical methods in polluting the environment, but also achieve efficient denitrification reaction.

[0006] The technical solution for achieving the purpose of the present invention is:

[0007] The application of human CYP2C9 recombinant enzyme in nitrocellulose denitrification, the amino acid sequence of the human CYP2C9 recombinant enzyme is shown in SEQ ID No.2.

[0008] In the present invention, the nucleotide sequence of the human CYP2C9 recombinase is shown as SEQ ID No.1.

[0009] Furthermore, the specific application method is: adding human CYP2C9 recombinant enzyme to the nitrocellulose solution, reducing some nitrate groups of the nitrocellulose, reducing the nitrate groups on the nitrocellulose, and achieving the effect of denitration.

[0010] Preferably, the concentration of the nitrocellulose solution is 10-20 g / L, the crude enzyme concentration of the human CYP2C9 recombinant enzyme is 1-10 g / L, the reaction temperature is 37° C., and the reaction time is 1-7 days.

[0011] Furthermore, after the reaction is completed, the nitrocellulose is re-extracted. The specific extraction method is as follows: the post-reaction solution is centrifuged, the precipitate is dried, the precipitate is dissolved in acetone, centrifuged, the supernatant is collected, 20% ethanol is added to precipitate the nitrocellulose, the precipitate is centrifuged, the process is repeated twice, the precipitate is resuspended with 1 mol / L NaCl, the precipitate is centrifuged, the process is repeated three times, the precipitate is resuspended with 20% ethanol and centrifuged once, and finally the precipitate is resuspended with pure water and centrifuged three times, the precipitate is collected, and the nitrocellulose is re-extracted by drying.

[0012] Preferably, the centrifugal speed is 7000 rpm, the centrifugal time is 15 min, and the drying temperature is 60°C.

[0013] Preferably, the human CYP2C9 recombinase is prepared by the following method: constructing a recombinant expression transformant containing a nucleotide fragment of the human CYP2C9 recombinase, and then transferring the recombinant expression transformant into a host microorganism for large-scale expression of the human CYP2C9 recombinase to obtain a crude human CYP2C9 recombinase.

[0014] In the present invention, the recombinant expression transformant, the host microorganism and the culture conditions of the host microorganism are not particularly limited and can be appropriately selected according to common knowledge in the art, as long as the recombinant expression transformant can grow normally and successfully express the recombinant CYP2C9 enzyme.

[0015] In a specific embodiment of the present invention, the preparation method of human CYP2C9 recombinant enzyme is as follows:

[0016] (1) Using the plasmids pET28a-CYP2C9-DH5α and pMalc5x-DH5α as templates, PCR amplification was performed to obtain the CYP2C9 insert and the pMalc5x linearized vector;

[0017] (2) The CYP2C9 insert fragment was ligated to the pMalc5x linearized vector using DNA ligase to construct the pMalc5x-CYP2C9 plasmid;

[0018] (3) Transform the pMalc5x-CYP2C9 plasmid into E. coli DH5α competent cells to amplify the recombinant plasmid;

[0019] (4) The pMalc5x-CYP2C9 plasmid was extracted from the competent cells of E. coli DH5α and transformed into competent cells of E. coli BL21 (DE3) to obtain recombinant E. coli containing the CYP2C9 gene. After culturing, the human CYP2C9 recombinant enzyme was obtained.

[0020] Furthermore, in step (4), the specific culture method is as follows: the recombinant Escherichia coli with the CYP2C9 gene is inoculated into LB medium containing ampicillin and cultured. When the OD600 of the culture solution reaches 0.6 to 0.8, IPTG with a final concentration of 0.5 to 1 mmol / L is added to induce the efficient expression of the human recombinant CYP2C9 enzyme.

[0021] Furthermore, in step (4), the method for collecting human CYP2C9 recombinant enzyme is as follows: the cells in the collected culture medium are resuspended in a PBS buffer solution with a pH of 6.0 to 7.0, subjected to ultrasonic cell wall breaking treatment, and the precipitate is collected to obtain crude human CYP2C9 recombinant enzyme.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention is the first to apply human CYP2C9 recombinant enzyme to the denitration of nitrocellulose, and the substrate does not need to be pretreated before denitration;

[0024] (2) The CYP2C9 recombinant enzyme provided by the present invention takes less time to denitrate nitrocellulose than traditional biological treatment. After 7 days of reaction, the nitrogen content of nitrocellulose with an original nitrogen content of 12.7% can be reduced to 11.585%;

[0025] (3) The CYP2C9 recombinant enzyme provided by the present invention does not require the use of toxic reagents when treating nitrocellulose, and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of plasmid construction of recombinant expression plasmid pMalc5x-CYP2C9;

[0027] Figure 2 The electrophoresis diagram of the PCR amplification products of the CYP2C9 insert fragment and the pMalc5x linearized vector, bands 1 and 2 are the insert fragments, and bands 3 and 4 are the linearized vectors;

[0028] Figure 3 The electrophoresis diagram of PCR amplification of 40 pMalc5x-CYP2C9-DH5α single colonies;

[0029] Figure 4 Polyacrylamide gel electrophoresis diagram for identifying the expression of recombinant protein CYP2C9, 1 is the crude enzyme solution of the recombinant expression body, 2 is the bacterial sludge resuspension solution of the recombinant expression body;

[0030] Figure 5 This is a diagram showing the nitrogen reduction effect of CYP2C9 recombinant enzyme on nitrocellulose;

[0031] Figure 6This is the FT-IR comparison diagram of nitrocellulose denitrification by CYP2C9 recombinant enzyme. DETAILED DESCRIPTION

[0032] The present invention is further described below through specific embodiments and drawings.

[0033] Example 1

[0034] Construction of pMalc5x-CYP2C9-BL21(DE3):

[0035] (1) Amplification of CYP2C9 insert and pMalc5x linearized vector

[0036] The plasmid pMalc5x-CYP2C9 was constructed using snapgene software, as shown in Figure 1 As shown, according to the constructed plasmid pMalc5x-CYP2C9, the primer sequences were designed as follows:

[0037] CYP2C9 upstream primer: ctcgggatcgagggaaggCATCATCATCATCATCATCACAGCAG (SEQ ID No. 3),

[0038] CYP2C9 downstream primer: ttcggatccgtcgacgatTCAGTGGTGGTGGTGGTGGTGC SEQ ID No.4,

[0039] pMalc5x upstream primer: atcgtcgacggatccgaat (SEQ ID No. 5),

[0040] pMalc5x downstream primer: ccttccctcgatcccgag (SEQ ID No. 6);

[0041] The CYP2C9 insert and pMalc5x linearized vector were obtained by PCR using pET28a-CYP2C9-DH5α and pMalc5x-DH5α plasmids as templates, respectively. The PCR system was: 2×Phanta Flash Master Mix (Dye Plus) 25μl, 2μl upstream primer and downstream primer, 2μl plasmid template and 19μl ddH2O. The PCR amplification steps were: (1) 98℃, pre-denaturation for 30sec; (2) 98℃, denaturation for 10sec; (3) 58℃ annealing for 5sec; (4) 72℃ extension at 4-5sec / kb; steps (2) to (4) were cycled 30 times; (5) 72℃, extension for 1min, cooled to 4℃ for storage. The PCR product was purified by agarose gel electrophoresis, and the target bands in the range of 1000-2000 bp (inserted fragment) and 5000-8000 bp (linearized vector) were recovered using an agarose gel electrophoresis DNA recovery kit. Figure 2 shown.

[0042] (2) Preparation of recombinant expression plasmid pMalc5x-CYP2C9

[0043] The recombination reaction system is: 2μl of purified insert fragment and linearized vector, 4μl of 5*CEⅡBuffer, 2μl of ExnaseⅡ and 10μl of ddH2O. Use a pipette to gently mix (do not oscillate), centrifuge briefly to collect the reaction solution to the bottom of the tube, react in a 37℃ water bath for 30min, and immediately cool on ice to obtain the recombinant expression plasmid pMalc5x-CYP2C9.

[0044] (3) Construction of the recombinant expression vector pMalc5x-CYP2C9-BL21(DE3)

[0045] The pMalc5x-CYP2C9 plasmid was transferred into E. coli DH5α competent cells and cultured overnight. The positive recombinants were screened on the resistance plate containing ampicillin. The positive clones were preliminarily identified by colony PCR. The primers used were the detection upper primer of the pMalc5x plasmid and the lower primer of the insert fragment. The band size should be 2987bp ( Figure 3 ), the plasmid obtained by amplifying the recombinant bacteria preliminarily identified as positive clones was sequenced and analyzed to further verify whether the constructed recombinant bacteria were correct, that is, to verify whether the insertion direction and reading frame of the target gene were correct, the correctly constructed recombinant bacteria were cultured, and the plasmid was extracted after plasmid amplification, and re-transformed into E. coli BL21 (DE3) competent cells to obtain the recombinant expression transformant pMalc5x-CYP2C9-BL21 (DE3).

[0046] Example 2

[0047] Preparation of recombinant CYP2C9 enzyme

[0048] The pMalc5x-CYP2C9-BL21 (DE3) obtained in Example 1 was inoculated into 10 ml LB medium containing ampicillin (final concentration 0.1 g / L), and cultured at 37°C with shaking until OD600 was between 0.6 and 0.8. The seed liquid was then inoculated into a 2L culture flask containing 500 ml LB medium at an inoculum amount of 2% (v / v), and cultured at 37°C with shaking until OD600 was between 0.6 and 0.8 for induction, i.e., IPTG was added at a final concentration of 1 mM, and cultured overnight in a shaking incubator at 18°C.

[0049] The obtained fermentation culture liquid was centrifuged at 6000rpm for 6min to remove the supernatant, and the wet cells were collected. The collected wet cells were washed once with PBS buffer solution of pH 7.0 (centrifuged), and the washed wet cells were resuspended in the buffer solution (the OD600 of the suspension was controlled to be 40) to obtain a suspension, and the suspension was treated with an ultrasonic cell crusher (power 40W, ultrasound 2s, interval 3s, time 10min), and centrifuged at 7000rpm for 15min. The supernatant was the crude enzyme solution, and the precipitate was cell fragments (hereinafter referred to as bacterial mud).

[0050] The protein concentration of the crude enzyme solution was determined by the Coomassie Brilliant Blue method using a spectrophotometer. The sample system to be tested is: 2100μl Coomassie Brilliant Blue, 60μl enzyme solution, which can be diluted with pure water; the control system is: 2100μl Coomassie Brilliant Blue, 60μl pure water. The reaction was carried out at room temperature for 5 minutes, and the absorbance at 595nm was measured using a spectrophotometer. The protein concentration in the crude enzyme solution can be calculated by substituting it into the standard equation. The crude enzyme solution and bacterial mud were analyzed by polyacrylamide gel electrophoresis ( Figure 4 ), the size of the recombinant protein is known to be 96kD. From the gel image, it can be seen that the recombinant expression body can be expressed correctly, and the recombinant protein is expressed in both soluble form and inclusion body.

[0051] Example 3

[0052] Denitrification of nitrocellulose by recombinant CYP2C9 enzyme

[0053] The crude enzyme solution obtained in Example 2 was diluted to 1 g / L with PBS buffer, the reaction system was controlled to be 10 ml, the final concentration of nitrocellulose was 10 g / L, and the nitrocellulose was denitrated in a shaker at 37° C. and 200 rpm for 64 h.

[0054] After the reaction is completed, the reaction solution is centrifuged at 7000rpm for 15min (the centrifugation conditions for re-extracting nitrocellulose are the same), the collected precipitate is resuspended in pure water, and then centrifuged. The collected precipitate is placed in a 60°C oven for drying for 2h, the dried precipitate is scraped into a small triangular bottle, an appropriate amount of acetone is added, and the precipitate is placed in a 45°C water bath for 2h to fully dissolve it, and then centrifuged. The supernatant is collected, 20% ethanol is added to precipitate nitrocellulose, and the precipitate is centrifuged and repeated twice. The precipitate is resuspended with 1mol / L NaCl and centrifuged to obtain the precipitate, repeated three times, then resuspended with 20% ethanol and centrifuged once, and finally resuspended with pure water and centrifuged three times. The precipitate is the re-extracted nitrocellulose, which is dried and analyzed for N content using an elemental analyzer with an instrument model of Elemantar: Vario EL Cube.

[0055] The change in nitrogen content of the original drug nitrocellulose after being treated with CYP2C9 recombinant enzyme for 64 hours was measured using an elemental analyzer, and it was found that it was reduced. In order to further verify its nitrogen reduction effect, repeated experiments were carried out and the culture time was extended to 7 days. It was found that CYP2C9 recombinant enzyme did have a nitrogen reduction effect on nitrocellulose, and the nitrogen reduction effect became more obvious as time went on. For details, see Figure 5 At the same time, the extracted nitrocellulose was subjected to infrared analysis, and it was found that the basic structure of nitrocellulose after recombinant enzyme treatment did not change. For details, see Figure 6 .

Claims

1. Application of human CYP2C9 recombinant enzyme in denitrification of nitrocellulose, characterized in that: The amino acid sequence of the human CYP2C9 recombinant enzyme is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene of the human CYP2C9 recombinase is shown in SEQ ID No.

1.

3. The use according to claim 1, characterized in that: The specific application method is: adding human CYP2C9 recombinant enzyme to the nitrocellulose solution, reducing some nitrate groups of the nitrocellulose, reducing the nitrate groups on the nitrocellulose, and achieving the effect of denitrification.

4. The use according to claim 3, characterized in that: The concentration of the nitrocellulose solution is 10~20g / L, the crude enzyme concentration of the human CYP2C9 recombinant enzyme is 1~10g / L, the reaction temperature is 37°C, and the reaction time is 1~7 days.

5. The use according to claim 3, characterized in that: After the reaction is completed, the nitrocellulose is re-extracted. The specific extraction method is as follows: the post-reaction solution is centrifuged, the precipitate is dried, the precipitate is dissolved in acetone, centrifuged, the supernatant is collected, 20% ethanol is added to precipitate the nitrocellulose, centrifuged to obtain the precipitate, repeated twice, and then the precipitate is resuspended with 1 mol / L NaCl, centrifuged to obtain the precipitate, repeated three times, then resuspended with 20% ethanol and centrifuged once, finally resuspended with pure water and centrifuged three times, the precipitate was collected, and dried to obtain the re-extracted nitrocellulose.

6. The use according to claim 5, characterized in that: The centrifugal speed is 7000 rpm, the centrifugal time is 15 min, and the drying temperature during drying the precipitate is 60°C.

7. The use according to claim 3, characterized in that: The human CYP2C9 recombinant enzyme is prepared by the following method: constructing a recombinant expression transformant containing a nucleotide fragment of the human CYP2C9 recombinant enzyme, and then transferring the recombinant expression transformant into a host microorganism to express a large amount of the human CYP2C9 recombinant enzyme to obtain a crude human CYP2C9 recombinant enzyme.

8. The use according to claim 7, characterized in that: The preparation method of human CYP2C9 recombinant enzyme is as follows: (1) Using the plasmids pET28a-CYP2C9-DH5α and pMalc5x-DH5α as templates, PCR amplification was performed to obtain the CYP2C9 insert and the pMalc5x linearized vector; (2) Use DNA ligase to connect the CYP2C9 insert fragment to the pMalc5x linearized vector to construct the pMalc5x-CYP2C9 plasmid; (3) Transform the pMalc5x-CYP2C9 plasmid into E. coli DH5α competent cells to amplify the recombinant plasmid; (4) The pMalc5x-CYP2C9 plasmid was extracted from the competent cells of E. coli DH5α and transformed into competent cells of E. coli BL21 (DE3) to obtain recombinant E. coli containing the CYP2C9 gene. After cultivation, the human CYP2C9 recombinant enzyme was obtained.

9. The use according to claim 8, characterized in that: In step (4), the specific culture method is as follows: the recombinant Escherichia coli with the CYP2C9 gene is inoculated into LB medium containing ampicillin and cultured. When the OD600 of the culture medium reaches 0.6-0.8, IPTG with a final concentration of 0.5-1 mmol / L is added to induce the efficient expression of the human recombinant CYP2C9 enzyme.

10. The use according to claim 9, characterized in that In step (4), the method for collecting human CYP2C9 recombinant enzyme is as follows: the cells in the collected culture medium are resuspended in a PBS buffer solution with a pH of 6.0 to 7.0, subjected to ultrasonic cell wall disruption treatment, and the precipitate is collected to obtain crude human CYP2C9 recombinant enzyme.

Images