CN107164350B - A kind of pyrazinamide hydrolase and its coding gene and application - Google Patents

Abstract

The present invention discloses a pyrazinamide hydrolase, its encoding gene and its application. The hydrolase has the amino acid sequence shown in SEQ ID NO.1, and the encoding gene has the nucleotide sequence shown in SEQ ID NO.2. DNA molecule. The pyrazinamide enzyme of the present invention has the advantages of high enzyme activity, good pH tolerance, etc., and provides a therapeutic direction for the combined use of pyrazinamide and pyrazinamide enzyme in treating patients with pyrazinamide drug resistance in vivo.

Description

A kind of pyrazinamide hydrolase and its coding gene and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a pyrazinamide hydrolase, its coding gene and application.

Background technique

Pyrazinamide is one of the first-line drugs recommended by the World Health Organization for the treatment of tuberculosis. The drug itself has no antibacterial activity, but is hydrolyzed into pyrazinoic acid in the pathogenic bacteria, so that it can continuously kill Mycobacterium tuberculosis existing in the crowd in an acidic pH environment, which can shorten the treatment time from 9-12 months to 6 months. months. Despite the recognized importance of pyrazinamide in the treatment of tuberculosis, its mechanism of action is perhaps the least understood of all anti-TB drugs.

Currently, the emergence of more and more drug-resistant tuberculosis strains has posed a major challenge to the antibacterial activity of pyrazinamide, a prodrug with no significant antibacterial activity that is metabolized to its active form, pyrazinic acid, It is hydrolyzed by the hydrolase activity encoded by the pyrazinamide hydrolase gene to produce pyrazinoic acid, which is used to kill Mycobacterium tuberculosis. Mutations in pyrazinamide hydrolase are currently the main mechanism of pyrazinamide resistance in Mycobacterium tuberculosis. Pyrazinamide hydrolase has been found in many microorganisms, such as Escherichia coli, Saccharomyces cerevisiae. This enzyme converts pyrazinamide to pyrazinoyl acid. The biochemical characterization of certain bacterial pyrazinamide hydrolases has been studied and the Mycobacterium tuberculosis pyrazinamide hydrolase MtPncA has been well characterized, but pyrazinamide hydrolysis in Pseudonocardia carboxydivorans The enzyme has not been characterized.

Contents of the invention

The present inventors discovered an open reading frame (ORF) in Pseudonocardia carboxydivorans, which has 62% sequence homology to MtPncA. The inventor cloned and expressed the gene, and unexpectedly found that it is a highly active pyrazinamide hydrolase, which is especially suitable for treating tuberculosis patients who have developed resistance to pyrazinamide.

Therefore, the main purpose of the present invention is to provide a new pyrazinamide hydrolase and its coding gene and application. In order to achieve the purpose of the present invention, the inventor has made unremitting efforts through a large number of experimental studies, and finally obtained the following technical solutions:

A pyrazinamide hydrolase, the hydrolase has the amino acid sequence shown in SEQ ID NO.1; or the hydrolase is deleted, replaced, inserted or/and added on the basis of the amino acid sequence shown in SEQ ID NO.1 A conservative variant with the activity of hydrolyzing pyrazinamide obtained by conservative mutation of one to several amino acids.

It should be noted that the pyrazinamide hydrolase provided by the present invention is a member of the cysteine hydrolase superfamily. The amino acid sequence shown in SEQ ID NO.1 consists of 191 amino acid residues.

In order to facilitate the purification of the above-mentioned protein, the tags shown in Table 1 can be attached to the amino-terminus or carboxy-terminus of the protein composed of the above-mentioned amino acid sequence.

Table 1 Sequence of tags

Label Residues amino acid sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The above-mentioned pyrazinamide hydrolase protein can be synthesized artificially, or its coding gene can be firstly synthesized and then biologically expressed. The protein-encoding gene mentioned above can also be deleted, substituted, inserted or added one to several in the amino acid sequence shown in SEQ ID NO. The coding sequence was obtained.

The present invention also provides a gene encoding pyrazinamide hydrolase, which encodes:

(a) a protein having the amino acid sequence shown in SEQ ID NO.1; or

(b) A protein having the amino acid sequence shown in SEQ ID NO. 1 derived from deletion, substitution, insertion or addition of one to several amino acids and having pyrazinamide hydrolase activity.

Further, the coding gene of the pyrazinamide hydrolase is the DNA molecule of (i), (ii) or (iii):

(i) DNA molecule with the nucleotide sequence shown in SEQ ID NO.2;

(ii) a DNA molecule that hybridizes to the nucleotide sequence described in (i) under stringent conditions and encodes a protein with pyrazinamide hydrolysis activity;

(iii) A DNA molecule having a nucleotide sequence that is 90% or more homologous to the nucleotide sequence of the DNA molecule described in (i) or (ii).

Further, the stringent conditions are that the reaction temperature is 50-68° C. in a solution with a sodium ion concentration of 50-300 mM.

For example: in the process of molecular hybridization, it can be hybridized at 65°C in a solution of 6×SSC and 0.5% SDS by mass fraction, and then use 2×SSC, 0.1% SDS by mass fraction and 1× The membrane was washed once with SSC and 0.1% SDS respectively. The Chinese name of SDS is sodium dodecyl sulfate, and 1×SSC includes 0.15mol/L NaCl and 0.015mol/L citric acid; SDS and SSC with different concentration multiples are commonly used reagents in this field.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing any of the above-mentioned coding genes also belong to the protection scope of the present invention.

The present invention provides a recombinant vector, including the gene encoding the above-mentioned pyrazinamide hydrolase. The recombinant vector is a recombinant expression vector obtained by inserting the above coding gene into the multiple cloning site of the departure vector (for example: pET28a). An existing expression vector can be used to construct a recombinant expression vector containing the gene. When using the gene to construct a recombinant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, and they can be used alone or in combination with other promoters; in addition, When using the gene of the present invention to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be consistent with the coding The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

The present invention also provides a transformant comprising the above-mentioned recombinant vector. The transformant can be a recombinant bacterium. For example, the recombinant expression vector obtained by inserting any of the above-mentioned coding genes into the multiple cloning site of the starting vector (for example: pET28a vector) is transformed into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium.

The present invention also provides a pair of primers for amplifying the full length of the gene encoding pyrazinamide hydrolase and any fragment thereof. For example: the sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4.

The application of any of the above-mentioned proteins, the above-mentioned coding genes, the above-mentioned recombinant expression vectors, the above-mentioned expression cassettes, transgenic cell lines or recombinant bacteria in the degradation of pyrazinamide and nicotinamide also belongs to the protection of the present invention scope.

In addition, the present invention can also combine pyrazinamide and pyrazinamide hydrolase to treat tuberculosis, especially for tuberculosis patients who have developed resistance to pyrazinamide. Therefore, the present invention also provides a pharmaceutical composition for treating tuberculosis, which comprises the above-mentioned pyrazinamide hydrolase. Further preferably, the pharmaceutical composition also contains pyrazinamide in addition to the above-mentioned pyrazinamide hydrolase.

A use of the above-mentioned pyrazinamide hydrolase in hydrolyzing amide bonds. For example: for the hydrolysis of pyrazinamide, nicotinamide, etc. In the process of specific application, the following method can be adopted: pyrazinamide and nicotinamide are used as substrates, and pyrazinamide and nicotinamide are enzymatically hydrolyzed by pyrazinamide hydrolase under the condition of neutral pH 8.5. The degradation conditions include: the temperature of the reaction system is 35-50°C, preferably 45°C, and the pH of the reaction system is 7.5-9.0, preferably 8.5.

The present invention also provides a method for producing pyrazinamide hydrolase, the method comprising culturing the above-mentioned transformant and collecting the pyrazinamide hydrolase from the culture product. The collected pyrazinamide hydrolase can be further purified.

The beneficial effects of the present invention are:

The protein of the invention has the activity of hydrolyzing pyrazinamide and belongs to amidohydrolase. And compared with the amino acid sequence of other characterized pyrazinamide hydrolase, the similarity of the protein is not more than 65%, and it belongs to a member of cysteine hydrolase superfamily. The optimum natural substrate of the pyrazinamide hydrolase in the present invention is nicotinamide, and has the characteristic of high activity under neutral and partial alkaline pH conditions. The present invention also has good enzymatic activity on pyrazinamide (PZA), and experiments show that the pyrazinamide hydrolase of the present invention uses PZA as a substrate and has the highest enzymatic activity at 45°C and pH 8.5 , is 17.20U/mg protein, and one unit of enzyme activity is the amount of enzyme required to generate 1 μmol of product per minute; the enzyme has high enzyme activity in the temperature range of 35-50°C; at pH 6.5 to pH 10.0 Within the range, the enzyme activity was high. The enzyme is maintained at pH 7.5-10.0 for 12 hours, still has more than 80% of the enzyme activity, and has good pH tolerance.

Description of drawings

Figure 1 is the SDS-PAGE electrophoresis before and after purification of PncA-Pse protein.

Figure 2 is the result of the activity of pyrazinamide hydrolase as a function of temperature.

Fig. 3 is the result of the change of the activity of pyrazinamide hydrolase with pH.

Detailed ways

The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, preparation and function of protein and gene

1. Extraction of total DNA of Pseudonocardia carboxydivorans:

Take 20 grams of fresh wet cells from Pseudonocardia carboxydivorans, suspend in 10 ml of 50 mM Tris buffer (pH 8.0), add a small amount of lysozyme and 8 ml of 0.25 mM EDTA (pH 8.0), mix and place at 37 ° C for 20 min; Then add 2 ml of SDS with a mass fraction of 10%, place at 55°C for 5 min, and extract once with equal volumes of phenol and chloroform respectively; take the last supernatant solution, add 2 times the volume of absolute ethanol, recover DNA, and use Washed twice with 70% absolute ethanol; the precipitate was vacuum-dried and dissolved in 0.5 ml TE buffer (pH 8.0, 10 mM Tris, 1 mM EDTA).

2. Synthesize the nucleotide sequence shown in SEQ ID NO.2

According to the nucleotide sequence shown in SEQ ID NO.2, the primer pair is designed as follows:

Forward primer: 5′-CGC GGATCC ATGGCACGAGCGTTGATCGTCGTC-3′, as shown in SEQ ID NO.3;

Reverse primer: 5'-GGG TTCGAA TCAGGCGGTGCGGAGCTCCACGC-3', as shown in SEQ ID NO.4;

The underlined part of the forward primer is the restriction site of BamHI, and the underlined part of the reverse primer is the restriction site of HindШ.

Using the total DNA of Pseudonocardia carboxydivorans as a template, PCR amplification was performed with the designed primer pairs.

PCR reaction system:

10× buffer 5μL dNTP 4μL exTaq DNA polymerase 0.5μL forward primer 1μL reverse primer 1μL template 0.5μL water 38μL

PCR reaction conditions: pre-denaturation at 95°C for 5 minutes, then denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, 30 cycles, and finally extension at 72°C for 10 minutes.

The yield and specificity of the PCR product were detected by agarose gel electrophoresis with a mass fraction of 0.7%, and purified with a DNA purification kit (ultra-thin spin column type, produced by Tiangen Company). The purified PCR product was sequenced, and the result showed that the sequence of the PCR product included positions 1-576 shown in SEQ ID NO.2, and it was named as PncA-Pse DNA fragment.

3. Construction of recombinant expression vector

1) Digest the above-mentioned PCR products or artificially synthesized fragments with correct sequencing with BamHI and HindШ, and recover the digested products by agarose electrophoresis.

2) Plasmid pET28a (Cat.N0 69864-3, Novogen) was double-digested with BamHI and HindШ, and the digested product was recovered by agarose electrophoresis.

3) Ligate the digested product of step 1) and the digested product of step 2), transform the ligated product into Escherichia coli DH5α by electroporation, spread it on an LB plate containing 50 μg/mL kanamycin, and culture it overnight at 37°C. The obtained transformant was subjected to colony PCR with the above-mentioned forward primer and reverse primer, and the recombinant bacteria containing the PncA-Pse gene were screened, the plasmid of the recombinant bacteria was extracted, and the sequencing verification was carried out. A PncA-Pse fragment was inserted between the sites, and the fragment included nucleotides 1 to 576 from the 5' end of SEQ ID NO.2, and the insertion direction was correct. The recombinant plasmid was named (pET28a-PncA- Pse).

4. Preparation of engineering bacteria

The plasmid pET28a-PncA-Pse was transformed into Escherichia coli Transetta (DE3) (Cat.NOCD801, Quanshijin Company) by electroporation, and spread on an LB plate containing 50 μg/mL kanamycin, and cultured overnight at 37°C to obtain pET28a- The engineering strain of PncA-Pse plasmid is recorded as Transetta/pET28a-PncA-Pse.

Replace pET28a-PncA-Pse with pET28a, transform Escherichia coli Transetta (DE3), and use the same procedure as above to obtain a recombinant bacterium containing pET28a as a control bacterium. The positive recombinant bacteria transformed into pET28a were recorded as Transetta/pET28a.

5. Preparation and purification of pyrazinamide hydrolase

His60Ni Superflow resin purification column was purchased from TaKaRa Company, the product catalog number is 635660.

GE HiTrap Desalting purification columns were purchased from GE Healthcare, and the product catalog numbers were 17-1408-01.

Culture the Transetta/pET28a-PncA-Pse-positive recombinant bacteria prepared in step 4 above in LB medium containing 50 μg/mL kanamycin, and culture at 37°C for 3 hours; when OD ₆₀₀ =0.7, add IPTG until it is cultured in LB The final concentration in the medium was 0.2mM, and the culture was continued at 18°C for 16h.

Collect the cells by centrifugation at 3800rpm for 15min, suspend in PBS solution (50mM Tris-HCl, pH8.0, 0.5M NaCl, 5mM imidazole), and sonicate in an ice bath (60w, 10min; sonicate for 1s, stop for 2s ), then centrifuge at 12000rpm for 10min to remove cell debris, and take the supernatant; pass the supernatant through a His60Ni Superflow resin purification column, rinse with 5mL ultrapure water, and then wash with 10mL solution A (50mM Tris-HCl, pH8.0, 0.5M NaCl, 25 mM imidazole), rinsed with 5 mL of solution B (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 500 mM imidazole), and collected the eluate. Then the eluate was desalted with a desalting column GE HiTrap Desalting, and eluted with solution C (50 mM Tris-HCl, pH 8.5) to obtain a pure enzyme solution of PncA-Pse.

The control bacteria prepared in step 4 were cultivated and purified in the same steps, and the obtained solution was used as the control enzyme solution.

SDS-PAGE electrophoresis showed that the molecular weight of the purified PncA-Pse protein was about 23kDa, which was in line with the theoretical deduction of 23kDa. The results are as shown in Figure 1, and in Figure 1, swimming lane M represents protein molecular weight standard (100,75,50,37,25kDa); Swimming lane 1 represents the supernatant after E. 2 represents PncA-Pse protein purified by Ni-NTA column; lane 3 represents PncA-Pse protein purified by GE Desalting column. It can be seen that the PncA-Pse protein has been obtained. At the same time, the experiment of the control group was carried out, but the control bacteria did not obtain the target protein.

Example 2. Using pyrazinamide as a substrate to verify protein function

The enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of pyrazinoic acid within 1 min.

(1) Optimum temperature

The PncA-Pse pure enzyme solution in Step 5 of Example 1 was diluted with 50 mM Tris-HCl buffer solution of pH 8.5, and the enzyme activity was measured with the diluted enzyme solution. The diluted enzyme solution was recorded as the diluted enzyme solution.

Composition of solution A: composed of 50 mM Tris-HCl buffer solution with pH 8.5 and pyrazinamide solution; the concentration of pyrazinamide in solution A is 100 μM.

Experimental group: The activity measurement reaction system is 0.5mL, dilute the enzyme solution from 0.475mL solution A and 0.025mL; the pH value of the reaction system is 8.5; 0.05mL of 10% trichloroacetic acid was used to terminate the reaction, passed through a 0.22μm organic filter, and then 20μL of the sample was loaded on HPLC to detect the amount of pyrazinic acid. Liquid phase conditions:

C18 chromatographic column;

Detection wavelength: 268nm Column temperature: 30°C

Flow rate: 0.5mL/min

Mobile phase: methanol: water (10:90)

The results are shown in Figure 2A. Figure 2A shows that pyrazinamide hydrolase has amidase activity. At 45°C, pyrazinamide hydrolase has the highest enzymatic activity; the peak area of the pyrazinoic acid product in the enzyme activity reaction system at this temperature is regarded as 100% of the relative activity, and the pyrazinoic acid product in the enzyme activity reaction system at other temperatures The ratio of the peak area of the highest enzyme activity system to the peak area of the pyrazinoic acid product was used as the relative activity. All have more than 50% activity at 30-50°C.

Control group: the above experiment was carried out with the protein obtained from the control bacteria Transetta/pET28a (referred to as the control enzyme solution). As a result, the control enzyme solution had no activity of hydrolyzing pyrazinamide no matter under which temperature conditions.

The experiment was repeated 3 times and the results were consistent.

(2) Optimum pH

The diluted enzyme solutions in the following groups were all obtained by diluting the PncA-Pse pure enzyme solution in Step 5 of Example 1 with the buffer solution in each group.

Experimental group: The activity assay reaction system is 0.5mL, which is composed of 0.475mL solution B (B1, B2, B3, B4, B5, B6, B7, B8 or B9) and 0.025mL diluted enzyme solution;

The composition of solution B1: 50NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer solution and pyrazinamide (PZA); the concentration of pyrazinamide in solution B1 is 100 μM; the pH of solution B1 is 5.5.

Composition of solution B2: the same composition as solution B1, the pH value of solution B2 is 6.0.

Composition of solution B3: same composition as solution B1, pH of solution B3 is 6.5.

Composition of solution B4: the same composition as solution B1, the pH value of solution B4 is 7.0.

Composition of solution B5: the same composition as solution B1, and the pH value of solution B5 is 7.5.

Composition of solution B6: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer was replaced by 50 mM Tris-HCl buffer. Solution B6 has a pH of 7.5.

Composition of solution B7: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ —Na ₂ HPO ₄ buffer was replaced by 50 mM Tris-HCl buffer. Solution B7 had a pH of 8.0.

Composition of solution B8: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ —Na ₂ HPO ₄ buffer was replaced by 50 mM Tris-HCl buffer. Solution B8 had a pH of 8.5.

Composition of solution B9: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer was replaced by 50 mM Tris-HCl buffer. Solution B9 had a pH of 9.0.

Composition of solution B10: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer was replaced by 50 mM glycine-NaOH buffer. Solution B10 had a pH of 9.0.

Composition of solution B11: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer was replaced by 50 mM glycine-NaOH buffer. with solution B11 having a pH of 9.5.

Composition of solution B12: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer was replaced by 50 mM glycine-NaOH buffer. Solution B12 had a pH of 10.0.

Composition of solution B13: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer was replaced by 50 mM glycine-NaOH buffer. Solution B13 had a pH of 10.5.

Composition of solution B13: the same composition as solution B1, except that 50 mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer was replaced by 50 mM glycine-NaOH buffer. Solution B13 had a pH of 11.0.

After incubating the reaction system at 45°C for 10 min, 0.05 mL of 10% trichloroacetic acid was added to terminate the reaction, passed through a 0.22 μm organic filter, and then 20 μL of the sample was loaded on HPLC, and the amount of the product pyrazinic acid was detected at 268 nm.

The experiment was repeated three times.

The results are shown in Figure 3A.

The pyrazinamide hydrolase has good pyrazinamide hydrolase activity under the condition of pH between 6.5 and 10.0, that is, it can degrade pyrazinamide.

It shows that pyrazinamide hydrolase has the highest enzyme activity at pH8.5. The peak area of the pyrazinoic acid product in this highest enzyme activity reaction system is regarded as relative activity 100%, and the ratio of the peak area of the enzyme activity reaction system pyrazinoic acid product to the peak area of this highest enzyme activity system pyrazinoic acid product under other pH is taken as relative activity.

Control group: the above experiment was carried out with the protein obtained from the control bacteria Transetta/pET28a (referred to as the control enzyme solution). As a result, the control enzyme solution had no activity of degrading pyrazinamide no matter under which pH conditions.

The experiment was repeated 3 times and the results were consistent.

(3) Enzyme thermostability

The PncA-Pse pure enzyme solution in Step 5 of Example 1 was diluted with 50 mM Tris-HCl buffer solution of pH 8.5, and the enzyme activity was measured with the diluted enzyme solution using pyrazinamide as a substrate. The diluted enzyme solution was recorded as the diluted enzyme solution.

The diluted enzyme solution was incubated in a 37°C water bath, and samples were taken every 5 minutes to determine the residual activity of the enzyme. The results showed that the enzyme could maintain 50% residual enzyme activity at 37°C for 10 minutes. The present invention measures the thermal stability of PncA-Pse at 37°C.

The results are shown in Figure 2B.

(4) pH tolerance

After the diluted enzyme solution was placed at pH 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0 for 12 hours, the remaining enzyme activity was determined using pyrazinamide as a substrate. The results show that the relative enzyme activity still has more than 60% under the condition of pH 7.5-10.0. It shows that the enzyme has good pH tolerance.

The results are shown in Figure 3B.

(5) Substrate specificity

The enzyme activity of the diluted enzyme solution was determined under the conditions of different substrates at the same concentration (100 μM molar concentration), and the substrates were pyrazinamide, nicotinamide, isonicotinamide, propionamide, isobutyramide, and benzamide .

Enzyme activity was measured with different amide substrates, only pyrazinamide and nicotinamide were tested for enzyme activity, and the enzyme activity for nicotinamide was the highest at 36.25U/mg, and the enzyme activity for pyrazinamide was 17.20U/mg. Therefore, the pyrazinamide hydrolase has strong substrate specificity for pyrazinamide and nicotinamide, and has high catalytic activity for these two heterocyclic amides substrates.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

SEQUENCE LISTING

<110> Hubei University

<120> A kind of pyrazinamide hydrolase and its coding gene and application

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 191

<212> PRT

<213> Pseudonocardia carboxydivorans WSC1212

<400> 1

Met Ala Arg Ala Leu Ile Val Val Asp Val Gln Asn Asp Phe Cys Glu

1 5 10 15

Gly Gly Ala Leu Ala Val Ala Gly Gly Ala Ala Val Ala Ala Gly Ile

20 25 30

Gly Pro Leu Ala Thr Gly Gly Arg Asp Gly Arg Val Tyr Asp His Val

35 40 45

Val Ala Thr Arg Asp Val His Val Asp Pro Gly Pro His Phe Ser Asp

50 55 60

Thr Pro Asp Phe Val Asp Ser Trp Pro Pro His Cys Arg Ala Gly Thr

65 70 75 80

Pro Gly Ala Ser Phe His Pro Ala Leu Asp Val Ala Pro Ile Glu Cys

85 90 95

Val Phe Asp Lys Gly Ala Tyr Thr Ser Ala Tyr Ser Gly Phe Glu Gly

100 105 110

Ala Ser Ala Asn Glu Ser Gly Ala Pro Leu Gly Asp Trp Leu Thr Glu

115 120 125

His Gly Val Thr Glu Val Asp Val Val Gly Leu Ala Thr Asp His Cys

130 135 140

Val Arg Ala Thr Ala Leu Asp Ala Ala Arg Leu Gly Leu Ala Thr Thr

145 150 155 160

Val Leu Leu His His Cys Ala Gly Val Ala Pro Glu Thr Thr Glu Arg

165 170 175

Ala Leu Ala Ala Leu Thr Glu Ala Gly Val Glu Leu Arg Thr Ala

180 185 190

<210> 2

<211> 576

<212>DNA

<213> Pseudonocardia carboxydivorans WSC1212

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atggcacgag cgttgatcgt cgtcgacgtc cagaacgact tctgcgaggg cggcgccctc 60

gcggtggccg gcggggccgc cgtggccgcc gggatcgggc cgctcgcgac cggtggccgc 120

gacgggcggg tctacgacca cgtcgtcgcc acccgggacg tgcacgtgga cccggggccg 180

cacttctccg acaccccgga cttcgtcgac tcctggccgc cgcactgccg cgcggggacg 240

ccgggcgcgt cgttccacccc ggcactggac gtggcgccga tcgagtgcgt cttcgacaag 300

ggcgcctaca ccagcgccta ctccgggttc gagggcgcga gcgcgaacga gtcgggcgcc 360

ccgctgggcg actggctcac cgagcacggg gtgaccgagg tcgacgtcgt cggcctggcg 420

accgaccact gcgtccgggc gaccgcgctc gacgcggccc ggctcgggct ggcgacgacg 480

gtgctgctgc accactgcgc cggggtggca ccggagacga cggagcgggc gctcgccgcg 540

ctgaccgagg ccggcgtgga gctccgcacc gcctga 576

<210> 3

<211> 33

<212>DNA

<213> Artificial sequence

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cgcggatcca tggcacgagc gttgatcgtc gtc 33

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<212>DNA

<213> Artificial sequence

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gggttcgaat caggcggtgc ggagctccac gc 32

Claims (10)

1. A pyrazinamide hydrolase, characterized in that the amino acid sequence of the hydrolase is as shown in SEQ ID NO.1. 2. A gene encoding pyrazinamide hydrolase, characterized in that the gene encodes a protein with the amino acid sequence shown in SEQ ID NO.1. 3. The gene encoding pyrazinamide hydrolase according to claim 2, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO.2. 4. A recombinant vector, characterized in that it comprises the gene encoding the pyrazinamide hydrolase according to claim 2 or 3. 5. A transformant, characterized in that it comprises the recombinant vector according to claim 4. 6. A pair of primers, characterized in that, it is used to amplify the full length of the gene encoding pyrazinamide hydrolase described in claim 2 or 3 and any fragment thereof, and the sequence of the pair of primers is as shown in SEQ ID NO.3 and shown in SEQ ID NO.4. 7. A method for producing pyrazinamide hydrolase, the method comprising culturing the transformant according to claim 5 and collecting the pyrazinamide hydrolase from the culture product. 8. A pharmaceutical composition for treating tuberculosis, characterized in that the pharmaceutical composition comprises a pyrazinamide hydrolase according to claim 1. 9. Use of the pyrazinamide hydrolase as claimed in claim 1 in converting pyrazinamide into pyrazinoic acid. 10. The application of the pyrazinamide hydrolase as claimed in claim 1 in the preparation of a medicament for the treatment of tuberculosis.