CN107312806A - A kind of carboxylic acid of 5 methylpyrazine of Production by Enzymes 2 - Google Patents

Abstract

The invention discloses an enzymatic method for producing 5-methylpyrazine-2-carboxylic acid. The present invention provides the application of specific protein in the preparation of 5-methylpyrazine-2-carboxylic acid; the specific protein is as follows (a1) or (a2): (a1) aldehyde dehydrogenase; (a2) in ( The fusion protein obtained by linking the N-terminus or/and C-terminus of a1). For the first time, the present invention utilizes a specific enzyme to efficiently realize the one-step production of 5-methylpyrazine-2-carboxylic acid. The process of the invention is simple, and all raw materials and enzymes can be reacted in one step to prepare 5-methyl-2-pyrazinic acid, without involving intermediate steps and reactions, which greatly simplifies the process flow, and the catalytic oxidation reaction basically does not Inhibited by the product 5-methyl-2-pyrazinoic acid, high-concentration conversion can obtain high yield, which simplifies the extraction process and is conducive to large-scale industrial production.

Description

An enzymatic method for producing 5-methylpyrazine-2-carboxylic acid

technical field

The invention relates to the field of biotechnology, in particular to an enzymatic method for producing 5-methylpyrazine-2-carboxylic acid.

Background technique

5-Methylpyrazine-2-carboxylic acid (5-Methylpyrazine-2-carboxylic acid) is an important pharmaceutical intermediate, mainly used in the synthesis of the second-generation sulfonylurea hypoglycemic drug Glipizide , a new generation of long-acting hypolipidemic drug Acipimox (Acipimox) and effective drug for the treatment of tuberculosis (PAE).

The existing preparation methods of 5-methylpyrazine-2-carboxylic acid include chemical synthesis, electrochemical synthesis and biosynthesis, among which the chemical synthesis has been industrialized.

Most of the current chemical synthesis methods use 2,5-dimethylpyrazine as a raw material, and then react with acetic anhydride to generate 2-acetoxy Methyl-5-methylpyrazine, followed by alkaline hydrolysis to obtain 2-hydroxymethyl-5-methylpyrazine, and finally oxidation by potassium permanganate to obtain the target product. The method has low yield and low operational safety.

CN 1141299 C "Method for preparing 5-methylpyrazine-2-carboxylic acid by one _- step oxidation with KMnO4" discloses a method for preparing ₅ -methylpyrazine-2-carboxylic acid by one-step oxidation with KMnO4. The method uses 2.5-dimethylpyrazine as a raw material, and in the presence of an inhibitor, _KMnO solution is added to react, and the filtrate that removes _MnO by hot filtration is concentrated, acidified, and filtered to obtain part of 5-methylpyrazine-2 -carboxylic acid. Because this method uses a large amount of high-valent metal salts in the preparation process, it is easy to generate a large amount of waste water, which does not meet the environmental protection requirements of modern chemical industry.

There is an urgent need in the art to develop new methods for efficiently producing 5-methylpyrazine-2-carboxylic acid.

Contents of the invention

The object of the present invention is to provide an enzymatic method for producing 5-methylpyrazine-2-carboxylic acid.

The present invention first protects the application of a specific protein in the preparation of 5-methylpyrazine-2-carboxylic acid;

The specific protein is (a1) or (a2) as follows:

(a1) aldehyde dehydrogenase;

(a2) A fusion protein obtained by attaching a tag to the N-terminal or/and C-terminal of (a1).

The labels are shown in Table 1.

Table 1 Sequence of tags

The present invention also protects the application of specific proteins in the preparation of 5-methylpyrazine-2-carboxylic acid from 5-methyl-2-pyrazinaldehyde;

The specific protein is (a1) or (a2) as follows:

(a1) aldehyde dehydrogenase;

(a2) A fusion protein obtained by attaching a tag to the N-terminal or/and C-terminal of (a1).

Any one of the above aldehyde dehydrogenases is as follows (b1) or (b2):

(b1) a protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing;

(b2) Substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence of Sequence 2 and having the same function as a protein derived from Sequence 2.

The present invention also protects a preparation method of 5-methylpyrazine-2-carboxylic acid, comprising the following steps: using the specific protein to catalyze the conversion of 5-methyl-2-pyrazinaldehyde into 5-methylpyrazine-2-carboxylic acid 2-Carboxylic acid.

The catalytic reaction system is composed of buffer, substrate, specific protein and NAD+.

The buffer is 0.5M Tris-HCl (pH 8.0).

The substrate is 5-methyl-2-pyrazinaldehyde, and the concentration of 5-methyl-2-pyrazinaldehyde in the reaction system is 20 mM.

The concentration of the specific protein in the reaction system is 1000U/L.

The concentration of NAD+ in the reaction system is 20mM.

The catalytic reaction conditions are 30° C., 200 rpm for 12 hours.

The present invention also protects a preparation method of 5-methylpyrazine-2-carboxylic acid, comprising the following steps:

(1) Inducing expression of the recombinant bacteria, then collecting the cells and crushing the cells, and then collecting the supernatant; the recombinant bacteria are recombinant bacteria having a gene encoding the specific protein;

(2) purifying the specific protein from the supernatant obtained in step (1);

(3) Using the specific protein obtained in step (2) to catalyze the conversion of 5-methyl-2-pyrazinal into 5-methylpyrazine-2-carboxylic acid.

The "gene encoding the specific protein" is the DNA molecule described in any of the following (c1)-(c3):

(c1) the DNA molecule whose coding region is shown in sequence 1 in the sequence listing;

(c2) a DNA molecule that hybridizes to the DNA sequence defined in (c1) and encodes aldehyde dehydrogenase under stringent conditions;

(c3) A DNA molecule having more than 90% homology with the DNA sequence defined in (c1) or (c2) and encoding an aldehyde dehydrogenase.

The recombinant bacteria are obtained by introducing the recombinant plasmid into the starting strain; the recombinant plasmid is a double-stranded DNA shown in sequence 1 of the sequence table by replacing the small fragment between the NdeI and XhoI restriction sites of the pET21a (+) vector Molecularly obtained recombinant plasmid; the starting bacteria is Escherichia coli. The starting bacteria can specifically be Escherichia coli BL21(DE3).

In the step (1), the "inducing expression of the recombinant bacteria" specifically includes: culturing the recombinant bacteria in LB liquid medium containing ampicillin until the OD _600nm of the bacterial solution = 1-2; Induced by adding IPTG. In the LB liquid medium, the concentration of ampicillin is 50 μg/mL. The culture condition is 37° C., 220 rpm. The concentration of the IPTG is 30ppm. The induction conditions are 30° C., 220 rpm for 16 hours.

In the step (1), the "collect the bacteria and crush the bacteria, and then collect the supernatant" specifically: collect the bacteria, resuspend the recombinant bacteria with phosphate buffer, and then sonicate the bacteria. The supernatant was collected after the broken product was centrifuged. The phosphate buffer is 20mM (pH 7.5) phosphate buffer. The power of the ultrasound is 250W, and the ultrasound time is 20 minutes (5 seconds of ultrasound, 10 seconds off). The condition of the centrifugation is 4° C., 12000×g for 1 h.

In the step (2), nickel column (GE Healthcare Bio-science AB, article number: 17-5248-01) and desalting column (GE Healthcare Bio-science AB, article number: 17-1408-01) are used for purification to realize.

In the step (3), the catalytic reaction system is composed of a buffer, a substrate, a specific protein and NAD+.

The buffer is 0.5M Tris-HCl (pH 8.0).

The substrate is 5-methyl-2-pyrazinaldehyde, and the concentration of 5-methyl-2-pyrazinaldehyde in the reaction system is 20 mM.

The concentration of the specific protein in the reaction system is 1000U/L.

The concentration of NAD+ in the reaction system is 20mM.

The catalytic reaction conditions are 30° C., 200 rpm for 12 hours.

The present invention also protects a kit comprising any of the above specific proteins and 5-methyl-2-pyrazinaldehyde; the purpose of the kit is to prepare 5-methylpyrazine-2-carboxylic acid. 0.5M Tris-HCl (pH 8.0) was also included in the kit. NAD+ is also included in the kit.

The present invention also protects a kit comprising any of the above recombinant bacteria and 5-methyl-2-pyrazinaldehyde; the purpose of the kit is to prepare 5-methylpyrazine-2-carboxylic acid. 0.5M Tris-HCl (pH 8.0) was also included in the kit. NAD+ is also included in the kit.

For the first time, the present invention utilizes specific enzymes to efficiently realize the one-step production of 5-methylpyrazine-2-carboxylic acid. The process of the invention is simple, all raw materials and enzymes can be reacted in one step to prepare 5-methyl-2-pyrazinic acid, no intermediate steps and reactions are involved, the process flow is greatly simplified, and the catalytic oxidation reaction basically does not Inhibited by the product 5-methyl-2-pyrazinoic acid, high-concentration conversion can obtain high yield, which simplifies the extraction process and is beneficial to large-scale industrial production.

Description of drawings

Figure 1 is the plasmid map of pET21a-xylc.

Fig. 2 is the liquid phase chromatogram of 5-methyl-2-pyrazinecarboxylic acid standard product.

Figure 3 is a standard curve of 5-methyl-2-pyrazinecarboxylic acid.

Figure 4 is a carbon nuclear magnetic resonance spectrum (CNMR) figure.

Fig. 5 is a hydrogen nuclear magnetic resonance spectrum (HNMR) diagram.

Figure 6 is the structural formula of the product 5-methyl-2-pyrazinecarboxylic acid.

detailed description

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The xylc gene derived from Pseudomonas putida is shown in sequence 1 of the sequence listing. The aldehyde dehydrogenase protein derived from pseudomonas pseudomonas putida is shown in sequence 2 of the sequence listing. The gene shown in sequence 1 encodes the protein described in sequence 2.

5-Methyl-2-pyrazinal: Beijing Yinuokai Technology Co., Ltd., CAS: 50866-30-3.

pET21a(+) plasmid: Novagen, catalog number: 69740-3CN.

Escherichia coli BL21(DE3): Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: CB105-02.

Embodiment 1, highly expressed pseudomonas pseudomonas putida source xylc gene recombinant expression vector

The small fragment between the NdeI and XhoI restriction sites of the pET21a(+) plasmid was replaced by the double-stranded DNA molecule shown in Sequence 1 of the sequence listing to obtain the recombinant expression vector pET21a-xylc (see Figure 1 for the plasmid map).

Embodiment 2, the preparation of aldehyde dehydrogenase

1. Transform Escherichia coli BL21(DE3) with the recombinant expression vector pET21a-xylc prepared in Example 1 to obtain recombinant bacteria. The aldehyde dehydrogenase protein whose C-terminus is fused with the His ₆ tag can be obtained through the expression of the recombinant bacteria.

2. Inoculate the recombinant bacteria obtained in step 1 in LB liquid medium containing 50 μg/mL ampicillin, cultivate at 37°C and 220rpm until the OD _600nm of the bacterial solution =1-2, add 30ppm IPTG to the culture system, and then 220rpm induced 16h.

3. After step 2 is completed, the culture system is centrifuged at 8000rpm to collect bacterial precipitates.

4. After completing step 3, resuspend the cells in 20mM (pH 7.5) phosphate buffer solution, and then sonicate. The sonication power is 250W, and the sonication time is 20min (sonication for 5s, stop for 10s).

5. After completing step 4, centrifuge the sonicated product at 4° C. at 12,000×g for 1 hour and collect the supernatant.

6. Purify the supernatant obtained in step 5 with a nickel column (GE Healthcare Bio-science AB, catalog number: 17-5248-01), load the supernatant onto the nickel column, and wash with mobile phase A and mobile phase B in sequence Then, the eluate was desalted with a desalting column (GE Healthcare Bio-science AB, catalog number: 17-1408-01), and eluted with mobile phase C, and finally the purified protein solution.

Mobile phase A: 20mM (pH 7.5) phosphate buffer (containing 20mM imidazole and 0.5M NaCl); mobile phase B: 20mM (pH7.5) phosphate buffer (containing 200mM imidazole and 0.5M NaCl); mobile phase C: 20mM (pH 7.5) phosphate buffer.

7. Perform protein quantification on the purified protein solution obtained in step 6, and the protein concentration is 6.4 mg/ml.

8. Perform aldehyde dehydrogenase enzyme activity detection on the purified protein solution obtained in step 6; add 5-methyl-2-pyrazinaldehyde, NAD+ and 1 μL of the protein solution to be tested in the reaction system, and dilute the volume with H ₂ O to 1ml. The concentration of 5-methyl-2-pyrazinaldehyde in the reaction system was 10 mM, and the concentration of NAD+ in the reaction system was 5 mM.

Protein solution to be tested: Dilute the protein solution obtained in step 6 to a concentration of 1 mg/mL with 20 mM (pH 7.5) phosphate buffer.

Spectrophotometer (instrument model: UV-1800PC) was used to monitor on-line for 5 minutes, and the specific activity of the enzyme was calculated as 5 U/mg based on the extinction coefficient of NADH at 340 nm (6.22 cm ^-1 ·mmol ^-1 ).

Enzyme Specific Activity (U/mg)=(OD _340nm ×1000)/(6.22cm ^-1 ·mmol ^-1 ×1cm×5min×1mg·mL ^-1 ×0.001mL)

Embodiment 3, aldehyde dehydrogenase prepares 5-methyl-2-pyrazinecarboxylic acid

The reaction system (1ml) consists of buffer, substrate, aldehyde dehydrogenase and NAD+; buffer: 0.5M Tris-HCl (pH 8.0); substrate: 5-methyl-2-pyrazinaldehyde, 5- The concentration of methyl-2-pyrazinaldehyde in the reaction system is 20mM; aldehyde dehydrogenase: the aldehyde dehydrogenase protein solution obtained in step 6 of Example 2, the concentration of aldehyde dehydrogenase in the reaction system is 1000U/L ; The concentration of NAD+ in the reaction system was 20mM.

Reaction conditions: 30°C, 200rpm for 12h.

After the reaction was completed, the reaction system was diluted 10 times, and the product concentration was detected by HPLC.

HPLC chromatographic conditions: instrument model: Agilent 1200Series; chromatographic column: Nucleosil 100 C18, 4.6×250mm, 5μm; column temperature: 25℃; UV detection wavelength: 275nm; collection time: 10min; injection volume: 5μL; flow rate: 1.00mL /min; the mobile phase is composed of phase A and phase B, phase A is 0.1% (volume percentage) TFA aqueous solution, phase B is acetonitrile, and the volume ratio of phase A and phase B is 80:20.

5-methyl-2-pyrazinecarboxylic acid was used as a standard (Shanghai Baishun Biotechnology Co., Ltd., CAS: 5521-55-1).

The chromatogram of the standard product is shown in Figure 2. The peak time of the 5-methyl-2-pyrazinecarboxylic acid standard product is 3.776 minutes; under the same conditions, the peak position is within ±0.5 min, which can be identified as the same substance.

The standard curve of 5-methyl-2-pyrazinecarboxylic acid is shown in Figure 3.

The elution peak corresponding to 5-methyl-2-pyrazinecarboxylic acid was collected, and the product characterization was verified by NMR in one step.

The carbon nuclear magnetic resonance spectrum (CNMR) figure is shown in Figure 4. The hydrogen nuclear magnetic resonance (HNMR) spectrum is shown in FIG. 5 .

Through the above detection, the results show that the product is 5-methyl-2-pyrazinecarboxylic acid (structural formula as shown in Figure 6); the substrate consumption of each reaction system is 88% (about 17.60mM), and the product 5- The amount of methyl-2-pyrazinecarboxylic acid was 17.02 mM.

SEQUENCE LISTING

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> An enzymatic method for the production of 5-methylpyrazine-2-carboxylic acid

<160> 2

<210> 1

<211> 1461

<212>DNA

<213> Pseudomonas putida

<400> 1

atgcgggaaa caaaagagca gcctatctgg tacgggaagg tgtttagttc taattgggta 60

gaggcgcggg gaggtgttgc caatgttgtc gatccgtcca atggagacat tcttggcatt 120

acgggtgttg ctaacggcga agatgtcgat gctgctgtga acgcagctaa gagagcgcaa 180

aaggaatggg ccgcaatacc atttagtgaa agagccgcca ttgtccgcaa ggctgccgaa 240

aaactaaagg agcgcgagta tgagttcgcc gattggaacg tacgggaatg cggcgcaatt 300

cgtccgaagg gcttatggga ggccggaatt gcgtatgagc aaatgcatca agctgcgggt 360

ctagcttctt tgcctaacgg tacattgttt ccatcggcag ttccaggggcg catgaatctt 420

tgtcagcgcg ttccagttgg cgtggtcggc gtaattgcac cttggaattt cccgttgttt 480

ctagcaatgc gttcggtagc accagcctta gcgttgggta atgcggtgat cttaaagccc 540

gaccttcaga ctgctgtcac cgggggggcg ctcattgccg aaatcttttc cgacgctggc 600

atgccggacg gtgttcttca cgttcttcct ggtggagcgg acgtaggaga gtcaatggtt 660

gcgaactccg gaattaacat gatttctttt accgggtcca cacaggtggg ccggttgatc 720

ggagagaaat gcgggagaat gctgaaaaag gttgcgcttg aactgggtgg taataatgtc 780

cacatcgtgttgcctgacgc cgattagaa ggggctgtca gctgcgctgc ttggggtacg 840

tttttgcatc agggccaagt gtgcatggcc gccggacgtc atttagtaca tagggacgtt 900

gctcagcaat atgcagagaa actggcgcta cgtgccaaga acttagtggt gggggaccca 960

aactcggatc aagtgcatct cggcccgctt atcaatgaga aacaggtagt tcgcgtccac 1020

gcgctcgttg aatctgcgca aagggccggt gctcaggttt tggcgggagg tacgtatcaa 1080

gatcgctact accaagctac cgtaatcatg gatgtgaagc cggagatgga ggttttcaaa 1140

tctgaaattt tcggcccggt ggctccgatc actgtatttg acagtattga agaggcgatt 1200

gaattggcaa actgttcgga gtatgggttg gccgcatcta tccatactag ggcgttggcg 1260

actggtctag acatcgcaaa gcgtctaaat accggtatgg tccatattaa tgaccagcca 1320

attaactgtg agccgcatgt tcccttcgga ggaatgggtg cctcgggtag cggaggccgg 1380

tttggcggac ctgcaagtat tgaagaattt actcaatctc aatggattag tatggttgag 1440

aagccagcta attacccatt t 1461

<210> 2

<211> 487

<212> PRT

<213> Pseudomonas putida

<400> 2

Met Arg Glu Thr Lys Glu Gln Pro Ile Trp Tyr Gly Lys Val Phe Ser

1 5 10 15

Ser Asn Trp Val Glu Ala Arg Gly Gly Val Ala Asn Val Val Asp Pro

20 25 30

Ser Asn Gly Asp Ile Leu Gly Ile Thr Gly Val Ala Asn Gly Glu Asp

35 40 45

Val Asp Ala Ala Val Asn Ala Ala Lys Arg Ala Gln Lys Glu Trp Ala

50 55 60

Ala Ile Pro Phe Ser Glu Arg Ala Ala Ile Val Arg Lys Ala Ala Glu

65 70 75 80

Lys Leu Lys Glu Arg Glu Tyr Glu Phe Ala Asp Trp Asn Val Arg Glu

85 90 95

Cys Gly Ala Ile Arg Pro Lys Gly Leu Trp Glu Ala Gly Ile Ala Tyr

100 105 110

Glu Gln Met His Gln Ala Ala Gly Leu Ala Ser Leu Pro Asn Gly Thr

115 120 125

Leu Phe Pro Ser Ala Val Pro Gly Arg Met Asn Leu Cys Gln Arg Val

130 135 140

Pro Val Gly Val Val Gly Val Ile Ala Pro Trp Asn Phe Pro Leu Phe

145 150 155 160

Leu Ala Met Arg Ser Val Ala Pro Ala Leu Ala Leu Gly Asn Ala Val

165 170 175

Ile Leu Lys Pro Asp Leu Gln Thr Ala Val Thr Gly Gly Ala Leu Ile

180 185 190

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

195 200 205

Leu Pro Gly Gly Ala Asp Val Gly Glu Ser Met Val Ala Asn Ser Gly

210 215 220

Ile Asn Met Ile Ser Phe Thr Gly Ser Thr Gln Val Gly Arg Leu Ile

225 230 235 240

Gly Glu Lys Cys Gly Arg Met Leu Lys Lys Val Ala Leu Glu Leu Gly

245 250 255

Gly Asn Asn Val His Ile Val Leu Pro Asp Ala Asp Leu Glu Gly Ala

260 265 270

Val Ser Cys Ala Ala Trp Gly Thr Phe Leu His Gln Gly Gln Val Cys

275 280 285

Met Ala Ala Gly Arg His Leu Val His Arg Asp Val Ala Gln Gln Tyr

290 295 300

Ala Glu Lys Leu Ala Leu Arg Ala Lys Asn Leu Val Val Gly Asp Pro

305 310 315 320

Asn Ser Asp Gln Val His Leu Gly Pro Leu Ile Asn Glu Lys Gln Val

325 330 335

Val Arg Val His Ala Leu Val Glu Ser Ala Gln Arg Ala Gly Ala Gln

340 345 350

Val Leu Ala Gly Gly Thr Tyr Gln Asp Arg Tyr Tyr Gln Ala Thr Val

355 360 365

Ile Met Asp Val Lys Pro Glu Met Glu Val Phe Lys Ser Glu Ile Phe

370 375 380

Gly Pro Val Ala Pro Ile Thr Val Phe Asp Ser Ile Glu Glu Ala Ile

385 390 395 400

Glu Leu Ala Asn Cys Ser Glu Tyr Gly Leu Ala Ala Ser Ile His Thr

405 410 415

Arg Ala Leu Ala Thr Gly Leu Asp Ile Ala Lys Arg Leu Asn Thr Gly

420 425 430

Met Val His Ile Asn Asp Gln Pro Ile Asn Cys Glu Pro His Val Pro

435 440 445

Phe Gly Gly Met Gly Ala Ser Gly Ser Gly Gly Arg Phe Gly Gly Pro

450 455 460

Ala Ser Ile Glu Glu Phe Thr Gln Ser Gln Trp Ile Ser Met Val Glu

465 470 475 480

Lys Pro Ala Asn Tyr Pro Phe

485

Claims (10)

1. application of the specified protein in 5-Methylpyrazine-2-carboxylic acid is prepared；

The specified protein is following (a1) or (a2)：

(a1) aldehyde dehydrogenase；

(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a1).

2. application as claimed in claim 1, it is characterised in that：The aldehyde dehydrogenase is following (b1) or (b2)：

(b1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；

(b2) by the amino acid sequence of sequence 2 by the substitution and/or missing and/or addition of one or several amino acid residues and With with identical function as derived from sequence 2 protein.

3. specified protein is preparing the application in 5-Methylpyrazine-2-carboxylic acid using 5- methyl -2- pyrazines aldehyde as raw material；

The specified protein is following (a1) or (a2)：

(a1) aldehyde dehydrogenase；

(a2) fused protein obtained in N-terminal or/and C-terminal the connection label of (a1).

4. application as claimed in claim 3, it is characterised in that：The aldehyde dehydrogenase is following (b1) or (b2)：

(b1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；

(b2) by the amino acid sequence of sequence 2 by the substitution and/or missing and/or addition of one or several amino acid residues and With with identical function as derived from sequence 2 protein.

5. a kind of preparation method of 5-Methylpyrazine-2-carboxylic acid, comprises the following steps：Using the spy described in claim 1 or 2 Determine protein catalysis 5- methyl -2- pyrazine aldehyde and be converted into 5-Methylpyrazine-2-carboxylic acid.

6. a kind of preparation method of 5-Methylpyrazine-2-carboxylic acid, comprises the following steps：

(1) recombinant bacterium is subjected to induced expression, then collects thalline and carry out bacterial cell disruption, then collect supernatant；It is described heavy Group bacterium is the recombinant bacterium of the gene with the specified protein described in coding claim 1 or 2；

(2) specified protein is purified in the supernatant obtained from step (1)；

(3) the specified protein catalysis 5- methyl -2- pyrazine aldehyde obtained using step (2) is converted into 5-Methylpyrazine-2-carboxylic acid.

7. method as claimed in claim 6, it is characterised in that：" the specific protein described in coding claim 1 or 2 The gene of matter " is any described DNA molecular in following (c1)-(c3)：

(c1) DNA molecular of the code area as shown in sequence 1 in sequence table；

(c2) the DNA sequence dna hybridization limited under strict conditions with (c1) and the DNA molecular of coding power aldehyde dehydrogenase；

(c3) DNA sequence dna limited with (c1) or (c2) has the DNA molecular of more than 90% homology and encoding aldehyde dehydrogenase.

8. method as claimed in claims 6 or 7, it is characterised in that：The recombinant bacterium is that recombinant plasmid is imported into starting strain Obtain；The recombinant plasmid is to replace the small fragment between NdeI the and XhoI restriction enzyme sites of pET21a (+) carrier for sequence The recombinant plasmid that double chain DNA molecule shown in the sequence 1 of list is obtained；It is described go out bacterium germination be Escherichia coli.

9. a kind of kit, including specified protein and 5- methyl -2- pyrazine aldehyde；The purposes of the kit is preparation 5- methyl Pyrazine -2- carboxylic acids.

10. a kind of kit, including recombinant bacterium and 5- methyl -2- pyrazine aldehyde；The purposes of the kit is preparation 5- methyl pyrroles Piperazine -2- carboxylic acids.