CN107312806B - Enzymatic production of 5-methylpyrazine-2-carboxylic acid - Google Patents
Enzymatic production of 5-methylpyrazine-2-carboxylic acid Download PDFInfo
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- CN107312806B CN107312806B CN201710645354.5A CN201710645354A CN107312806B CN 107312806 B CN107312806 B CN 107312806B CN 201710645354 A CN201710645354 A CN 201710645354A CN 107312806 B CN107312806 B CN 107312806B
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
Abstract
The invention discloses an enzymatic method for producing 5-methylpyrazine-2-carboxylic acid. The invention provides application of a specific protein in preparation of 5-methylpyrazine-2-carboxylic acid; the specific protein is (a1) or (a2) as follows: (a1) an aldehyde dehydrogenase; (a2) and (a1) wherein a tag is attached to the N-terminus or/and the C-terminus of the protein. The invention realizes the one-step production of 5-methylpyrazine-2-carboxylic acid for the first time by using specific enzyme. The method has simple process, can prepare the 5-methyl-2-pyrazinoic acid by one-step reaction of all raw materials and enzyme in one reactor, does not relate to intermediate steps and reaction, greatly simplifies the process flow, basically does not suffer from the inhibition of the product 5-methyl-2-pyrazinoic acid by catalytic oxidation reaction, can obtain high yield by high-concentration conversion, simplifies the extraction process, and is beneficial to large-scale industrial production.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to an enzymatic method for producing 5-methylpyrazine-2-carboxylic acid.
Background
5-Methylpyrazine-2-carboxylic acid (5-Methylpyrazine-2-carboxylic acid) is an important drug intermediate, and is mainly used for synthesizing a second generation of sulfonylurea hypoglycemic agent Glipizide (Glipizide), a new generation of long-acting hypolipidemic agent Acipimox (Acipimox) and an effective drug (PAE) for treating tuberculosis.
The existing preparation methods of 5-methylpyrazine-2-carboxylic acid include chemical synthesis methods, electrochemical synthesis methods and biological synthesis methods, wherein the chemical synthesis methods are industrialized.
The existing chemical synthesis methods mostly adopt 2, 5-dimethyl pyrazine as a raw material, N-oxo-2, 5-dimethyl pyrazine is obtained by oxidation of hydrogen peroxide and then reacts with acetic anhydride to generate 2-acetoxymethyl-5-methyl pyrazine, then 2-hydroxymethyl-5-methyl pyrazine is obtained by alkaline hydrolysis, and finally a target product is obtained by oxidation of potassium permanganate. The method has low yield and low operational safety.
CN 1141299C used KMnO4A method for preparing 5-methylpyrazine-2-carboxylic acid by one-step oxidation discloses KMnO4A method for preparing 5-methylpyrazine-2-carboxylic acid by one-step oxidation. The method takes 2, 5-dimethyl pyrazine as raw material, and KMnO is added in the presence of inhibitor4Solution reaction, hot filtration to remove MnO2The filtrate is concentrated, acidified and filtered to obtain partial 5-methylpyrazine-2-carboxylic acid. The method uses a large amount of high-price metal salt in the preparation process, so that a large amount of wastewater is easily generated, and the method does not meet the environmental protection requirement of modern chemical engineering.
There is an urgent need in the art to develop a novel method for efficiently producing 5-methylpyrazine-2-carboxylic acid.
Disclosure of Invention
The invention aims to provide an enzymatic method for producing 5-methylpyrazine-2-carboxylic acid.
The invention firstly protects the application of specific protein in the preparation of 5-methylpyrazine-2-carboxylic acid;
the specific protein is (a1) or (a2) as follows:
(a1) an aldehyde dehydrogenase;
(a2) and (a1) wherein a tag is attached to the N-terminus or/and the C-terminus of the protein.
The labels are shown in table 1.
TABLE 1 sequences of tags
The invention also protects the application of the specific protein in the preparation of 5-methylpyrazine-2-carboxylic acid by taking 5-methyl-2-pyrazinaldehyde as a raw material;
the specific protein is (a1) or (a2) as follows:
(a1) an aldehyde dehydrogenase;
(a2) and (a1) wherein a tag is attached to the N-terminus or/and the C-terminus of the protein.
The aldehyde dehydrogenase described above is (b1) or (b2) as follows:
(b1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;
(b2) and (b) the amino acid sequence of the sequence 2 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is similar to the protein which has the same function and is derived from the sequence 2.
The invention also provides a preparation method of the 5-methylpyrazine-2-carboxylic acid, which comprises the following steps: the specific protein is adopted to catalyze the 5-methyl-2-pyrazinaldehyde to be converted into 5-methylpyrazine-2-carboxylic acid.
The catalytic reaction system consists of buffer solution, substrate, specific protein and NAD +.
The buffer was 0.5M Tris-HCl (pH 8.0).
The substrate is 5-methyl-2-pyrazinaldehyde, and the concentration of the 5-methyl-2-pyrazinaldehyde in the reaction system is 20 mM.
The concentration of the specific protein in the reaction system was 1000U/L.
The concentration of NAD + in the reaction system was 20 mM.
The catalytic reaction conditions are 30 ℃ and 200rpm for 12 hours.
The invention also provides a preparation method of the 5-methylpyrazine-2-carboxylic acid, which comprises the following steps:
(1) carrying out induction expression on the recombinant bacteria, then collecting the bacteria, carrying out bacteria crushing, and then collecting supernatant; the recombinant bacterium is a recombinant bacterium with a gene for coding the specific protein;
(2) purifying the specific protein from the supernatant obtained in step (1);
(3) and (3) catalyzing the 5-methyl-2-pyrazinaldehyde to be converted into 5-methylpyrazine-2-carboxylic acid by using the specific protein obtained in the step (2).
The "gene encoding the specific protein" is a DNA molecule as described in any one of (c1) to (c3) below:
(c1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;
(c2) a DNA molecule which hybridizes with the DNA sequence defined in (c1) under stringent conditions and encodes a furfural dehydrogenase;
(c3) and (c) a DNA molecule which has more than 90% homology with the DNA sequence defined in (c1) or (c2) and encodes aldehyde dehydrogenase.
The recombinant strain is obtained by introducing a recombinant plasmid into an original strain, wherein the recombinant plasmid is obtained by replacing a small fragment between NdeI and XhoI enzyme cutting sites of a pET21a (+) vector with a double-stranded DNA molecule shown as a sequence 1 in a sequence table, the outgoing strain is escherichia coli, and the outgoing strain can be specifically escherichia coli B L21 (DE 3).
In the step (1), the step of inducing and expressing the recombinant bacteria is to culture the recombinant bacteria in L B liquid culture medium containing ampicillin to bacterial liquid OD600nm1-2, adding IPTG to the culture system for induction, wherein the concentration of ampicillin in the L B liquid culture medium is 50 mu g/m L, the culture conditions are 37 ℃ and 220rpm, the concentration of IPTG is 30ppm, and the induction conditions are 30 ℃ and 220rpm for 16 h.
In the step (1), "collecting and crushing the thalli, and then collecting the supernatant" specifically includes collecting the thalli, resuspending the recombinant thalli with phosphate buffer solution, performing ultrasonic treatment, centrifuging the ultrasonically-crushed product, and then collecting the supernatant, wherein the phosphate buffer solution is 20mM (pH7.5) phosphate buffer solution, the ultrasonic power is 250W, the ultrasonic time is 20min (ultrasonic treatment is 5s, stopping 10s), and the centrifuging condition is 4 ℃, 12000 × g, and centrifuging for 1 h.
In the step (2), purification is carried out by using a nickel column (GE Healthcare Bio-science AB, cat # 17-5248-01) and a desalting column (GE Healthcare Bio-science AB, cat # 17-1408-01).
In the step (3), the catalytic reaction system is composed of a buffer solution, a substrate, a specific protein and NAD +.
The buffer was 0.5M Tris-HCl (pH 8.0).
The substrate is 5-methyl-2-pyrazinaldehyde, and the concentration of the 5-methyl-2-pyrazinaldehyde in the reaction system is 20 mM.
The concentration of the specific protein in the reaction system was 1000U/L.
The concentration of NAD + in the reaction system was 20 mM.
The catalytic reaction conditions are 30 ℃ and 200rpm for 12 hours.
The invention also provides a kit, which comprises any one of the specific proteins and 5-methyl-2-pyrazinaldehyde; the kit is used for preparing 5-methylpyrazine-2-carboxylic acid. The kit also comprises 0.5M Tris-HCl (pH 8.0). The kit also comprises NAD +.
The invention also provides a kit, which comprises any one of the recombinant bacteria and 5-methyl-2-pyrazinaldehyde; the kit is used for preparing 5-methylpyrazine-2-carboxylic acid. The kit also comprises 0.5M Tris-HCl (pH 8.0). The kit also comprises NAD +.
The invention realizes the one-step production of 5-methylpyrazine-2-carboxylic acid for the first time by using specific enzyme. The method has simple process, can prepare the 5-methyl-2-pyrazinoic acid by one-step reaction of all raw materials and enzyme in one reactor, does not relate to intermediate steps and reaction, greatly simplifies the process flow, basically does not suffer from the inhibition of the product 5-methyl-2-pyrazinoic acid by catalytic oxidation reaction, can obtain high yield by high-concentration conversion, simplifies the extraction process, and is beneficial to large-scale industrial production.
Drawings
FIG. 1 is a map of pET21a-xylc plasmid.
FIG. 2 is a liquid chromatogram of a 5-methyl-2-pyrazinecarboxylic acid standard.
FIG. 3 is a standard curve of 5-methyl-2-pyrazinecarboxylic acid.
FIG. 4 is a nuclear magnetic resonance carbon spectrum (CNMR) chart.
FIG. 5 is a Hydrogen Nuclear Magnetic Resonance (HNMR) chart.
FIG. 6 shows the structural formula of the product 5-methyl-2-pyrazine carboxylic acid.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The xylc gene derived from Pseudomonas putida is shown in sequence 1 of the sequence table. The aldehyde dehydrogenase protein derived from Pseudomonas putida is shown in sequence 2 of the sequence table. The gene shown in sequence 1 encodes the protein shown in sequence 2.
5-methyl-2-pyrazinal: beijing Yinoka science and technology, Inc., CAS: 50866-30-3.
pET21a (+) plasmid: novagen, catalog No.: 69740-3 CN.
Escherichia coli B L21 (DE3) manufactured by Tiangen Biochemical technology, Inc., having a cargo number of CB 105-02.
Example 1 recombinant expression vector for highly expressing Pseudomonas putida-derived xylc Gene
The double-stranded DNA molecule shown in the sequence 1 of the sequence table is used for replacing a small segment between NdeI and XhoI enzyme cutting sites of pET21a (+) plasmid to obtain a recombinant expression vector pET21a-xylc (a plasmid map is shown in figure 1).
Example 2 preparation of aldehyde dehydrogenase
1. The recombinant expression vector pET21a-xylc prepared in example 1 is transformed into Escherichia coli B L21 (DE3) to obtain recombinant bacteria, and His fused at the C terminal can be obtained through expression of the recombinant bacteria6A labeled aldehyde dehydrogenase protein.
2. The recombinant strain obtained in step 1 was inoculated into L B solution containing 50. mu.g/m L ampicillinCulturing in culture medium at 37 deg.C and 220rpm until bacterial liquid OD600nmTo the culture system, 30ppm of IPTG was added and induced at 30 ℃ and 220rpm for 16 hours, that is, 1-2.
3. After completion of step 2, the culture system was centrifuged at 8000rpm, and the pellet was collected.
4. After the step 3 is completed, the cells are resuspended in 20mM (pH7.5) phosphate buffer and then sonicated, wherein the sonication power is 250W and the sonication time is 20min (sonication for 5s, and cessation of 10 s).
5. After completion of step 4, the sonicated product was centrifuged at 12000 × g for 1h at 4 ℃ and the supernatant was collected.
6. Purifying the supernatant obtained in the step 5 by using a nickel column (GE Healthcare Bio-science AB, the product number is 17-5248-01), loading the supernatant into the nickel column, eluting by using a mobile phase A and a mobile phase B in sequence, and collecting the eluent of the mobile phase B; desalting the eluate with desalting column (GE Healthcare Bio-science AB, cat # 17-1408-01), and eluting with mobile phase C to obtain purified protein solution.
Mobile phase A: 20mM (pH7.5) 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 (pH7.5) phosphate buffer.
7. And (4) quantifying the protein of the purified protein solution obtained in the step (6), wherein the protein concentration is 6.4 mg/ml.
8. Performing aldehyde dehydrogenase enzyme activity detection on the purified protein solution obtained in the step 6, and adding 5-methyl-2-pyrazinaldehyde, NAD + and a protein solution to be detected, namely 1 mu L, H into a reaction system2O is added to the volume of 1 ml. The concentration of 5-methyl-2-pyrazinaldehyde in the reaction system was 10mM, and the concentration of NAD + in the reaction system was 5 mM.
Test protein solution the protein solution obtained in step 6 was diluted to a concentration of 1mg/m L with 20mM (pH7.5) phosphate buffer.
Monitoring with spectrophotometer (model: UV-1800PC type) for 5min, and adjusting extinction coefficient (6.22 cm) at 340nm according to NADH-1·mmol-1) The specific activity of the enzyme was calculated to be 5U/mg.
Ratio of enzymesVitality (U/mg) ═ OD340nm×1000)/(6.22cm-1·mmol-1×1cm×5min×1mg·mL-1×0.001mL)
Example 3 preparation of 5-methyl-2-pyrazinecarboxylic acid by aldehyde dehydrogenase
The reaction system (1ml) consisted of buffer, substrate, aldehyde dehydrogenase and NAD +, the buffer was 0.5M Tris-HCl (pH 8.0), the substrate was 5-methyl-2-pyrazinaldehyde, and the concentration of 5-methyl-2-pyrazinaldehyde in the reaction system was 20mM, the aldehyde dehydrogenase was the aldehyde dehydrogenase protein solution obtained in step 6 of example 2, and the concentration of aldehyde dehydrogenase in the reaction system was 1000U/L, and the concentration of NAD + in the reaction system was 20 mM.
Reaction conditions are as follows: the reaction was carried out at 30 ℃ and 200rpm for 12 hours.
After the reaction, the reaction system was diluted 10 times, and then the product concentration was measured with HP L C.
HP L C chromatographic conditions comprise an instrument model of Agilent 1200Series, a chromatographic column of Nucleosil 100C 18, 4.6 × 250mm and 5 mu m, a column temperature of 25 ℃, an ultraviolet detection wavelength of 275nm, a collection time of 10min, a sample injection amount of 5 mu L, a flow rate of 1.00m L/min, a mobile phase consisting of an A phase and a B phase, wherein the A phase is a 0.1 percent (volume percentage content) TFA aqueous solution, the B phase is acetonitrile, and the volume ratio of the A phase to the B phase is 80: 20.
5-methyl-2-pyrazinecarboxylic acid was used as a standard (Shunhun biotech, Inc., CAS: 5521-55-1).
The chromatogram of the standard is shown in FIG. 2, and the peak-off time of 5-methyl-2-pyrazine carboxylic acid standard is 3.776 min; under the same conditions, the peak positions are within +/-0.5 min, and the same substances can be identified.
The standard curve of 5-methyl-2-pyrazinecarboxylic acid is shown in FIG. 3.
And collecting an elution peak corresponding to the 5-methyl-2-pyrazine carboxylic acid, and verifying the product characterization through nuclear magnetism one-step.
The Carbon Nuclear Magnetic Resonance (CNMR) chart is shown in FIG. 4. The Hydrogen Nuclear Magnetic Resonance (HNMR) spectrum is shown in FIG. 5.
The detection result shows that the product is 5-methyl-2-pyrazine carboxylic acid (the structural formula is shown in figure 6); the substrate consumption per reaction system was 88% (about 17.60mM), giving an amount of the product 5-methyl-2-pyrazinecarboxylic acid of 17.02 mM.
SEQUENCE LISTING
<110> institute of microbiology of Chinese academy of sciences
<120> an enzymatic method for producing 5-methylpyrazine-2-carboxylic acid
<160>2
<210>1
<211>1461
<212>DNA
<213> Pseudomonas (pseudomonas putida)
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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 ttccagggcg 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
cacatcgtgt tgcctgacgc cgatttagaa 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
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aagccagcta attacccatt t 1461
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<213> Pseudomonas (pseudomonas putida)
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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 (7)
1. The application of specific protein in the preparation of 5-methylpyrazine-2-carboxylic acid by taking 5-methyl-2-pyrazinaldehyde as a raw material;
the specific protein is (a1) or (a2) as follows:
(a1) an aldehyde dehydrogenase;
the amino acid sequence of the aldehyde dehydrogenase is shown as SEQ ID NO: 2 is shown in the specification;
(a2) his attached to the C-terminus of (a1)6And labeling the resulting fusion protein.
2. A preparation method of 5-methylpyrazine-2-carboxylic acid comprises the following steps: the use of a specific protein as claimed in claim 1 for catalyzing the conversion of 5-methyl-2-pyrazinal to 5-methylpyrazine-2-carboxylic acid.
3. A preparation method of 5-methylpyrazine-2-carboxylic acid comprises the following steps:
(1) carrying out induction expression on the recombinant bacteria, then collecting the bacteria, carrying out bacteria crushing, and then collecting supernatant; the recombinant bacterium is a recombinant bacterium having a gene encoding the specific protein according to claim 1;
(2) purifying the specific protein from the supernatant obtained in step (1);
(3) and (3) catalyzing the 5-methyl-2-pyrazinaldehyde to be converted into 5-methylpyrazine-2-carboxylic acid by using the specific protein obtained in the step (2).
4. The method of claim 3, wherein: the "gene encoding the specific protein according to claim 1" is a gene represented by SEQ ID NO: 1.
5. The method of claim 3 or 4, wherein: the recombinant strain is obtained by introducing a recombinant plasmid into an original strain; the recombinant plasmid is obtained by replacing a small fragment between NdeI and XhoI enzyme cutting sites of pET21a (+) vector by a fragment shown in SEQ ID NO: 1, a recombinant plasmid obtained from the double-stranded DNA molecule shown in the figure; the outbreak bacteria is escherichia coli.
6. A kit comprising a specific protein as described in claim 1 and 5-methyl-2-pyrazinal; the kit is used for preparing 5-methylpyrazine-2-carboxylic acid.
7. A kit comprising the recombinant bacterium according to any one of claims 3 to 5 and 5-methyl-2-pyrazinaldehyde; the kit is used for preparing 5-methylpyrazine-2-carboxylic acid.
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| CN107974428A (en) * | 2017-12-13 | 2018-05-01 | 迪沙药业集团有限公司 | A kind of recombination bacillus coli and the method for converting production 5-Methylpyrazine-2-carboxylic acid |
| CN112094879B (en) * | 2019-06-17 | 2022-05-06 | 中国科学院微生物研究所 | Enzyme-catalyzed C-terminal selective amidation and hydrazidation modification method for polypeptide |
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| CN105647844A (en) * | 2016-03-02 | 2016-06-08 | 中国科学院青岛生物能源与过程研究所 | Recombinant bacteria using xylose to produce glycollic acid and building method and application of recombinant bacteria |
| CN106434434A (en) * | 2016-09-12 | 2017-02-22 | 迪沙药业集团有限公司 | Strain Arthrobacter woluwensis for producing xylene monooxygenase and application thereof |
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| US7723498B2 (en) * | 2004-06-04 | 2010-05-25 | University Of Connecticut | Directed evolution of recombinant monooxygenase nucleic acids and related polypeptides and methods of use |
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