CN101481713B - Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof - Google Patents
Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof Download PDFInfo
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- IPEHBUMCGVEMRF-UHFFFAOYSA-N pyrazinecarboxamide Chemical compound NC(=O)C1=CN=CC=N1 IPEHBUMCGVEMRF-UHFFFAOYSA-N 0.000 title claims abstract description 54
- PMSVVUSIPKHUMT-UHFFFAOYSA-N cyanopyrazine Chemical compound N#CC1=CN=CC=N1 PMSVVUSIPKHUMT-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 238000006555 catalytic reaction Methods 0.000 title claims description 12
- 230000001580 bacterial effect Effects 0.000 title description 39
- 238000004519 manufacturing process Methods 0.000 title description 9
- 241000607715 Serratia marcescens Species 0.000 claims abstract description 35
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 17
- 230000003197 catalytic effect Effects 0.000 claims abstract description 5
- 238000006703 hydration reaction Methods 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 19
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 16
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention provides a method of producing 2-cyanopyrazine by biocatalysis to produce pyrazinamide and a new strain, Serratia marcescens ZJB-09104, which is used in the preparation process. In the method, the pyrazinamide is obtained through the catalytic hydration reaction under the conditions that the pH is 6.0-8.0 and the temperature is 20-40 DEG C in a reaction system which uses 2-cyanopyrazine as a substrate and nitrile hydratase which is obtained by the culturing of the Serratia marcescens as a catalyst. The method applies the Serratia marcescens ZJB-09104 to the experiments producing pyrazinamide by biocatalysis. Results show that the strain has a wide application prospect in the field of producing pyrazinamide by biocatalysis as the strain can effectively produce the pyrazinamide by biocatalysis and the transformation ratio of the 2-cyanopyrazine.attains 83.8 percent within 12h.
Description
(1) technical field
The present invention relates to a kind of method of producing aldinamide by biological catalysis of 2-cyano pyrazine, and the new bacterial strain of a used strain in the preparation process---serratia marcescens (Serratia marcescens) ZJB-09104.
(2) background technology
Pyrazinoic acid amide is an a kind of important line antitubercular agent, is mainly used in chronic pulmonary tuberculosis, heating, cough, many phlegm etc.Tubercule bacillus has restraining effect in its pair cell, can advance in people's cell and see through hemato encephalic barrier to advance human brain tissue and kill tubercule bacillus; The slow metabolic semidormancy bacterium of scavenger cell is had unique sterilization functions, is one of main medicine of tuberculosis SCC.Because pyrazinoic acid amide has some advantages that are different from other anti-mycobacterium tuberculosis medicine, therefore in recent years the research of pyrazinoic acid amide is become focus gradually.
Pyrazinoic acid amide is except well-known with antitubercular agent, and it also is the important intermediate of other pharmaceutical prod.Its derivative 2-pyrazinoic acid amide-4-oxide compound is a kind of Claritin.Pyrazinoic acid amide can form the amorphous state complex compound with metal ion, can be applicable to analytical chemistry field or ultimate analysis, hot analysis, infrared spectra and magnetic field measuring etc.Pyrazinoic acid amide also can be used for the sensitive materials aspect, but the material processing imaging that contains silver halide photosensitivity emulsion layer and non-photosensitivity hydrophilic colloid layer and pyrazinoic acid amide class nucleophilic reagent, this material has very high photosensitivity and good storage stability.Simultaneously, the polypyrazine acid amides also is a kind of highly transparent, has good operation performance, and the water-soluble material of energy impedance.It has good price and performance potential, and it can also provide good bond properties and gloss characteristic, and has the good metal characteristic.China once adopted pyrazine-2, and the 3-dicarboxylic acid is the raw material production pyrazinoic acid amide, because factors such as quality product, cost begin to use instead 2 cyano pyrazine and make raw material the nineties in 20th century.Because China's 2 cyano pyrazine synthetic technology is more backward, up to 20 the end of the century 2 cyano pyrazine rely on import to solve always.Since the domestic still incompetent producer that stablizes ordinary production, and the import supply of material does not have guarantee, and internal price and world market valency are synchronous, need the particularly thorny production that seriously restricts China's antitubercular agent pyrazinoic acid amide because of producing.
In the later stage eighties 20th century, the application of biocatalysis in organic synthesis is subjected to chemist's favor day by day.Nitrile hydratase is a kind of protein that certain micro-organisms produces in carrying out oxime or urea metabolic process, utilize its can with the itrile group of vinyl cyanide (AN) (CN) catalytic hydration be acid amides (CONH2).1980, the people such as Yamada of Japan find the deleterious acetonitrile of microorganism Rhodocococcus sp.N2774 Nitrile hydratase degradable first, soon this bacterial classification successfully is applied to the industrial production acrylamide, and this technology has realized industrialization in China, Japan, Russia.And utilize Nitrile hydratase to produce pyrazinoic acid amide, do not see the industrialization report both at home and abroad as yet.
The present invention is intended to screening and obtains having the active bacterial strain of 2 cyano pyrazine hydratase, is leading indicator with the pyrazinoic acid amide productive rate, sets up a new pyrazinoic acid amide biosynthesis process.Biological process is produced pyrazinoic acid amide, and for the cleaner production pyrazinoic acid amide provides powerful technical support, the technology of utilizing microorganism Nitrile hydratase cleaner production pyrazinoic acid amide that exploitation has independent intellectual property right is imperative.
(3) summary of the invention
The object of the invention provides a kind of method of producing aldinamide by biological catalysis of 2-cyano pyrazine, and the new bacterial strain of a used strain in the preparation process---serratia marcescens (Serratiamarcescens) ZJB-09104.
The technical solution used in the present invention is:
A kind of method of producing aldinamide by biological catalysis of 2-cyano pyrazine, described method comprises: at the Nitrile hydratase that is substrate with the 2 cyano pyrazine, obtains with serratia marcescens (Serratia marcescens) cultivation is in the reaction system of catalyzer, under 6.0~8.0,20~40 ℃ of pH, carry out the catalytic hydration reaction, make described pyrazinoic acid amide.Described Nitrile hydratase consumption also can be excessive so that 2 cyano pyrazine fully reacts is advisable.
Preferably, described serratia marcescens is serratia marcescens (Serratia marcescens) ZJB-09104, is preserved in Chinese typical culture collection center, the address: Chinese Wuhan Wuhan University 430072, preservation date on November 18th, 2008, deposit number CCTCC No.M 208231.
Preferably, in the described reaction system, described substrate 2 cyano pyrazine starting point concentration is 10~50g/L.
Described Nitrile hydratase contains the enzyme cell from what serratia marcescens cultivate to obtain, contains the enzyme final concentration of cells in the described reaction system and counts 1~50g/L to contain the enzyme wet cell weight.
The described enzyme cell that contains is by can following method preparing: serratia marcescens is seeded in the fermention medium that is applicable to serratia marcescens, concussion is cultured to the thalline logarithmic phase under 20 ℃~40 ℃, initial pH 4.0~8.0 conditions, centrifugation obtains the described enzyme cell that contains.
The described fermention medium that is applicable to serratia marcescens is applicable to the fermention medium of serratia marcescens for this area routine, among the present invention, and described fermention medium (g/L) composed as follows: glucose 5~50, yeast extract paste 1~20, urea 1~20, monosodium glutamate 0.1~5, K
2HPO
40.1~20, KH
2PO
40.1~5, MgSO
40.1~20, CoCl
2Or FeSO
40.05~1, solvent is a water, pH value 4.0~8.0.
Concrete, described method is as follows:
(1) serratia marcescens ZJB-09104 is seeded to seed culture medium, cultivates 18~24 hours, and obtains seed liquor for 28~32 ℃; Described seed culture medium (g/L) composed as follows: sucrose 5~80, yeast powder 1~20, NaCl 0.1~5, K
2HPO
40.1~1, MgSO
40.1~1, solvent is a water, pH value 4.0~8.0;
(2) step (1) seed liquor is seeded to fermention medium with 1~10% volume ratio, and concussion is cultured to the thalline logarithmic phase under 20 ℃~40 ℃, initial pH 4.0~8.0 conditions, and centrifugation obtains thalline and contains the enzyme cell; Described fermention medium (g/L) composed as follows: glucose 5~50, yeast extract paste 1~20, urea 1~20, monosodium glutamate 0.1~5, K
2HPO
40.1~20, KH
2PO
40.1~5, MgSO
40.1~20, CoCl
2Or FeSO
40.05~1, solvent is a water, pH value 6.0~7.0;
(3) step (2) thalline contains the enzyme cell and is added in the reaction system that contains 2 cyano pyrazine, the solvent of transformation system is selected pure water or phosphate buffered saline buffer, to contain the enzyme cell be 1~20g/L to contain enzyme wet cell weight concentration to thalline in the described reaction system, 2 cyano pyrazine concentration is 10~30g/L, under 6.0~8.0,20~40 ℃ of pH, carry out catalytic hydration reaction 20~30h, make described pyrazinoic acid amide.
The mensuration of pyrazinoic acid amide concentration in the reaction solution: adopt the pyrazinoic acid amide Determination on content in the reaction solution that efficient liquid-phase chromatography method obtains step (4): the 1mL conversion fluid, centrifugal, supernatant liquor carries out gas chromatographic analysis: the GC-14C gas chromatograph, the stainless steel packed column, filler is Porapak Q, long 2m, internal diameter 4mm, 220 ℃ of column temperatures, 160 ℃ of sample introduction temperature, 220 ℃ of detected temperatures, carrier gas is a nitrogen, flow velocity 30ml/min, sample size are 0.8 μ l.Wherein, the pyrazinoic acid amide appearance time is 4.2min-5.2min, determines the content of pyrazinoic acid amide in the sample.
The invention still further relates to the new bacterial strain of a strain that screens---serratia marcescens (Serratiamarcescens) ZJB-09104, be preserved in Chinese typical culture collection center, address: China. Wuhan. Wuhan University 430072, preservation date on November 18th, 2008, deposit number CCTCCNo.M 208231.
This bacterial strain is obtained by following method screening:
From near 10 parts of the soil samplings pharmaceutical factory, Hangzhou, Zhejiang province city, got back rapid strain screening within 12h, the concrete grammar of screening is: sample behind the enrichment culture 24h, is coated on the screening culture medium flat board in enrichment medium again; Through plate screening, but be divided into from the bacterial strain that obtains 5 strain producing aldinamide by biological catalysis of 2-cyano pyrazine; Through further sieving the final aimed strain ZJB-09104 that gets again.
The bacterial strain ZJB-09104 that the present invention's screening obtains, Physiology and biochemistry by " common bacteria system identification handbook " and " uncle's Jie Shi Bacteria Identification handbook (the 8th edition) " is identified, the form of bacterial strain is and is approximately the spheric rod-short, wide 1.15 μ m~1.26 μ m, long 1.15 μ m~1.73 μ m, thalline motion, Gram-negative, whole body flaggellation, cultivate 24h for 30 ℃ on the LB flat board, bacterium colony is rounded, neat in edge, projection slightly, white, opaque, matt, easy picking, in 30 ℃ of constant temperature culture 24h, the about 1~2mm of single colony diameter;
16S rDNA sequential analysis: with the total DNA of the cell that extracts is template, utilize primer: the 16S rDNA gene of p16S-8:5 '-aga gtt tga tcc tgg ctc ag-3 ' and p16S-1541:5 '-aag gag gtg atc cagccg ca-3 ' amplification bacterial strain, gene product is connected with the T carrier, confirm that through order-checking this fragment physical length is 1534bp, (submitted this sequence to GenBank, the GenBank registration number is FJ479790) with GenBank in related data carry out similarity analysis and find, the highest (the homology of homology of this bacterium and serratia marcescens (Serratia marcescens), 99%/1543bps, based on 16S rDNA).
Identify according to above microbial characteristic, this new bacterial strain ZJB-09104 belongs to serratia marcescens (Serratia marcescens), this strain microorganism does not belong to any of now own openly bacterial classification, this strain microorganism has the ability of producing aldinamide by biological catalysis of 2-cyano pyrazine, can be used for the production of pyrazinoic acid amide.
Beneficial effect of the present invention is mainly reflected in: (1) but, the present invention screens from micropopulation and obtains the serratia marcescens (Serratia marcescens) that biocatalysis is produced pyrazinoic acid amide: CCTCC No.M 208231., it can effectively produce pyrazinoic acid amide under the condition of being fit to; (2), the present invention is used for the experiment of biocatalysis production pyrazinoic acid amide with serratia marcescens bacterial strain ZJB-09104, the result shows, this bacterium effectively biocatalysis produces pyrazinoic acid amide, the 2 cyano pyrazine transformation efficiency reaches 83.8% (seeing embodiment 3,4) in the 12h, so this bacterium is with a wide range of applications in the field of biocatalysis production pyrazinoic acid amide.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the screening of new bacterial strain ZJB-09104
The present invention obtains 10 parts of soil samples near the pharmaceutical factory, Hangzhou, Zhejiang province city, strain screening therefrom rapidly within 12h; The concrete grammar of screening is: earlier sample behind the enrichment culture 24h, is coated on the screening culture medium flat board that contains 2 cyano pyrazine in enrichment medium again; Through plate screening, but be divided into from the bacterial strain that obtains 5 strain producing aldinamide by biological catalysis of 2-cyano pyrazine; This 5 strain bacterial strain is first respectively by behind the 5% inoculum size input fermention medium cultivation 24h, centrifugal acquisition thalline.The 0.1g wet thallus is added in the reaction solution of 2 cyano pyrazine that final concentration is 10g/L, react after 24 hours, measure the content of pyrazinoic acid amide respectively, therefrom filter out and produce the strongest bacterial strain ZJB-09104 of pyrazinoic acid amide ability, as the starting strain of further research.
Described enrichment culture based formulas is: sucrose 2g, yeast powder 10g, NaCl 2g, K
2HPO
40.5g, MgSO
40.5g, complement to 1000mL with tap water, pH value 4.0~8.0.
Described screening culture medium is: sucrose 20g, yeast powder 10g, NaCl 2g, K
2HPO
40.5g, MgSO
40.5g, 2 cyano pyrazine 10g, agar powder 20g complements to 1000mL with tap water, pH value 7.0.
Described fermentative medium formula is: glucose 30g, yeast extract paste 20g, urea 10g, monosodium glutamate 3g, K
2HPO
40.5g, KH
2PO
43g, MgSO
45g, CoCl
20.5g, complement to 1000mL with tap water, pH value 7.0.
Embodiment 2: the evaluation of new bacterial strain ZJB-09104
The bacterial strain ZJB-09104 that 1 screening obtains to embodiment is by " uncle's Jie Shi Bacteria Identification handbook (the 8th edition) ", and " common bacteria system identification handbook " carries out physio-biochemical characteristics evaluations (seeing Table 1):
This bacterial strain bacterium colony is rounded, neat in edge, and projection slightly, white, opaque, matt, easy picking, in 30 ℃ of constant temperature culture 24h, the about 1~2mm of single colony diameter, thalline is rod-short;
On the basis of the above, further bacterial strain ZJB-09104 is pressed fine works molecular biology experiment guide method and extract chromosomal DNA, with the total DNA of the cell that extracts is template, utilize primer: the 16S rDNA gene of p16S-8:5 '-aga gtt tga tcc tgg ctc ag-3 ' and p16S-1541:5 '-aag gag gtg atc cag ccg ca-3 ' amplification bacterial strain, after gene product connected with the T carrier, entrust Dalian precious biotechnology company to this bacterium 16S rDNA amplification and order-checking, after obtaining the 16S rDNA sequence of this bacterial strain, on the NCBI website, retrieve the 16S rDNA gene order of relevant bacterial strain among the GenBank, and carry out the homology comparison with BLAST; Evaluation based on form, physiological and biochemical property and aspects such as 16S rDNA sequence and phylogenetic systematics analysis, ZJB-09104 is accredited as serratia marcescens with bacterial strain, intend called after serratia marcescens (Serratia marcescens) ZJB-09104, this bacterial strain is deposited in Chinese typical culture collection center on November 17th, 2008, and deposit number is CCTCC No.M208231.。
Table 1: the evaluation contrast of bacterial strain ZJB-09104 and serratia part member physio-biochemical characteristics
Physiological and biochemical property | Serratia marcescens | ZJB-09104 |
Gramstaining | - | - |
Catalase | + | + |
Oxydase | - | - |
4 ℃ of growths | + | + |
41 ℃ of growths | + | + |
Arginine dihydrolase | - | - |
Methyl red | - | - |
Phenol red | - | + |
V-P | + | + |
Gelatine liquefication | + | + |
Utilize: | ||
Starch | - | - |
Glucose | + | + |
D-N.F,USP MANNITOL | + | + |
The D-wood sugar | - | - |
L-arabinose | - | - |
The D-seminose | + | + |
D-sucrose | + | + |
The D-trehalose | + | + |
The D-cellobiose | - | - |
The D-raffinose | - | - |
The D-melibiose | - | - |
Lactose | - | - |
Inositol | + | + |
Propanedioic acid | - | - |
The KCN growth | + | + |
H 2S | - | - |
Annotate: positive more than+90%, negative more than-90%.
The 16S rDNA sequence of bacterial strain ZJB-09104 CCTCC No.M 208231 is as follows:
AAGGAGGTGA TCCAGCCGCA GGTTCCCCTA CGGTTACCTT GTTACGACTT CACCCCAGTCATGAATCACA AAGTGGTAAG CGCCCTCCCG AAGGTTAAGC TACCTACTTC TTTTGCAACCCACTCCCATG GTGTGACGGG CGGTGTGTAC AAGGCCCGGG AACGTATTCA CCGTAGCATTCTGATCTACG ATTACTAGCG ATTCCGACTT CATGGAGTCG AGTTGCAGAC TCCAATCCGGACTACGACGT ACTTTATGAG GTCCGCTTGC TCTCGCGAGG TCGCTTCTCT TTGTATACGCCATTGTAGCA CGTGTGTAGC CCTGCTCGTA AGGGCCATGA TGACTTGACG TCATCCCCACCTTCCTCCAG TTTATCACTG GCAGTCTCCT TTGAGTTCCC GGCCGAACCG CTGGCAACAAAGGATAAGGG TTGCGCTCGT TGCGGGACTT AACCCAACAT TTCACAACAC GAGCTGACGACAGCCATGCA GCACCTGTCT CAGAGTTCCC GAAGGCACCA ATCCATCTCT GGAAAGTTCTCTGGATGTCA AGAGTAGGTA AGGTTCTTCG CGTTGCATCG AATTAAACCA CATGCTCCACCGCTTGTGCG GGCCCCCGTC AATTCATTTG AGTTTTAACC TTGCGGCCGT ACTCCCCAGGCGGTCGATTT AACGCGTTAG CTCCGGAAGC CACGCCTCAA GGGCACAACC TCCAAATCGACATCGTTTAC AGCGTGGACT ACCAGGGTAT CTAATCCTGT TTGCTCCCCA CGCTTTCGCACCTGAGCGTC AGTCTTCGTC CAGGGGGCCG CCTTCGCCAC CGGTATTCCT CCAGATCTCTACGCATTTCA CCGCTACACC TGGAATTCTA CCCCCCTCTA CGAGACTCTA GCTTGCCAGTTTCAAATGCA GTTCCCAGGT TGAGCCCGGG GATTTCACAT CTGACTTAAC AAACCGCCTGCGTGCGCTTT ACGCCCAGTA ATTCCGATTA ACGCTTGCAC CCTCCGTATT ACCGCGGCTGCTGGCACGGA GTTAGCCGGT GCTTCTTCTG CGAGTAACGT CAATTGATGA ACGTATTAAGCTCACCACCT TCCTCCTCGC TGAAAGTGCT TTACAACCCG AAGGCCTTCT TCACACACGCGGCATGGCTG CATCAGACTT GCGCCCATTG TGCAATATTC CCCACTGCTG CCTCCCGTAGGAGTCTGGAC CGTGTCTCAG TTCCAGTGTG GCTGGTCATC CTCTCAGACC AGCTAGGGATCGTCGCCTAG GTGAGCCATT ACCCCACCTA CTAGCTAATC CCATCTGGGC ACATCTGATGGCAAGAGGCC CGAAGGTCCC CCTCTTTGGT CTTGCGACGT TATGCGGTAT TAGCTACCGT TTCCAGTAGT TATCCCCCTC CATCAGGCAG TTTCCCAGAC ATTACTCACC CGTCCGCCGCTCGTCACCCA GGGAGCAAGC TCCCCTGTGC TACCGCTCGA CTTGCATGTG TTAAGCCTGCCGCCAGCGTT CAATCTGAGC CAGGATCAAA CTCT。
Embodiment 3: utilize bacterial strain ZJB-09104 biocatalysis to produce pyrazinoic acid amide and carry out according to the following steps:
(1) preparation of slant medium: sucrose 10g, yeast powder 10g, NaCl 5g, K
2HPO
40.1g, MgSO
40.3g agar powder 20g complements to 1000mL with tap water, pH value 6; The preparation of seed culture medium (g/L): sucrose 10, yeast powder 5, NaCl 1, K
2HPO
40.5, MgSO
40.1 solvent is a water, pH value 4.0~8.0.
(2) activation culture of bacterial classification: adopt step (1) slant medium, with the new bacterial strain ZJB-09104CCTCC No.M 208231 of serratia marcescens (Serratia marcescens), after 30 ℃ of activation, cultivating 18h, standby;
(3) preparation of seed liquor: microorganism 2 articulatings after step (2) activation are gone into described in (1) in the seed culture medium, and 30 ℃ of culture condition, 200r/min cultivates 24h down.
(4) fermentation culture of bacterial classification: step (3) strain liquid is inserted in the fermention medium by 5% volume ratio inoculum size,, under 200r/min, cultivate 36h,, carry out centrifugation, obtain thalline to thalline logarithmic growth after date 30 ℃ of culture condition; Fermentative medium formula is: glucose 20g, yeast extract paste 10g, urea 15g, monosodium glutamate 1g, K
2HPO
40.2g, KH
2PO
42g, MgSO
43g, CoCl
20.2g, complement to 1000mL with tap water, pH value 7.0.
(5) producing aldinamide by biological catalysis of 2-cyano pyrazine: the wet thallus 0.1g that step (4) is obtained joins 10ml and contains in 0.1M phosphate buffered saline buffer (pH8.0) reaction system of 0.2g 2 cyano pyrazine, and 30 ℃, 150r/min reacts 24h;
(6) pyrazinoic acid amide Determination on content in the conversion fluid: the 1mL conversion fluid, centrifugal, supernatant liquor carries out gas chromatographic analysis: the GC-14C gas chromatograph, the stainless steel packed column, filler is Porapak Q, long 2m, internal diameter 4mm, 220 ℃ of column temperatures, 160 ℃ of sample introduction temperature, 220 ℃ of detected temperatures, carrier gas is a nitrogen, flow velocity 30mL/min, sample size are 0.8 μ L.Wherein, the pyrazinoic acid amide appearance time is 4.2min~5.2min, and the content of determining pyrazinoic acid amide in the sample is 16.7g/L, and transformation efficiency is 71.4%.
Embodiment 4: utilize bacterial strain ZJB-09104 biocatalysis to produce pyrazinoic acid amide
(1) preparation of slant medium: the component of this substratum and the weight percent content of each component are: sucrose 1.5%, and yeast powder 0.5%, NaCl 0.5%, K
2HPO
40.01%, MgSO
40.03%, agar powder 2% complements to 100% with tap water, pH value 6;
(2) activation culture of bacterial classification: adopt step (1) substratum, after new 208231, the 30 ℃ of activation of bacterial strain ZJB-09104CCTCC No.M of serratia marcescens (Serratiamarcescens), cultivating 18h, standby;
(3) preparation of seed liquor: microorganism 2 articulatings after step (2) activation are gone into described in (1) in the seed culture medium,, under 200r/min, cultivate 24h 30 ℃ of culture condition.
(4) fermentation culture of bacterial classification: step (3) strain liquid is inserted in the fermention medium by 5% volume ratio inoculum size,, under 200r/min, cultivate 36h,, carry out centrifugation, obtain thalline to thalline logarithmic growth after date 28 ℃ of culture condition; Fermentative medium formula is (g/L): glucose 15, yeast extract paste 20, urea 10, monosodium glutamate 3, K
2HPO
40.5, KH
2PO41, MgSO45, FeSO
40.5, complement to 1000mL with tap water, pH value 7.0.
(5) producing aldinamide by biological catalysis of 2-cyano pyrazine: the wet thallus 0.1g that step (4) is obtained joins 10mL and contains in 0.1M phosphate buffered saline buffer (pH 8.0) reaction system of 0.2g 2 cyano pyrazine, and 30 ℃, 150r/min reacts 24h;
(6) pyrazinoic acid amide Determination on content in the conversion fluid: with embodiment 3.Pyrazinoic acid amide content in the conversion fluid is 19.6g/L after measured, and transformation efficiency is 83.8%.
Sequence table _ ST25.txt
SEQUENCE LISTING
<110〉Zhejiang Polytechnical University
<120〉method of producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof
<130>
<160>3
<170>PatentIn version 3.4
<210>1
<211>20
<212>DNA
<213>Unknown
<220>
<223〉artificial sequence
<400>1
agagtttgat cctggctcag 20
<210>2
<211>20
<212>DNA
<213>Unknown
<220>
<223〉artificial sequence
<400>2
aaggaggtga tccagccgca 20
<210>3
<211>1534
<212>DNA
<213>Serratia marcescens
<400>3
aaggaggtga tccagccgca ggttccccta cggttacctt gttacgactt caccccagtc 60
atgaatcaca aagtggtaag cgccctcccg aaggttaagc tacctacttc ttttgcaacc 120
cactcccatg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgtagcatt 180
ctgatctacg attactagcg attccgactt catggagtcg agttgcagac tccaatccgg 240
actacgacgt actttatgag gtccgcttgc tctcgcgagg tcgcttctct ttgtatacgc 300
cattgtagca cgtgtgtagc cctgctcgta agggccatga tgacttgacg tcatccccac 360
cttcctccag tttatcactg gcagtctcct ttgagttccc ggccgaaccg ctggcaacaa 420
aggataaggg ttgcgctcgt tgcgggactt aacccaacat ttcacaacac gagctgacga 480
cagccatgca gcacctgtct cagagttccc gaaggcacca atccatctct ggaaagttct 540
ctggatgtca agagtaggta aggttcttcg cgttgcatcg aattaaacca catgctccac 600
cgcttgtgcg ggcccccgtc aattcatttg agttttaacc ttgcggccgt actccccagg 660
cggtcgattt aacgcgttag ctccggaagc cacgcctcaa gggcacaacc tccaaatcga 720
catcgtttac agcgtggact accagggtat ctaatcctgt ttgctcccca cgctttcgca 780
cctgagcgtc agtcttcgtc cagggggccg ccttcgccac cggtattcct ccagatctct 840
acgcatttca ccgctacacc tggaattcta cccccctcta cgagactcta gcttgccagt 900
ttcaaatgca gttcccaggt tgagcccggg gatttcacat ctgacttaac aaaccgcctg 960
cgtgcgcttt acgcccagta attccgatta acgcttgcac cctccgtatt accgcggctg 1020
ctggcacgga gttagccggt gcttcttctg cgagtaacgt caattgatga acgtattaag 1080
ctcaccacct tcctcctcgc tgaaagtgct ttacaacccg aaggccttct tcacacacgc 1140
ggcatggctg catcagactt gcgcccattg tgcaatattc cccactgctg cctcccgtag 1200
gagtctggac cgtgtctcag ttccagtgtg gctggtcatc ctctcagacc agctagggat 1260
cgtcgcctag gtgagccatt accccaccta ctagctaatc ccatctgggc acatctgatg 1320
gcaagaggcc cgaaggtccc cctctttggt cttgcgacgt tatgcggtat tagctaccgt 1380
ttccagtagt tatccccctc catcaggcag tttcccagac attactcacc cgtccgccgc 1440
tcgtcaccca gggagcaagc tcccctgtgc taccgctcga cttgcatgtg ttaagcctgc 1500
cgccagcgtt caatctgagc caggatcaaa ctct 1534
Claims (2)
1. the method for a producing aldinamide by biological catalysis of 2-cyano pyrazine is characterized in that described method is as follows:
(1) serratia marcescens CCTCC No.M 208231 is seeded to seed culture medium, cultivates 24 hours, and obtains seed liquor for 30 ℃; Described seed culture medium is composed as follows: sucrose 10g/L, yeast powder 5g/L, NaCl 1g/L, K
2HPO
40.5g/L, MgSO
40.1g/L solvent is a water, pH value 4.0~8.0;
(2) step (1) seed liquor is seeded to fermention medium with 5% volume ratio, and concussion is cultured to the thalline logarithmic phase under 30 ℃ of conditions, and centrifugation obtains thalline and contains the enzyme cell; Described fermention medium is composed as follows: glucose 20g/L, yeast extract paste 10g/L, urea 15g/L, monosodium glutamate 1g/L, K
2HPO
40.2g/L, KH
2PO
42g/L, MgSO
43g/L, CoCl
20.2g/L solvent is a water, pH value 7.0;
(3) step (2) thalline contains in the phosphate buffered saline buffer reaction system that the enzyme cell is added into the 0.1M, the pH8.0 that contain 2 cyano pyrazine, to contain the enzyme cell be 10g/L to contain enzyme wet cell weight concentration to thalline in the described reaction system, 2 cyano pyrazine concentration is 20g/L, under pH8.0,30 ℃, carry out catalytic hydration reaction 24h, make described pyrazinoic acid amide.
2. serratia marcescens (Serratia marcescens) ZJB-09104, be preserved in Chinese typical culture collection center, address: China. Wuhan. Wuhan University 430072, be preserved in Chinese typical culture collection center, preservation date on November 18th, 2008, deposit number CCTCC No.M208231.
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CN101886096B (en) * | 2010-06-21 | 2013-07-24 | 浙江工业大学 | Method for preparing 2-amino-2, 3-dimethyl butamide by microbial catalysis method and strain |
CN102776252B (en) * | 2010-06-21 | 2014-07-02 | 浙江工业大学 | Method for preparing 2-amino-2, 3-dimethylbutyramide by catalyzing of microorganism and bacterial strain |
CN104498466B (en) * | 2014-12-11 | 2017-07-07 | 浙江大学 | nitrile hydratase and its application |
CN107118977B (en) * | 2017-03-31 | 2020-02-21 | 浙江工业大学 | Serratia marcescens and application thereof |
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