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
- Publication number
- CN101481713B CN101481713B CN2008101637505A CN200810163750A CN101481713B CN 101481713 B CN101481713 B CN 101481713B CN 2008101637505 A CN2008101637505 A CN 2008101637505A CN 200810163750 A CN200810163750 A CN 200810163750A CN 101481713 B CN101481713 B CN 101481713B
- Authority
- CN
- China
- Prior art keywords
- serratia marcescens
- thalline
- bacterial strain
- producing
- acid amide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 13
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 239000004202 carbamide Substances 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 claims description 6
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 6
- 239000004223 monosodium glutamate Substances 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 4
- 239000002953 phosphate buffered saline Substances 0.000 claims description 4
- 230000009514 concussion Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 108010024026 Nitrile hydratase Proteins 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 8
- 230000009466 transformation Effects 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 3
- 239000000758 substrate Substances 0.000 abstract description 3
- 229960005206 pyrazinamide Drugs 0.000 abstract 5
- 101000794822 Serratia marcescens Anthranilate synthase component 1 Proteins 0.000 abstract 1
- 101000847781 Serratia marcescens Anthranilate synthase component 2 Proteins 0.000 abstract 1
- 239000003054 catalyst Substances 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 238000012216 screening Methods 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 11
- 108020004465 16S ribosomal RNA Proteins 0.000 description 9
- 244000005700 microbiome Species 0.000 description 7
- 229960004793 sucrose Drugs 0.000 description 7
- 239000008399 tap water Substances 0.000 description 7
- 235000020679 tap water Nutrition 0.000 description 7
- 230000004913 activation Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 239000012530 fluid Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- -1 2-pyrazinoic acid amide-4-oxide compound Chemical class 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 206010034972 Photosensitivity reaction Diseases 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000036211 photosensitivity Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 229940098362 serratia marcescens Drugs 0.000 description 3
- 239000000814 tuberculostatic agent Substances 0.000 description 3
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 201000008827 tuberculosis Diseases 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108010082340 Arginine deiminase Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- GUBGYTABKSRVRQ-UHFFFAOYSA-N D-Cellobiose Natural products OCC1OC(OC2C(O)C(O)C(O)OC2CO)C(O)C(O)C1O GUBGYTABKSRVRQ-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 230000001355 anti-mycobacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229940088529 claritin Drugs 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- HCPOCMMGKBZWSJ-UHFFFAOYSA-N ethyl 3-hydrazinyl-3-oxopropanoate Chemical compound CCOC(=O)CC(=O)NN HCPOCMMGKBZWSJ-UHFFFAOYSA-N 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- JCCNYMKQOSZNPW-UHFFFAOYSA-N loratadine Chemical compound C1CN(C(=O)OCC)CCC1=C1C2=NC=CC=C2CCC2=CC(Cl)=CC=C21 JCCNYMKQOSZNPW-UHFFFAOYSA-N 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- DLRVVLDZNNYCBX-ABXHMFFYSA-N melibiose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-ABXHMFFYSA-N 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000012434 nucleophilic reagent Substances 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000005527 soil sampling Methods 0.000 description 1
- 239000002195 soluble material Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 229960003487 xylose Drugs 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101637505A CN101481713B (en) | 2008-12-30 | 2008-12-30 | Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101637505A CN101481713B (en) | 2008-12-30 | 2008-12-30 | Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101481713A CN101481713A (en) | 2009-07-15 |
| CN101481713B true CN101481713B (en) | 2011-12-21 |
Family
ID=40879003
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008101637505A Active CN101481713B (en) | 2008-12-30 | 2008-12-30 | Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101481713B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776253B (en) * | 2010-06-21 | 2014-08-06 | 浙江工业大学 | Method for bio-catalytically preparing 2-amino-2, 3-dimethyl butyramide and strain |
| 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 |
| CN108660131B (en) * | 2018-04-27 | 2020-07-21 | 浙江工业大学 | Preparation method of immobilized nitrile hydratase and (S) -N-ethylpyrrolidine-2-formamide |
| CN113106130B (en) * | 2020-06-23 | 2023-03-14 | 核芯生物医药科技(杭州)有限公司 | Application of nitrile hydratase derived from staphylococcus aggregatus in preparation of amide pyrazine compounds |
| CN113817650B (en) * | 2021-10-26 | 2023-05-30 | 贵州大学 | Pyrazine-producing high-temperature actinomycetes, separation screening and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1032436A (en) * | 1987-09-18 | 1989-04-19 | 山田秀明 | The method of biological production of amides |
-
2008
- 2008-12-30 CN CN2008101637505A patent/CN101481713B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1032436A (en) * | 1987-09-18 | 1989-04-19 | 山田秀明 | The method of biological production of amides |
Non-Patent Citations (1)
| Title |
|---|
| 邓林，刘延岭，王忠彦，胡承，孙勇，段洪武.《腈水合酶产生菌的分离、鉴定及部分酶学性质研究》.《四川大学学报（自然科学版）》.2005,第42卷(第1期),第167-170页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101481713A (en) | 2009-07-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101481713B (en) | Method for producing aldinamide by biological catalysis of 2-cyano pyrazine and bacterial strain thereof | |
| CN103865855B (en) | Bacillus subtilis strain and application thereof | |
| CN103224898B (en) | Marine bacillus and its polypeptide with antitumor activity | |
| CN101591628B (en) | Acinetobacter juni. X8 and application thereof in preparing algin lyase | |
| CN103451133A (en) | Bacillus circulans and application for same in preparation for ferulic acid decarboxylase | |
| CN103710292A (en) | Pseudomonas aeruginosa and application of pseudomonas aeruginosa in aspect of degrading aflatoxin | |
| CN104762238A (en) | Lactic acid bacteria not generating amino acid decarboxylase high-yield urease and application of lactic acid bacteria | |
| CN105420128B (en) | A kind of selenium-rich rhodotorula mucilaginosa bacterial strain FXY-7 and its cultural method and the application in shrimp feed additive | |
| CN106755186A (en) | A kind of middle anthropi exocellular polysaccharide and its application in terms of soil improvement | |
| CN102363757A (en) | Screening method for 2-keto-D-gluconic acid high-yield bacterial strain, and fermentation method of such bacterial strain | |
| CN113265337A (en) | Marine aspergillus versicolor and isolated culture method and application thereof | |
| CN102911896B (en) | Bacillus subtilis for producing gamma-polyglutamic acid by high-temperature fermentation and application of bacillus subtilis for producing gamma-polyglutamic acid by high-temperature fermentation | |
| CN103981134A (en) | Pseudomonas aeruginosa and application thereof in degrading zearalenone | |
| CN101701243B (en) | Method for producing R-mandelic acid and derivates thereof by biocatalysis | |
| CN102433289B (en) | Strain for producing citrulline and method for biologically synthesizing citrulline with same | |
| CN113337432B (en) | Methylophilus for producing pyrroloquinoline quinone and application thereof | |
| CN101886096B (en) | Method for preparing 2-amino-2, 3-dimethyl butamide by microbial catalysis method and strain | |
| CN112641032B (en) | Application of cryptococcus rhodochrous Y3-based intracellular enzyme in degradation of ochratoxin A | |
| CN105886407B (en) | Radiation hardness filamentous fungi F35 and its application in Adsorption of Lead biological treatment | |
| CN103923853A (en) | Paenibacillus sp. and method for preparing k-carrageenanase by using same | |
| CN101857889B (en) | Method for biocatalystic production of 2-chloronicotinic acid from 2-cloro-3-cyanopyridine and strain thereof | |
| CN108587923B (en) | Method for improving malic acid fermentation performance | |
| CN102776252B (en) | Method for preparing 2-amino-2, 3-dimethylbutyramide by catalyzing of microorganism and bacterial strain | |
| CN102559540B (en) | Flavobacterium aquati and application thereof in preparation of L-tryptophan by microbial transformation | |
| CN102433290A (en) | Strain for producing citrulline and method for biologically synthesizing citrulline with same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |