CN101857889B - Method for biocatalystic production of 2-chloronicotinic acid from 2-cloro-3-cyanopyridine and strain thereof - Google Patents
Method for biocatalystic production of 2-chloronicotinic acid from 2-cloro-3-cyanopyridine and strain thereof Download PDFInfo
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- CN101857889B CN101857889B CN 200910154891 CN200910154891A CN101857889B CN 101857889 B CN101857889 B CN 101857889B CN 200910154891 CN200910154891 CN 200910154891 CN 200910154891 A CN200910154891 A CN 200910154891A CN 101857889 B CN101857889 B CN 101857889B
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- chloronicotinic acid
- chloro
- nicotinonitrile
- chlorine apellagrin
- rhodococcus erythropolis
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title abstract description 15
- IBRSSZOHCGUTHI-UHFFFAOYSA-N 2-chloropyridine-3-carboxylic acid Chemical compound OC(=O)C1=CC=CN=C1Cl IBRSSZOHCGUTHI-UHFFFAOYSA-N 0.000 title abstract 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 239000002904 solvent Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 7
- 230000004151 fermentation Effects 0.000 claims abstract description 7
- 230000003197 catalytic effect Effects 0.000 claims abstract description 5
- 239000000758 substrate Substances 0.000 claims abstract description 5
- 239000000460 chlorine Substances 0.000 claims description 53
- 229910052801 chlorine Inorganic materials 0.000 claims description 53
- JAUPUQRPBNDMDT-UHFFFAOYSA-N 2-chloropyridine-3-carbonitrile Chemical compound ClC1=NC=CC=C1C#N JAUPUQRPBNDMDT-UHFFFAOYSA-N 0.000 claims description 29
- 239000002609 medium Substances 0.000 claims description 29
- 241000187561 Rhodococcus erythropolis Species 0.000 claims description 27
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 11
- JBKVHLHDHHXQEQ-UHFFFAOYSA-N epsilon-caprolactam Chemical compound O=C1CCCCCN1 JBKVHLHDHHXQEQ-UHFFFAOYSA-N 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 150000002825 nitriles Chemical class 0.000 claims description 10
- 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 9
- 239000008103 glucose Substances 0.000 claims description 9
- 108010051210 beta-Fructofuranosidase Proteins 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000001573 invertase Substances 0.000 claims description 8
- 235000011073 invertase Nutrition 0.000 claims description 8
- 238000011218 seed culture Methods 0.000 claims description 7
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 6
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products 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-PICCSMPSSA-N 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 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
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000002953 phosphate buffered saline Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 3
- 230000002210 biocatalytic effect Effects 0.000 abstract 4
- 239000003054 catalyst Substances 0.000 abstract 1
- 239000008363 phosphate buffer Substances 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 40
- 241000894006 Bacteria Species 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 230000004913 activation Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 108020004465 16S ribosomal RNA Proteins 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000575 pesticide Substances 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 239000003905 agrochemical Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- ITQTTZVARXURQS-UHFFFAOYSA-N 3-methylpyridine Chemical compound CC1=CC=CN=C1 ITQTTZVARXURQS-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical class OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
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- 239000000284 extract Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 239000000645 desinfectant Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
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- 230000007613 environmental effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002917 insecticide Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- -1 nitrile compound Chemical class 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- QNZRJGJNLOMEGJ-UHFFFAOYSA-N 2-chloro-n,n-dimethylpyridine-3-carboxamide Chemical compound CN(C)C(=O)C1=CC=CN=C1Cl QNZRJGJNLOMEGJ-UHFFFAOYSA-N 0.000 description 1
- WYKHFQKONWMWQM-UHFFFAOYSA-N 2-sulfanylidene-1h-pyridine-3-carboxylic acid Chemical compound OC(=O)C1=CC=CN=C1S WYKHFQKONWMWQM-UHFFFAOYSA-N 0.000 description 1
- GZPHSAQLYPIAIN-UHFFFAOYSA-N 3-pyridinecarbonitrile Chemical compound N#CC1=CC=CN=C1 GZPHSAQLYPIAIN-UHFFFAOYSA-N 0.000 description 1
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 1
- 239000005586 Nicosulfuron Substances 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- KXIRHWPVRUKGFS-UHFFFAOYSA-N [N].C(C1=CN=CC=C1)(=O)O Chemical compound [N].C(C1=CN=CC=C1)(=O)O KXIRHWPVRUKGFS-UHFFFAOYSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
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- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
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- 230000002950 deficient Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- ZIUSEGSNTOUIPT-UHFFFAOYSA-N ethyl 2-cyanoacetate Chemical compound CCOC(=O)CC#N ZIUSEGSNTOUIPT-UHFFFAOYSA-N 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
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- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
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- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- RTCOGUMHFFWOJV-UHFFFAOYSA-N nicosulfuron Chemical compound COC1=CC(OC)=NC(NC(=O)NS(=O)(=O)C=2C(=CC=CN=2)C(=O)N(C)C)=N1 RTCOGUMHFFWOJV-UHFFFAOYSA-N 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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- 239000002351 wastewater Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for the biocatalytic production of 2-chloronicotinic acid from 2-cloro-3-cyanopyridine and a strain of the 2-chloronicotinic acid. The method comprises a step of producing the 2-chloronicotinic acid by performing a catalytic hydrolysis reaction in a reaction system which takes the 2-cloro-3-cyanopyridine as a substrate, nitrile-converting enzyme obtained by fermentation culture of Rhodococus erythropolis as a catalyst and water or a phosphate buffer as a solvent under pH of 7.0 to 9.0 and at the temperature of 25 to 60 DEG C. The method has the following advantages that: (1), the Rhodococus erythropolis (CCTCC, No: M 2091194) which can be used for biocatalytic production of the 2-chloronicotinic acid is obtained by sorting from microorganisms, and the 2-chloronicotinic acid can be effectively generated under proper conditions; (2), and a Rhodococus erythropolis strain ZJB-09149 is applied to a 2-chloronicotinic acid biocatalystic production experiment. The result shows that the Rhodococus erythropolis can be used for effective biocatalytic production of the 2-chloronicotinic acid and that the conversion rate of the 2-cloro-3-cyanopyridine reaches 60to 100 percent in 1 to 2 hours, so the Rhodococus erythropolis has wide application prospect in the field of 2-chloronicotinic acid biocatalytic production.
Description
(1) technical field
The present invention relates to a kind of method of utilizing nitrile invertase biocatalysis 2-chloro-nicotinonitrile to produce the 2-chlorine apellagrin, and the new bacterial strain that can be used for producing nitrile invertase---rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-09149.
(2) background technology
Because along with the development of agricultural chemicals and medical science, nicotinic acid series chemical is paid close attention to more and more widely and is used in recent years.2 chlorine substituent 2-chlorine apellagrins of nicotinic acid are then because its special physiologically active, be widely used at medicine, pesticide field, it can be used for synthetic many medical pain killers or anti-inflammatory agent, medicine and disinfectant use in agriculture, sterilant and the weedicide etc. of medical antibacterial element, Cardiovarscular, particularly at pesticide field, it is particularly outstanding that the application of 2-chlorine apellagrin seems.Agricultural chemicals of new generation take pyridine and its derivatives as intermediate has efficiently, low toxicity, characteristics that Environmental compatibility is good, is becoming the exploitation focus.Be the novel pesticide of parent exploitation by the 2-chlorine apellagrin, not only toxicity of pesticide is low, and active high, and China is a large agricultural country, improves and improve the people's standard of living, produces how better agricultural chemicals and is very important to guarantee stable good harvest.In addition its derivative 2-chlorine apellagrin methyl esters, 2-chloro-N, N-dimethyl nicotinamide and 2-sulfydryl nicotinic acid also are important medicine and pesticide intermediates.Set out by them, also can synthesize medicine and disinfectant use in agriculture, sterilant and the weedicide etc. of many medical antibacterial elements, Cardiovarscular.In view of the 2-chlorine apellagrin has become important agricultural chemicals and medicine intermediate, application prospect is very wide.2002, the whole world is about 300t to the demand of 2-chlorine apellagrin, and reached more than the 600t in 2003, growth is up to 100%, along with the exploitation of 2-chlorine apellagrin as the new pharmaceutical prod of raw material, nicosulfuron patent expired, and the medication chemistry industry of development, 2008 annual requirements are more than 1500t.And now output in domestic is roughly about 600～700t, and the market value of 2-chlorine apellagrin reaches about 140,000 now originally at 128000 yuan/t, and market outlook are considerable.But owing to lack suitable production line, worldwide supply falls short of demand for the 2-chlorine apellagrin, seriously restricting the research and development of new pharmaceutical and novel pesticide, show particularly outstandingly in China, the market requirement that this and 2-chlorine apellagrin sharply increase is runed counter to, simultaneously also show preferably 2-chlorine apellagrin synthetic route of present shortage, therefore, the production of further investigation 2-chlorine apellagrin has great economic benefit and social benefit.
The 2-chlorine apellagrin mainly is the method acquisition by chemosynthesis at present.Its synthetic method mainly contains: (1) nicotinic acid nitrogen oxidation-cyaniding-hydrolysis method: the method cost of material is more expensive, prepared product cost is higher, economic benefit is relatively poor, easily produce 2,6 isomers in this external rear two-step reaction, not easily separated, product purity is also lower, do not meet the requirement of medicine and agricultural chemicals, total yield is lower, and environmental pollution is large; (2) nicotinonitrile Sohio process for acrylonitrile Sohio-chloride-hydrolysis: the method is domestic comparatively ripe, but total reaction yield is lower, long reaction time, and poor operability needs expensive catalyzer and produces a large amount of acid waste waters, and environmental pollution is large; (3) 3-picoline cyaniding-oxidation style: the raw materials used 3-picoline of the method is domestic not to have suitability for industrialized production, and the chlorination reaction condition is harsh, and produces a large amount of isomer, separation difficulty, and production cost is large; (4) ethyl cyanacetate cyaniding-propenal Michael addition-hydrolysis method: the shortcoming of this reaction is that reaction raw materials propenal toxicity is large, and consumption is also larger, and is volatile, and whole operational path is loaded down with trivial details, operation steps is tediously long, uses solvent many in the reaction, reclaims difficulty.Therefore these chemical process defectives are more, have on the one hand a large amount of spent acid to discharge, and cause serious environmental pollution, and equipment requirements is high on the other hand, severe reaction conditions, and cost is higher.Because pollution is large, cost is high, these methods all are not suitable for large-scale industrial production.So efficient, the clean preparation method of 2-chlorine apellagrin have great society and ecological benefits.
The in recent years breakthrough of biological technical field makes biocatalysis increasingly important in chemosynthesis.Biocatalysis is a kind of novel transformation technology, is particularly suitable for the synthetic of active compound in pharmacy and the related industries thereof.Society is more and more higher to the cry of " Sustainable development ", " Green Chemistry " and " environmental friendliness manufacturing ", therefore biocatalysis and biotransformation are subject to unprecedented attention, be generally considered the third wave of biotechnology, greatly promote the development of biological industry, having great social effect and economic benefit.Utilizing the nitrile invertase biocatalysis to produce acid amides, carboxylic acid and derivative thereof is a kind of very effective production method, can greatly reduce energy consumption, conservation, minimizing pollution, with the Synthesis of biotechnology applications in nitrile compound, people's great attention and concern have been caused.Everything development of producing the 2-chlorine apellagrin to biological process has brought opportunity.
(3) summary of the invention
The object of the invention provides a kind of method of utilizing nitrile invertase biocatalysis 2-chloro-nicotinonitrile to produce the 2-chlorine apellagrin, and the new bacterial strain that can be used for producing nitrile invertase---rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-09149.
The technical solution used in the present invention is:
A kind of biocatalysis 2-chloro-nicotinonitrile is produced the method for 2-chlorine apellagrin, described method comprises: the nitrile invertase that obtains through fermentation culture take 2-chloro-nicotinonitrile as substrate, take rhodococcus erythropolis (Rhodococcus erythropolis) as catalyzer, take water or phosphate buffered saline buffer in the reaction system of solvent, under pH7.0～9.0,25～60 ℃, carry out catalytic hydrolysis reaction, make described 2-chlorine apellagrin.Described nitrile invertase consumption also can be excessive so that till 2-chloro-nicotinonitrile fully reacts.
Described nitrile invertase contains the enzyme cell from rhodococcus erythropolis CCTCC NO:M 209194 through what fermentation culture obtained, contains the enzyme cell concn in the described reaction system and counts 10～20g/l to contain the enzyme wet cell weight.
In the described reaction system, substrate 2-chloro-nicotinonitrile starting point concentration is 1.0～5.0g/l.
The described enzyme cell that contains prepares by the following method: rhodococcus erythropolis is seeded in the fermention medium that is applicable to rhodococcus erythropolis, 48～72h is cultivated in concussion under the condition of 25～30 ℃ of temperature, initial pH 6.5～7.0, shaking speed 100～300rpm, centrifugation obtains the described enzyme cell that contains.
The described fermention medium that is applicable to rhodococcus erythropolis is the conventional liquid nutrient medium that is applicable to rhodococcus erythropolis, the final concentration of fermention medium described in the present invention is composed as follows: maltose 5.0～15.0g/l, yeast powder 7.0～18.0g/l, hexanolactam 4.0～8.0g/l, MgSO
47H
2O 0.1～0.5g/l, K
2HPO
43H
2O 0.1～0.5g/l, KH
2PO
40.1～0.5g/l, FeSO
47H
2O 0.01～0.02g/l, solvent are water, pH value 6.5～7.0.
Concrete, described method is as follows:
(1) rhodococcus erythropolis CCTCC NO:M 209194 is seeded to seed culture medium, cultivates 16～20h, obtains seed liquor for 25～30 ℃; Described seed culture medium final concentration is composed as follows: glucose 10.0～20.0g/l, peptone 5.0～10.0g/l, hexanolactam 4.0～8.0g/l, MgSO
47H
2O 0.2～0.5g/l, K
2HPO
43H
2O 0.2～0.5g/l, KH
2PO
40.2～0.5g/l, FeSO
47H
2O 0.01～0.02g/l, solvent are water, pH value 6.5～7.0;
(2) step (1) seed liquor is seeded to fermention medium with 3～10% volume ratios, and 48～72h is cultivated in concussion under 25～30 ℃, initial pH6.5～7.0 conditions, and centrifugation obtains thalline and contains the enzyme cell; Described fermention medium final concentration is composed as follows: maltose 5.0～15.0g/l, yeast powder 7.0～18.0g/l, hexanolactam 4.0～8.0g/l, MgSO
47H
2O0.1～0.5g/l, K
2HPO
43H
2O 0.1～0.5g/l, KH
2PO
40.1～0.5g/l, FeSO
47H
2O 0.01～0.02g/l, solvent are water, pH value 6.5～7.0.
(3) step (2) thalline being contained the enzyme cell is added in the reaction system that contains 2-chloro-nicotinonitrile, thalline contains the enzyme cell to contain enzyme wet cell weight concentration as 10～20g/l in the described reaction system, 2-chloro-nicotinonitrile starting point concentration is 1.0～5.0g/l, under pH7.0～9.0,25～60 ℃, carry out catalytic hydrolysis reaction 10～12h, make described 2-chlorine apellagrin.
The mensuration of 2-chlorine apellagrin concentration in the reaction solution: centrifugal to the reaction solution 1ml that step (3) obtains, supernatant liquor carries out the content that liquid-phase chromatographic analysis is measured substrate.
The condition of liquid chromatography: Japanese Shimadzu liquid chromatograph, stainless stee l packed column, filler are C
18, long 0.25m, internal diameter 4.6mm, flow velocity 1ml/min, column temperature room temperature, moving phase are acetonitrile: (25V: 75V), sample size is 10 μ l to 0.1% phosphate aqueous solution.Wherein, 2-chlorine apellagrin appearance time is 4.2min.Adopt external standard method to determine the content of 2-chlorine apellagrin in the sample.
The invention still further relates to new bacterial strain---rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-09149 of the product nitrile invertase that a strain screens, be preserved in Chinese Typical Representative culture collection center, address: Wuhan, China Wuhan University, 430072, preservation date: on September 4th, 2009, deposit number CCTCC NO:M 209194.
Bacterial strain involved in the present invention screens by the following method and obtains:
(1) will be inoculated in the enrichment medium by soil sample and the sewage sample of insecticide factory in all parts of the country annex collection, at 30 ℃, on the shaking table of 150rpm behind the enrichment culture 72h, get again the 1ml nutrient solution and be inoculated on the fresh enrichment medium and cultivate 72h.So repeat 3 circulations.The prescription of enrichment medium following (final concentration): glucose 5～10g/l, K
2HPO
40.2～0.5g/l, KH
2PO
40.2～0.5g/l, MgSO
40.2～0.5g/l, NaCl 1～2g/l, FeSO
40.01～0.02g/l, 2-chloro-nicotinonitrile 0.7～1.4g/l, pH 6.5～7.0.
(2) be applied on the plate culture medium after the last nutrient solution dilution, picking list bacterium colony is to slant preservation.Plate culture medium is the agar that adds 1.5-2.0% in the enrichment medium.
(3) single colony inoculation of picking is to fermention medium, component concentration following (final concentration): glucose 10.0～20.0g/l, peptone 5.0～10.0g/l, hexanolactam 4.0～8.0g/l, MgSO
47H
2O 0.2～0.5g/l, K
2HPO
43H
2O 0.2～0.5g/l, KH
2PO
40.2～0.5g/l, FeSO
47H
2O 0.01～0.02g/l, solvent are water, pH value 6.5～7.0; 48～72h is cultivated in concussion under 25～30 ℃, initial pH6.5～7.0 conditions, and centrifugation obtains thalline and contains the enzyme cell; Be suspended in after centrifugal in the phosphoric acid buffer system.
(4) carry out transformation experiment by adding 2-chloro-nicotinonitrile, but be divided into the bacterial strain for preparing the 2-chlorine apellagrin from acquisition 3 strain biocatalysis; Through further sieving again, finally obtain aimed strain ZJB-09149.
The bacterial strain ZJB-09149 that the present invention's screening obtains, by " Physiology and biochemistry of common bacteria system identification handbook and " uncle's Jie Shi Bacteria Identification handbook (the 8th edition) " is identified, bacterial strain is Gram-positive, it is shaft-like that form is approximately, give birth in pairs, wide about 0.5～1.0 μ m, be about 1.0～1.5 μ m, cultivate 36h for 30 ℃ on the screening flat board, colony morphology characteristic is rounded, neat in edge, middle slightly projection, easy picking, bacterium colony pinkiness, in 30 ℃ of constant temperature culture 24h, the about 2～3mm of single colony diameter.
16S rDNA sequential analysis: take the total DNA of the cell that extracts as 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 1434bp, related data among this sequence and the GenBank is carried out similarity analysis find that the homology of this bacterium and rhodococcus erythropolis (Rhodococcus erythropolis) is the highest.
Identify according to above microbial characteristic, this new bacterial strain ZJB-09149 belongs to rhodococcus erythropolis (Rhodococcus erythropolis), this strain microorganism does not belong to now, and oneself discloses any of bacterial classification, this strain microorganism has the ability that biocatalysis 2-chloro-nicotinonitrile is produced the 2-chlorine apellagrin, can be used for the production of 2-chlorine apellagrin.
Beneficial effect of the present invention is mainly reflected in: (1) but the present invention from micropopulation, screen and obtain the rhodococcus erythropolis ZJB-09149 (Rhodococcuserythropolis ZJB-09149) that biocatalysis is produced the 2-chlorine apellagrin: CCTCC NO:M 209194.It can effectively produce the 2-chlorine apellagrin under the condition of being fit to; (2) the present invention is used for the experiment of biocatalysis production 2-chlorine apellagrin with rhodococcus erythropolis bacterial strain ZJB-09149, the result shows, this bacterium effectively biocatalysis produces the 2-chlorine apellagrin, 2-chloro-nicotinonitrile transformation efficiency reaches 60～100% (seeing embodiment 3～5) in the 12h, so this bacterium is with a wide range of applications in the field of biocatalysis production 2-chlorine apellagrin.
(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-09149
From being inoculated into the enrichment medium by soil sample and the sewage sample of insecticide factory in all parts of the country annex collection; At 30 ℃, on the shaking table of 150rpm behind the enrichment culture 72h, get again the 1ml nutrient solution and be inoculated on the fresh enrichment medium and cultivate 72h.So repeat 3 circulations.Be applied on the plate culture medium after the last nutrient solution dilution, picking list bacterium colony is to slant preservation.Single colony inoculation of picking is to fermention medium, and 48～72h is cultivated in concussion under 25～30 ℃, initial pH6.5～7.0 conditions, and centrifugation obtains thalline and contains the enzyme cell; Be suspended in the phosphoric acid buffer system, carry out transformation experiment by adding 2-chloro-nicotinonitrile.The 0.1g wet thallus is added in the reaction solution of 2-chloro-nicotinonitrile that final concentration is 1.38g/l, behind the reaction 12h, measure respectively the content of 2-chlorine apellagrin, therefrom filter out and produce the strongest bacterial strain ZJB-09149 of 2-chlorine apellagrin ability, as the starting strain of further research.
Described enrichment culture based formulas is (final concentration): glucose 5g/l, K
2HPO
43H
2O 0.5g/l, KH
2PO
40.5g/l, MgSO
47H
2O 0.5g/l, NaCl 1g/l, FeSO
47H
2O 0.02g/l, 2-chloro-nicotinonitrile 0.7g/l, pH 7.0, and solvent is water.
Described fermentative medium formula is (final concentration): glucose 20g/l, peptone 5g/l, caprolactam 4g/l, K
2HPO43H
2O 0.5g/l, KH
2PO
40.5g/l, MgSO
47H
2O 0.5g/l, FeSO
47H
2O 0.02g/l, pH 7.0, and solvent is water.
Embodiment 2: the evaluation of new bacterial strain ZJB-09149
The bacterial strain ZJB-09149 that 1 screening obtains to embodiment is by " uncle's Jie Shi Bacteria Identification handbook (the 8th edition) ", and " common bacteria system identification handbook is carried out physio-biochemical characteristics and identified.
This bacterial strain bacterium colony is rounded, neat in edge, and middle slightly projection, easy picking, the bacterium colony pinkiness, in 30 ℃ of constant temperature culture 24h, the about 2～3mm of single colony diameter.On the basis of the above, further bacterial strain ZJB-09149 is pressed fine works molecular biology experiment guide method and extract chromosomal DNA, take the total DNA of the cell that extracts as 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 Shanghai white good development in science and technology company limited to this bacterium 16SrDNA 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 with BLAST, and carry out sequence analysis; Evaluation based on form, physiological and biochemical property and the aspects such as 16S rDNA sequence and phylogeny Epidemiological Analysis, bacterial strain ZJB-09149 is accredited as rhodococcus erythropolis, intend called after rhodococcus erythropolis ZJB-09149 (Rhodococcus erythropolis ZJB-09149), this bacterial strain is deposited in Chinese Typical Representative culture collection center on September 4th, 2009, and deposit number is CCTCC NO:M 209194.Bacterial strain ZJB-09149, the 16S rDNA sequence of CCTCC NO:M 209194 is as follows:
TTGTTACGACTTCGTCCCAATCGCCGATCCCACCTTCGACGGCTCCCTCC
CACAAGGGGTTAAGCCACCGGCTTCGGGTGTTACCGACTTTCATGACGTG
ACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCGTTGCTGA
TCTGCGATTACTAGCGACTCCGACTTCACGGGGTCGAGTTGCAGACCCCG
ATCCGAACTGAGACCAGCTTTAAGGGATTCGCTCCACCTCACGGTCTCGC
AGCCCTCTGTACTGGCCATTGTAGCATGTGTGAAGCCCTGGACATAAGGG
GCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAG
TCTCTTACGAGTCCCCACCATAACGTGCTGGCAACATAAGATAGGGGTTG
CGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAG
CCATGCACCACCTGTATACCGACCACAAGGGGGGCCACATCTCTGCAGCT
TTCCGGTATATGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTA
ATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTT
TAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACG
GCACGGATTCCGTGGAAGGAACCCACACCTAGCGCCCACCGTTTACGGCG
TGGACTACCAGGGTATCTAATCCTGTTCGCTACCCACGCTTTCGTTCCTC
AGCGTCAGTTACTGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTG
ATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTGCAGT
ACTCAAGTCTGCCCGTATCGCCTGCAAGCCAGCAGTTGAGCTGCTGGTTT
TCACAAACGACGCGACAAACCGCCTACGAACTCTTTACGCCCAGTAATTC
CGGACAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTA
GCCGGTGCTTCTTCTGCAGGTACCGTCACTTGCGCTTCGTCCCTGCTGAA
AGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATC
AGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGT
CTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCTCTCAGGTCGGCT
ACCCGTCGTCGCCTTGGTAGGCCATTACCCCACCAACAAGCTGATAGGCC
GCGGGCCCATCCTGCACCGATAAATCTTTCCACCACCCACCATGCGATAG
GAGGTCATATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCGAAGTGC
AGGGCAGATCACCCACGTGTTACTCACCCGTTCGCCGCTCGTGTACCCCG
AAAGGCCTTACCGCTCGACTTGCATGTGTAAGCA
Embodiment 3: utilize bacterial strain ZJB-09149 biocatalysis to produce the 2-chlorine apellagrin
Carry out according to the following steps:
(1) preparation of slant medium: glucose 10g/l, yeast extract paste 3g/l, NaCl 1g/l, K
2HPO
43H
2O 0.2g/l, MgSO
47H
2O 0.2g/l, agar 20g/l, pH 7.0, and solvent is water;
(2) activation culture of bacterial classification: adopt step (1) slant medium, with new bacterial strain ZJB-09149, after 30 ℃ of activation, cultivating 24h, for subsequent use;
(3) preparation of seed liquor: bacterial classification 1 articulating after step (2) activation is entered in the seed culture medium, 30 ℃ of culture condition, cultivate 20h under the 200r/min.
(4) fermentation culture of bacterial classification: step (3) strain liquid by in the 10% volume ratio inoculum size access fermention medium, 30 ℃ of culture condition, is cultivated 72h under the 200r/min, carry out centrifugation, obtain thalline; Fermentative medium formula is (final concentration): maltose 5.0g/l, yeast powder 7.0g/l, hexanolactam 8.0g/l, MgSO
47H
2O 0.5g/l, K
2HPO
43H
2O 0.5g/l, KH
2PO
40.5g/l, FeSO
47H
2O 0.02g/l, solvent are water, pH value 6.5～7.0.
(5) biocatalysis 2-chloro-nicotinonitrile is produced the 2-chlorine apellagrin: the wet thallus 0.1g that step (4) is obtained joins in phosphate buffered saline buffer (pH7.0) reaction system that 10ml contains 0.0138g 2-chloro-nicotinonitrile, 30 ℃, 150r/min reacts 12h;
(6) mensuration of 2-chlorine apellagrin content in the conversion fluid: get the 1ml conversion fluid, centrifugal, supernatant liquor carries out liquid-phase chromatographic analysis: Japanese Shimadzu liquid chromatograph, stainless stee l packed column, filler are C
18, long 0.25m, internal diameter 4.6mm, flow velocity 1ml/min, column temperature room temperature, moving phase are acetonitrile: 0.1% phosphate aqueous solution (25V: 75V) sample size be 10 μ l wherein, 2-chlorine apellagrin appearance time is 4.2min, and the content of determining 2-chlorine apellagrin in the sample is 0.95g/l, and molar yield is 60%.
Embodiment 4: utilize bacterial strain ZJB-09149 biocatalysis to produce the 2-chlorine apellagrin
(1) preparation of slant medium: glucose 10g/l, yeast extract paste 3g/l, NaCl 1g/l, K
2HPO
43H
2O 0.2g/l, MgSO
47H
2O 0.2g/l, agar 20g/l, pH 7.0, and solvent is water;
(2) activation culture of bacterial classification: adopt step (1) slant medium, with new bacterial strain ZJB-09149, after 30 ℃ of activation, cultivating 24h, for subsequent use;
(3) preparation of seed liquor: bacterial classification 1 articulating after step (2) activation is entered in the seed culture medium, 30 ℃ of culture condition, cultivate 20h under the 200r/min.
(4) fermentation culture of bacterial classification: step (3) strain liquid by in the 10% volume ratio inoculum size access fermention medium, 30 ℃ of culture condition, is cultivated 72h under 200r/min, carry out centrifugation, obtain thalline; Fermentative medium formula is: maltose 5.0g/l, yeast powder 7.0g/l, hexanolactam 8.0g/l, MgSO
47H
2O 0.5g/l, K
2HPO
43H
2O 0.5g/l, KH
2PO
40.5g/l, FeSO
47H
2O 0.02g/l, solvent are water, pH value 6.5～7.0.
(5) biocatalysis 2-chloro-nicotinonitrile is produced the 2-chlorine apellagrin: biocatalysis 2-chloro-nicotinonitrile is produced the 2-chlorine apellagrin: the wet thallus 0.2g that step (4) is obtained joins in phosphate buffered saline buffer (pH7.0) reaction system that 10ml contains 0.0138g 2-chloro-nicotinonitrile, 30 ℃, 150r/min reacts 12h;
(6) mensuration of 2-chlorine apellagrin content in the conversion fluid: with embodiment 3.The content of determining 2-chlorine apellagrin in the sample is 1.6g/l, and transformation efficiency is 99%.
Embodiment 5: utilize bacterial strain ZJB-09149 biocatalysis to produce the 2-chlorine apellagrin
(1) preparation of slant medium: glucose 10g/l, yeast extract paste 3g/l, NaCl 1g/l, K
2HPO
43H
2O 0.2g/l, MgSO
47H
2O 0.2g/l, agar 20g/l, pH 7.0, and solvent is water;
(2) activation culture of bacterial classification: adopt step (1) slant medium, with new bacterial strain ZJB-09149, after 30 ℃ of activation, cultivating 24h, for subsequent use;
(3) preparation of seed liquor: bacterial classification 1 articulating after step (2) activation is entered in the seed culture medium, 30 ℃ of culture condition, cultivate 20h under the 200r/min.
(4) fermentation culture of bacterial classification: step (3) strain liquid by in the 10% volume ratio inoculum size access fermention medium, 30 ℃ of culture condition, is cultivated 72h under the 200r/min, carry out centrifugation, obtain thalline; Fermentative medium formula is: maltose 5.0g/l, yeast powder 7.0g/l, hexanolactam 8.0g/l, MgSO
47H
2O 0.5g/l, K
2HPO
43H
2O 0.5g/l, KH
2PO
40.5g/l, FeSO
47H
2O 0.02g/l, solvent are water, pH value 6.5～7.0.
(5) biocatalysis 2-chloro-nicotinonitrile is produced the 2-chlorine apellagrin: biocatalysis 2-chloro-nicotinonitrile is produced the 2-chlorine apellagrin: the wet thallus 0.1g that step (4) is obtained joins in phosphoric acid salt (pH7.0) reaction system that 10ml contains 0.0138g 2-chloro-nicotinonitrile, 35 ℃, 150r/min reacts 12h;
(6) mensuration of 2-chlorine apellagrin content in the conversion fluid: with embodiment 3.The content of determining 2-chlorine apellagrin in the sample is 1.3g/l, and transformation efficiency is 84%.
SEQUENCE LISTING
＜110〉Zhejiang Polytechnical University
＜120〉a kind of biocatalysis 2-chloro-nicotinonitrile is produced method and the bacterial strain of 2-chlorine apellagrin
<130>
<160>1
<170>PatentIn version 3.4
<210>1
<211>1434
<212>DNA
<213>Rhodococcus erythropolis
<400>1
ttgttacgac ttcgtcccaa tcgccgatcc caccttcgac ggctccctcc cacaaggggt 60
taagccaccg gcttcgggtg ttaccgactt tcatgacgtg acgggcggtg tgtacaaggc 120
ccgggaacgt attcaccgca gcgttgctga tctgcgatta ctagcgactc cgacttcacg 180
gggtcgagtt gcagaccccg atccgaactg agaccagctt taagggattc gctccacctc 240
acggtctcgc agccctctgt actggccatt gtagcatgtg tgaagccctg gacataaggg 300
gcatgatgac ttgacgtcgt ccccaccttc ctccgagttg accccggcag tctcttacga 360
gtccccacca taacgtgctg gcaacataag ataggggttg cgctcgttgc gggacttaac 420
ccaacatctc acgacacgag ctgacgacag ccatgcacca cctgtatacc gaccacaagg 480
ggggccacat ctctgcagct ttccggtata tgtcaaaccc aggtaaggtt cttcgcgttg 540
catcgaatta atccacatgc tccgccgctt gtgcgggccc ccgtcaattc ctttgagttt 600
tagccttgcg gccgtactcc ccaggcgggg cgcttaatgc gttagctacg gcacggattc 660
cgtggaagga acccacacct agcgcccacc gtttacggcg tggactacca gggtatctaa 720
tcctgttcgc tacccacgct ttcgttcctc agcgtcagtt actgcccaga gacccgcctt 780
cgccaccggt gttcctcctg atatctgcgc atttcaccgc tacaccagga attccagtct 840
cccctgcagt actcaagtct gcccgtatcg cctgcaagcc agcagttgag ctgctggttt 900
tcacaaacga cgcgacaaac cgcctacgaa ctctttacgc ccagtaattc cggacaacgc 960
ttgcacccta cgtattaccg cggctgctgg cacgtagtta gccggtgctt cttctgcagg 1020
taccgtcact tgcgcttcgt ccctgctgaa agaggtttac aacccgaagg ccgtcatccc 1080
tcacgcggcg tcgctgcatc aggctttcgc ccattgtgca atattcccca ctgctgcctc 1140
ccgtaggagt ctgggccgtg tctcagtccc agtgtggccg gtcaccctct caggtcggct 1200
acccgtcgtc gccttggtag gccattaccc caccaacaag ctgataggcc gcgggcccat 1260
cctgcaccga taaatctttc caccacccac catgcgatag gaggtcatat ccggtattag 1320
acccagtttc ccaggcttat cccgaagtgc agggcagatc acccacgtgt tactcacccg 1380
ttcgccgctc gtgtaccccg aaaggcctta ccgctcgact tgcatgtgta agca 1434
Claims (2)
1. a biocatalysis 2-chloro-nicotinonitrile is produced the method for 2-chlorine apellagrin, described method be the nitrile invertase that obtains through fermentation culture take 2-chloro-nicotinonitrile as substrate, take rhodococcus erythropolis (Rhodococcus erythropolis) CCTCC NO:M 209194 as catalyzer, take water or phosphate buffered saline buffer in the reaction system of solvent, under pH7.0,35 ℃, carry out catalytic hydrolysis reaction, make described 2-chlorine apellagrin; Described method is as follows:
(1) rhodococcus erythropolis CCTCC NO:M 209194 is seeded to seed culture medium, cultivates 16 ~ 20h, obtains seed liquor for 25 ~ 30 ℃; Described seed culture medium final concentration is composed as follows: glucose 10.0 ~ 20.0 g/l, peptone 5.0 ~ 10.0 g/l, hexanolactam 4.0 ~ 8.0 g/l, MgSO
47H
2O 0.2 ~ 0.5 g/l, K
2HPO
43H
2O 0.2 ~ 0.5 g/l, KH
2PO
40.2 ~ 0.5 g/l, FeSO
47H
2O 0.01 ~ 0.02 g/l, solvent are water, pH value 6.5 ~ 7.0;
(2) step (1) seed liquor is seeded to fermention medium with 3 ~ 10% volume ratios, 25 ~ 30 ℃, initial pH6.5 ~ 7.0
Shake under the conditionCultivate 48 ~ 72h, centrifugation obtains thalline and contains the enzyme cell; Described fermention medium final concentration is composed as follows: maltose 5.0 ~ 15.0 g/l, yeast powder 7.0 ~ 18.0 g/l, hexanolactam 4.0 ~ 8.0 g/l, MgSO
47H
2O 0.1 ~ 0.5 g/l, K
2HPO
43H
2O 0.1 ~ 0.5 g/l, KH
2PO
40.1 ~ 0.5 g/l, FeSO
47H
2O 0.01 ~ 0.02 g/l, solvent are water, pH value 6.5 ~ 7.0;
(3) step (2) thalline being contained the enzyme cell is added in the reaction system that contains 2-chloro-nicotinonitrile, thalline contains the enzyme cell to contain enzyme wet cell weight concentration as 10 ~ 20g/l in the described reaction system, 2-chloro-nicotinonitrile starting point concentration is 1.0 ~ 5.0g/l, in pH7.0,35 ℃, carry out catalytic hydrolysis reaction 12h under the 150r/min, make described 2-chlorine apellagrin.
2. rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-09149, be preserved in Chinese Typical Representative culture collection center, the address: Wuhan, China Wuhan University, 430072, preservation date: on September 4th, 2009, deposit number CCTCC NO:M 209194.
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| 徐赛珍 郑裕国.选择性腈水解酶在生物催化中的应用.《浙江化工》.2009,13-18. * |
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