CN101886096B - Method for preparing 2-amino-2, 3-dimethyl butamide by microbial catalysis method and strain - Google Patents
Method for preparing 2-amino-2, 3-dimethyl butamide by microbial catalysis method and strain Download PDFInfo
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Abstract
The invention discloses a method for producing an imidazolinone type ultra-high efficient herbicide intermediate-2-amino-2, 3-dimethyl butamide by the microbial method and a strain used during the process. The method comprises the steps of taking 2-amino-2, 3-dimethyl butyronitrile as raw material, and taking nitrile hydratase produced by fermentation culture of microbes of Rhodococcus qingshengii CCTCC NO: M 2010050, Nocardia globerula CCTCC NO: M 209214 or Rhodococcus erythropolis CCTCC NO: M 209244 as a catalyst for producing the 2-amino-2, 3-dimethyl butamide. The method has the advantages of mild reaction conditions, low waste water discharge, environment-friendly property, high yield, short conversion time, low cost and easy realization of industrial production.
Description
(1) technical field
The present invention relates to the standby 2-amino-2 of a kind of microorganism catalysis legal system, the method for 3-amide dimethyl butyrate, and the new bacterial strain of using in process of production.
(2) background technology
2-amino-2, the 3-amide dimethyl butyrate, English name 2-amino-2,3-dimethylbutyamide, outward appearance is the white plates crystal, fusing point is 84 ℃, can water-soluble and most of organic solvents.Be the general intermediate of imidazolone type ultra-high efficiency weedicide as imazethapyr, imazaquin and imazamox etc., the market requirement is wide.Wherein imazethapyr and imazaquin once occupied U.S. soybean weedicide market dominant position for 10 years; And China is since nineteen ninety import imazethapyr in enormous quantities since Heilongjiang Province's large-area applications, consumption continues to increase.After particularly this weedicide product comes out at home, because it is cheap, herbicidal effect is good, so used, becomes the leading kind of Heilungkiang and Inner Mongolia soybean field herbicide on a large scale.
2-amino-2, the 3-amide dimethyl butyrate is at present mainly by chemical method production.Its production process can be divided into two parts: 3-methyl-2-butanone obtains amino-nitrile by Strake (Strecker) reaction, and the latter is hydrolyzed and obtains product (seeing following formula) under acid or base catalysis.
(Northwest University's journal, 1994,24 (3): the synthetic method of 227-234) having reported this amino-nitrile such as Sun Xiaohong, after first ammonium chloride is water-soluble, add sodium cyanide and ammoniacal liquor, after stirring, add a small amount of benzyltriethylammoinium chloride, drip methyl isopropyl ketone, stirring and refluxing 4～6 hours, after purifying with methylene dichloride, obtain colourless transparent liquid, purity is 95%, and yield is 90%.
(US 6339158) such as Peter J. have been reported by 2-amino-2, the 3-nitrile dimethyl prepares 2-amino-2, the method of 3-dimethylformamide, at first add the 29.7ml vitriol oil, slowly add 11.8g 2-amino-2, the 3-nitrile dimethyl, guarantee that temperature of reaction system is no more than 25 ℃, be heated to 100 ℃ and react 1h at this temperature, reaction is cooling after finishing, add the 85ml strong aqua, in process, hierarchy of control temperature is no more than 75 ℃, then uses dichloromethane extraction, crystallization, finally obtains 11.2g 2-amino-2, the 3-dimethylformamide, yield is 81.7%.Lv Xiaodong etc. (Liaoning chemical industry, 2001,30 (10): 455-456) report is amino-2 by 2-, and the 3-nitrile dimethyl is added dropwise in 95% the vitriol oil at a certain temperature, heats up, stoichiometric number hour.After completion of the reaction, be cooled to room temperature, drip strong aqua and be neutralized to weakly alkaline, add extraction agent, stir certain hour, filter, leach thing and wash with extraction agent, and washing lotion is integrated with to filtrate.Filtrate is separated, and organic layer steams extraction agent, and bottoms adds sherwood oil, and freezing and crystallizing obtains 2-amino-2 after the solid drying leached, the 3-dimethylformamide, and ultimate yield is 95%, product purity reaches 96%.
As from the foregoing, chemical method is produced 2-amino-2, and the main drawback of 3-dimethylformamide is: the conditions such as reaction needed high temperature, strong acid, and energy consumption raw material consumption is high, product is subject to by-product contamination, and purification step is loaded down with trivial details, and the cycle is long, yield is low, produces a large amount of nitrile sewage that contains, and does not meet environmental requirement etc.
Nitrile hydratase (Nitrile Hydratase) is a kind of microbial enzyme be widespread in nature, but the multiple nitrile compound hydrolysis of its catalysis generates acid amides.Microorganism Nitrile Hydratase can be widely used in the synthetic of amino acid, acid amides, carboxylic acid and derivative thereof.1980, (the Agricultural andBiological Chemistry such as Asano, 1982,46 (5): 1183-1189) find first the poisonous acetonitrile of microorganism Rhodocococcus sp.N-774 degradable, successfully be applied to the industrial production acrylamide soon.In recent years, Nitrile hydratase also is used to prepare chiral drug, as (Applied Microbiology and Biotechnology such as Gilligan, 1993,39:720-725) successfully use Rhodocococcus equi TG328 Nitrile hydratase and Ntn hydrolase catalysis racemize 2-phenyl propionitrile stereo selective hydrolysis, generated non-steroidal anti-inflammatory drugs S-(+)-2-phenylpropionic acid.Existing research shows, the reaction of Nitrile hydratase catalysis has highly selective, high efficiency, mild condition, environmental pollution is little, cost is low, product optical purity advantages of higher, the developing direction that meets atom economy and Green Chemistry, the unrivaled superiority of chemical process is arranged, thereby promoted development and the exploitation of fine chemical product and chiral drug.Therefore utilize the characteristics such as high efficiency, highly selective and reaction conditions gentleness of Nitrile hydratase to carry out amino-2, the 3-amide dimethyl butyrate of catalytic production 2-, can improve productive rate and quality product, and reduce production costs and reduce environmental pollution.
It is reported, alpha-aminonitriles is extremely unstable in the aqueous solution, and easily spontaneous hydrolysis is produced the materials (Engineering in Life Sciences, 2004.4:547-556) such as HCN and ketone (aldehyde).These by products particularly HCN are the strongly inhibited agent of Nitrile hydratase, and it is by the metal cations Fe with the nitrile hydratase activity center
2+or Co
2+complexing and make the Nitrile hydratase inactivation.(the EuropeanJournal of Biochemistry such as Nagasawa, 1991.196:581-589) report, the Nitrile hydratase of producing as the effective catalyst Rhodococcus rhodochrous J1 that produces acrylamide, when cyanide ion concentration is 0.01mM, enzyme is lived and is lost 62%.Therefore screening obtains tolerating the Nitrile hydratase of cryanide ion, will be conducive to realize serialization production 2-amino-2, and the 3-amide dimethyl butyrate, improve production concentration.
(3) summary of the invention
For overcoming the defect of above-mentioned chemical synthesis, the invention provides a kind of microorganism catalysis hydration 2-amino-2, the 3-nitrile dimethyl prepares 2-amino-2, the method for 3-dimethylformamide (ADBA), and new bacterial strain used in preparation process.
The technical solution used in the present invention is:
The standby 2-amino-2 of a kind of microorganism catalysis legal system, the method of 3-amide dimethyl butyrate, described method comprises: with 2-amino-2, the 3-nitrile dimethyl is substrate, with celebrating sheng, a reed pipe wind instrument rhodococcus (Rhodococcusqingshengii) CCTCC No:M 2010050, in the reaction system that the Nitrile hydratase that globule nocardia (Nocardia globerula) CCTCC No:M 209214 or rhodococcus erythropolis (Rhodococcus erythropolis) CCTCC No:M 209244 fermentations produce is catalyzer, in pH6.0～10.0, carry out the catalytic hydration reaction under 20～40 ℃, make described 2-amino-2, the 3-amide dimethyl butyrate.
Described Nitrile hydratase is present in somatic cells, during concrete preparation, the fermented liquid of the mycetome cell that can directly fermentation be obtained participates in reaction as catalyzer, after also somatic cells can being separated, participate in reaction as catalyzer, described Nitrile hydratase is for catalysis 2-amino-2, and the 3-nitrile dimethyl prepares 2-amino-2, and the reaction principle of 3-dimethylformamide is as shown in following reaction formula:
Described reaction is carried out in citric acid-sodium citrate damping fluid, phosphate buffered saline buffer, Tris-HCl damping fluid or the glycine-sodium hydrate buffer solution of water or pH6.0～10.0.
In described reaction system, initial substrate concentration is 0.03～0.3M.In reaction process, when concentration of substrate during lower than 0.01M, can continue to fill into substrate 2-amino-2,3-nitrile dimethyl to concentration is 0.03～0.3M, to reach the quantity-produced effect.
Preferably, described Nitrile hydratase from celebrating sheng, a reed pipe wind instrument rhodococcus CCTCC No:M 2010050, globule nocardia CCTCC No:M 209214 or rhodococcus erythropolis CCTCC No:M 209244, through fermentation, obtain containing the enzyme somatic cells, described containing the enzyme somatic cells in reaction system addition containing the enzyme wet cell weight, to count 5～50g/L.The described enzyme somatic cells that contains can, by above-mentioned three kinds of bacterial strains in the substratum that is applicable to bacterial classification celebrating sheng, a reed pipe wind instrument rhodococcus, globule nocardia and rhodococcus erythropolis, obtain through fermentation culture.The described the present invention of being applicable to celebrates sheng, a reed pipe wind instrument rhodococcus, globule nocardia and rhodococcus erythropolis substratum, can be some conventional mediums that contain the nutritive substance that can be utilized by celebrating sheng, a reed pipe wind instrument rhodococcus, globule nocardia and rhodococcus erythropolis.Rationally allocate carbon source that mentioned microorganism can utilize, nitrogenous source, inorganic salt etc. in substratum into.As having of carbon source: sucrose, glucose, lactose, Trisodium Citrate, starch; As having of nitrogenous source: yeast extract paste, peptone, extractum carnis, soybean cake powder, corn steep liquor, ammonium salt etc.; Separately can add: amino acids (as glycine, Methionin, L-glutamic acid, methionine(Met), proline(Pro), aspartic acid etc.), VITAMIN are (as VB
1, VB
2, VB
12, V
c, V
e, nicotinic acid etc.), nucleic acid (as purine, pyrimidine and derivative thereof etc.).Regulating the pH value with 1.0M HCl or NaOH solution is 6.0～8.0.For improve producing enzyme efficiency, can in substratum, add inductor, it is one of following that described inductor can be: Sodium Glutamate, 2,2-dimethyl cyclopropanecarboxamide, hexanolactam, acrylamide, ethanamide, urea.Fermentation culture can be shake-flask culture or ventilation stir culture; Can in shaking flask, carry out also can in fermentor tank, carrying out.
Shake-flask culture: in the triangle shaking flask, be respectively charged into appropriate seed culture medium and culture medium (10～40%, v/v), 121 ℃ of sterilizing 20min.Choose into a certain amount of slant strains with transfering loop, in the access seed culture medium, under 20～40 ℃, cultivate 24～48h, obtain seed liquor; Seed liquor is contained in the fermention medium of inductor with 2.5% volume ratio access, in 20～40 ℃, under rotating speed 100～300rpm, cultivate 48～96h, obtain the fermented liquid containing Nitrile hydratase.
Fermentor cultivation: (30～65%, v/v), a certain amount of slant strains of access after real tank sterilization, 20～40 ℃ of temperature, cultivate 24～48h under mixing speed 100～300rpm to the appropriate fermention medium of packing in the 5L seeding tank, obtains seed liquor; The appropriate culture medium (30～65% of packing in the 50L fermentor tank, v/v), the seed liquor of access 2.5% volume ratio after real tank sterilization, at ventilation ratio 0.1～1.0: 1 ratio of nutrient solution volume (volume of air of passing through in per minute with), 20～40 ℃ of temperature, carry out fermentation culture 48～96h under the condition of mixing speed 100～300rpm, obtain the fermented liquid containing Nitrile hydratase.
Obtain can adopting the modes such as centrifugal or filtration to isolate somatic cells after bacterial culture fluid, utilize this wet thallus or immobilized cell or the broken apart Nitrile hydratase catalysis 2-gone out amino-2 from this cell, the 3-nitrile dimethyl prepares 2-amino-2, the 3-amide dimethyl butyrate.
Fermented liquid is centrifugal under 12000rpm, 4 ℃ of conditions, abandons supernatant, centrifugal collection thalline again after 0.9% physiological saline washing for cell; Perhaps fermented liquid carries out filtering separation with membrane filtration system, and adds 0.9% physiological saline washing of 2 times of fermentating liquid volumes, collects thalline.Membrane filtration system wherein can be hollow-fibre membrane or rolled film or ceramic membrane or ultra-filtration membrane.
Preferably, the described enzyme somatic cells that contains prepares by the following method:
(1) will be in the celebrating sheng, a reed pipe wind instrument rhodococcus CCTCC of slant activation No:M 2010050, globule nocardia CCTCC No:M 209214 or rhodococcus erythropolis CCTCC No:M 209244 access seed culture mediums, cultivate 24～48h under 20～40 ℃, obtain seed liquor; Described seed culture medium final concentration is composed as follows: glucose 5～20.0g/L, yeast extract powder 2～8.0g/L, peptone 1～4.0g/L, KH
2pO
40.5～1.5g/L, K
2hPO
40.5～1.5g/L, NaCl 0.5～3.0g/L, hexanolactam 0.5～2.0g/L, MgSO
40.05～0.2g/L, CoCl
20.01～0.05g/L, FeSO
40.01～0.05g/L, solvent is water, and pH 6.0～8.0;
(2) seed liquor is accessed in culture medium with 1～10% volume ratio, under 20～40 ℃, cultivate 48～96h, the fermented liquid that cultivation obtains, through centrifugal or membrane sepn, obtains described containing the enzyme somatic cells; Described culture medium final concentration is composed as follows: sucrose 5.0～10.0g/L, Trisodium Citrate 2.0～5.0g/L, extractum carnis 2.0～8.0g/L, yeast powder 2.0～8.0g/L, sodium-chlor 0.5～2.0g/L, potassium primary phosphate 0.5～2.5g/L, cobalt chloride 0.005～0.01g/L, iron(ic) chloride 0.005～0.01g/L, Manganous chloride tetrahydrate 0.005～0.01g/L, inductor 1.0～3.0g/L, solvent is water, pH 6.0～8.0; Described inductor is one of following: Sodium Glutamate, 2,2-dimethyl cyclopropanecarboxamide, hexanolactam, acrylamide, ethanamide or urea.
Described method is as follows: by described containing the enzyme somatic cells with after physiological saline washing, be suspended in citric acid-sodium citrate damping fluid, phosphate buffered saline buffer, Tris-HCl damping fluid or the glycine-sodium hydrate buffer solution of water or pH6.0～10.0, add substrate 2-amino-2,3-nitrile dimethyl to concentration of substrate is 0.03～0.3M, under 15～40 ℃, 100～200rpm, reacts 10～60min; After reaction finishes, conversion fluid obtains described 2-amino-2,3-amide dimethyl butyrate through separation and purification.
Preferably, described separation purification method is as follows: reaction is got the centrifugal 10min of conversion fluid 12000rpm after finishing, and collects supernatant liquor; Add Powdered Activated Carbon, after heated and stirred decolouring 30min, proceed to vacuum filtration device suction filtration, obtain the clear liquid that decolours; The decolouring clear liquid proceeds to Rotary Evaporators, at vacuum tightness-0.1Mpa, under 40～60 ℃ of water bath heating temperatures, rotating speed 60～100rpm condition, reduction vaporization is removed substrate 2-amino-2,3-nitrile dimethyl and water, cooling 2-amino-2 that obtain, 3-amide dimethyl butyrate crude product; By 2-amino-2,3-amide dimethyl butyrate crude product is dissolved in the ethyl acetate of 20～65 ℃, and adding volume is ethyl acetate volume 10% salting-out agent normal hexane, in 0～4 ℃ of insulation crystallization; Filter, get filter residue and wash with normal hexane, drying obtains 2-amino-2,3-amide dimethyl butyrate crystal.
The invention still further relates to the new bacterial strain of 3 strains used in preparation process, the Nitrile hydratase of this generation all has good cryanide ion tolerance, in KCN concentration, during up to 10mM, transforms vinyl cyanide and still can keep the Nitrile hydratase vigor more than 80%:
Celebrating sheng, a reed pipe wind instrument rhodococcus (Rhodococcus qingshengii) ZJB-09153, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number CCTCCNo:M 2010050, preservation date on March 10th, 2010.
Globule nocardia (Nocardia globerula) ZJB-09141 is preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number CCTCCNo:M 209214, preservation date on September 27th, 2009.
Rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-0910, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, 430072, deposit number CCTCC No:M 209244, preservation date on October 26th, 2009.This bacterial strain is formerly being protected as new bacterial strain in Chinese patent application 201010107481.8.
Above-mentioned three bacterial strains separate and obtain from soil by bio-engineering research institute of Zhejiang Polytechnical University.
Enzyme is lived and defined: under 30 ℃, per minute catalysis generates the 2-amino-2 of 1 μ mol, and the required enzyme amount of 3-amide dimethyl butyrate is defined as an enzyme activity unit (U).The vigor of Nitrile hydratase means (U/g dcw) with the contained enzyme activity unit of every gram stem cell.
2-amino-2 in the present invention, 3-nitrile dimethyl and 2-amino-2, the content of 3-amide dimethyl butyrate passes through gas chromatography determination.
Beneficial effect of the present invention is mainly reflected in:
(1) reaction conditions is gentle, the time is short.Chemical hydrolysis need to carry out 100 ℃ of left and right, must be equipped with heating and temperature control unit, severe reaction conditions, and energy consumption is high and high to equipment requirements, expends time in and reaches a couple of days even a few hours; And biological catalysis carries out at normal temperatures, the reaction conditions gentleness, reaction can complete within 1h;
(2) waste water output is few.Can discharge a large amount of acid-bearing wastewaters with chemical hydrolysis, and also contain (NH in waste water
4)
2sO
4, Na
2sO
4etc. unmanageable salt; Produce 2-amino-2 with the Nitrile hydratase biological catalysis, the 3-amide dimethyl butyrate, in waste water, unmanageable foreign matter content is few, is more conducive to realize cleaner production;
(3) cost is low.The raw material of chemical method is except 2-amino-2, and the 3-nitrile dimethyl also has the vitriol oil, strong aqua, Na outward
2cO
3with NaOH etc., desired raw material is many, and cost is high.And biological catalysis generally only need amino-2, the 3-nitrile dimethyl of 2-as raw material and nitrile hydratase production cell as catalyzer, transformation efficiency and productive rate all can reach more than 95%, raw material is simple and easy to get, cost is low.
(4) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the shake flask fermentation of globule nocardia ZJB-09141 (CCTCC No:M 209214) and rhodococcus erythropolis ZJB-0910 (CCTCC No:M 209244) is cultivated
Preparation seed culture medium: glucose 10.0g/L, yeast extract powder 5.0g/L, peptone 2.0g/L, KH
2pO
41.0g/L, K
2hPO
41.0g/L, NaCl 1.0g/L, hexanolactam 1.0g/L, MgSO
40.1g/L, CoCl
20.01g/L, FeSO
40.01g/L solvent is water, regulating pH with 1.0M HCl or NaOH solution is 7.5,121 ℃ of sterilizing 20min.Access slant strains after being cooled to room temperature, 30 ℃ of temperature, under mixing speed 150rpm, cultivate 24h, obtain seed liquor.
Prepare respectively shaking flask culture medium A, B, C, D, E, its medium component is as follows:
A: sucrose 4.0g/L, Trisodium Citrate 2.0g/L, extractum carnis 3.0g/L, yeast powder 3.0g/L, hexanolactam 0.5g/L, sodium-chlor 1.0g/L, potassium primary phosphate 0.5g/L, cobalt chloride 0.005g/L, iron(ic) chloride 0.005g/L, Manganous chloride tetrahydrate 0.005g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 6.0;
B: sucrose 5.0g/L, Trisodium Citrate 2.5g/L, extractum carnis 4.0g/L, yeast powder 4.0g/L, hexanolactam 1.0g/L, sodium-chlor 1.0g/L, potassium primary phosphate 1.0g/L, cobalt chloride 0.01g/L, iron(ic) chloride 0.01g/L, Manganous chloride tetrahydrate 0.01g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 6.5;
C: sucrose 6.0g/L, Trisodium Citrate 3.0g/L, extractum carnis 5.0g/L, yeast powder 5.0g/L, hexanolactam 1.5g/L, sodium-chlor 1.0g/L, potassium primary phosphate 1.5g/L, cobalt chloride 0.015g/L, iron(ic) chloride 0.015g/L, Manganous chloride tetrahydrate 0.015g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 7.0;
D: sucrose 7.0g/L, Trisodium Citrate 3.5g/L, extractum carnis 6.0g/L, yeast powder 6.0g/L, hexanolactam 2.0g/L, sodium-chlor 1.0g/L, potassium primary phosphate 2.0g/L, cobalt chloride 0.02g/L, iron(ic) chloride 0.02g/L, Manganous chloride tetrahydrate 0.02g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 7.5;
E: sucrose 8.0g/L, Trisodium Citrate 4.0g/L, extractum carnis 7.0g/L, yeast powder 7.0g/L, hexanolactam 2.5g/L, sodium-chlor 1.0g/L, potassium primary phosphate 2.5g/L, cobalt chloride 0.025g/L, iron(ic) chloride 0.025g/L, Manganous chloride tetrahydrate 0.025g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 8.0.
Respectively the above-mentioned substratum prepared is poured in 5 250ml shaking flasks into to 121 ℃ of sterilizing 20min with the 40ml/ bottle.Be cooled to after room temperature the volume ratio with 2.5% respectively and access above-mentioned seed liquor, in 30 ℃, under the 150rpm rotating speed, cultivate 72h.Fermentation culture is collected respectively fermented liquid, the centrifugal collection thalline of 8000rpm after finishing.Taking respectively the 0.05g wet thallus is suspended in the 5ml deionized water, add 2-amino-2 after preheating 5min under 30 ℃ of 180rpm rotating speeds, 3-nitrile dimethyl (final concentration is 0.1M) starts reaction, and after 20min, sampling 1000 μ l, add 30 μ l 5M HCl termination reactions.By the amino of the 2-in the gas chromatography determination conversion fluid-2,3-amide dimethyl butyrate content, calculate enzyme and live, and result is as shown in table 1:
Table 1: the enzyme of shake flask fermentation nutrient solution is lived
Embodiment 2: the ferment tank of celebrating sheng, a reed pipe wind instrument rhodococcus ZJB-09153 (CCTCC No:M 2010050) is cultivated
Preparation seed culture medium: glucose 10.0g/L, yeast extract powder 5.0g/L, peptone 2.0g/L, KH
2pO
41.0g/L, K
2hPO
41.0g/L, NaCl 1.0g/L, hexanolactam 1.0g/L, MgSO
40.1g/L, CoCl
20.01g/L, FeSO
40.01g/L solvent is water, regulating pH with 1.0M HCl or NaOH solution is 7.5.Add the 3L seed culture medium in the 5L fermentor tank, access bacterial classification after real tank sterilization, 30 ℃ of temperature, cultivate 24h under the condition of mixing speed 150rpm, obtains seed liquor.
Prepare respectively fermentor tank culture medium A, B, C, D, E, its medium component is as follows:
A: sucrose 4.0g/L, Trisodium Citrate 2.0g/L, extractum carnis 3.0g/L, yeast powder 3.0g/L, hexanolactam 0.5g/L, sodium-chlor 1.0g/L, potassium primary phosphate 0.5g/L, cobalt chloride 0.005g/L, iron(ic) chloride 0.005g/L, Manganous chloride tetrahydrate 0.005g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 6.0;
B: sucrose 5.0g/L, Trisodium Citrate 2.5g/L, extractum carnis 4.0g/L, yeast powder 4.0g/L, hexanolactam 1.0g/L, sodium-chlor 1.0g/L, potassium primary phosphate 1.0g/L, cobalt chloride 0.01g/L, iron(ic) chloride 0.01g/L, Manganous chloride tetrahydrate 0.01g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 6.5;
C: sucrose 6.0g/L, Trisodium Citrate 3.0g/L, extractum carnis 5.0g/L, yeast powder 5.0g/L, hexanolactam 1.5g/L, sodium-chlor 1.0g/L, potassium primary phosphate 1.5g/L, cobalt chloride 0.015g/L, iron(ic) chloride 0.015g/L, Manganous chloride tetrahydrate 0.015g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 7.0;
D: sucrose 7.0g/L, Trisodium Citrate 3.5g/L, extractum carnis 6.0g/L, yeast powder 6.0g/L, hexanolactam 2.0g/L, sodium-chlor 1.0g/L, potassium primary phosphate 2.0g/L, cobalt chloride 0.02g/L, iron(ic) chloride 0.02g/L, Manganous chloride tetrahydrate 0.02g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 7.5;
E: sucrose 8.0g/L, Trisodium Citrate 4.0g/L, extractum carnis 7.0g/L, yeast powder 7.0g/L, hexanolactam 2.5g/L, sodium-chlor 1.0g/L, potassium primary phosphate 2.5g/L, cobalt chloride 0.025g/L, iron(ic) chloride 0.025g/L, Manganous chloride tetrahydrate 0.025g/L, solvent is water, regulating pH with 1.0M HCl or NaOH solution is 8.0.
Add respectively the above culture medium 30L that respectively organizes in the 50L fermentor tank, the sterilization of real tank, be cooled to after room temperature the volume ratio with 2.5% respectively and access above-mentioned seed liquor.30 ℃ of temperature, mixing speed 150rpm, ventilation ratio 0.5: 1, cultivate under the condition of tank pressure 0.06MPa.After cultivating 36h, collect respectively fermented liquid every 12h, the centrifugal collection thalline of 8000rpm.Press the method mensuration enzyme of embodiment 1 and live, result is as shown in table 2:
Table 2: the enzyme of ferment tank nutrient solution is lived
Embodiment 3: containing the collection of enzyme cell
Get according to the fermented liquid 1L that in embodiment 1 and 2, the fermentation of C substratum obtains, processed by following solid-liquid separation method:
A: low-temperature and high-speed is centrifugal, at 4 ℃, and centrifugal 10min under the 12000rpm rotating speed;
B: rolled film filters, flow 120ml/min, pressure 12Kg/cm
2;
C: ceramic membrane filter, flow 120ml/min, pressure 4Kg/cm
2;
D: tubular fibre membrane filtration, flow 120ml/min, pressure 1.0Kg/cm
2;
E: flat plate ultrafiltration membrane filtration, flow 120ml/min, pressure 3Kg/cm
2.
Then use the physiological saline washed cell of 3L 0.9%, collect wet thallus, press the method mensuration enzyme of embodiment 1 and live, result is as shown in table 3:
Table 3: the cellular enzymes after the different modes isolated cell is lived
Embodiment 4:
The celebrating sheng, a reed pipe wind instrument rhodococcus that the rhodococcus erythropolis that the globule nocardia obtained according to low-temperature and high-speed centrifugal method in embodiment 3, rolled film filter method obtain and low-temperature and high-speed centrifugal method obtain, get respectively in the Tris-HCl damping fluid that wet thallus 0.05g is suspended in 5ml pH8.9, add the 2-amino-2 that the substrate final concentration is 0.09M, the 3-nitrile dimethyl, at 15,20,25,30,35,40,45 ℃, under rotating speed 150rpm condition, transform respectively.Press the method mensuration enzyme of embodiment 1 and live, result is as shown in table 4:
Table 4: the catalytic activity of resting cell under differing temps
Embodiment 5:
The celebrating sheng, a reed pipe wind instrument rhodococcus that the rhodococcus erythropolis that the globule nocardia obtained according to embodiment 3 low-temperature and high-speed centrifugal methods, rolled film filter method obtain and low-temperature and high-speed centrifugal method obtain, get respectively in the damping fluid that wet thallus 0.05g is suspended in the different pH values of 5ml.PH value and the type of damping fluid are as shown in table 5:
Table 5: pH value and the type of damping fluid
| Damping fluid | Citrate trianion | Phosphoric acid salt | Tris-HCl | Glycine-sodium hydroxide |
| pH | 5，5.6，6.2 | 6.6，7.2，7.8 | 8.0，8.4，8.8 | 8.9，9.4 |
Add substrate 2-amino-2, the 3-nitrile dimethyl, make 2-amino-2, and the final concentration of 3-nitrile dimethyl is 0.09M.Press the method mensuration enzyme of embodiment 1 and live, result is as shown in table 6:
Table 6: the catalytic activity of resting cell under condition of different pH
Embodiment 6:
The globule nocardia obtained according to embodiment 3 low-temperature and high-speed centrifugal methods, get in the phosphate buffered saline buffer that wet thallus 3.0g is suspended in 180ml pH7.8, at 30 ℃, preheating 5min under the condition of rotating speed 180rpm, add substrate 2-amino-2, the 3-nitrile dimethyl, make its final concentration reach 0.2M, reaction 40min, the sampling gas phase is measured product 2-amino-2 in conversion fluid, the concentration of 3-amide dimethyl butyrate.Detected result 2-amino-2,3-amide dimethyl butyrate concentration 0.191M, productive rate is 95.5%.
Embodiment 7:
The rhodococcus erythropolis obtained according to embodiment 3 rolled film filter methods, get in the phosphate buffered saline buffer that wet thallus 3.0g is suspended in 200ml pH7.2, at 35 ℃, preheating 5min under the condition of rotating speed 150rpm, add substrate 2-amino-2, the 3-nitrile dimethyl, make its final concentration reach 0.15M, reaction 60min, the sampling gas phase is measured product 2-amino-2 in conversion fluid, the concentration of 3-amide dimethyl butyrate.Detected result 2-amino-2,3-amide dimethyl butyrate concentration 0.133M, 2-amino-2,3-diformazan basic point acid amides productive rate is 88.67%.
Embodiment 8:
The celebrating sheng, a reed pipe wind instrument rhodococcus obtained according to embodiment 3 low-temperature and high-speed centrifugal methods, get wet thallus 20g and be suspended in 1L deionized water or damping fluid, pack in three mouthfuls of reaction flasks of 2L, add 10ml substrate 2-amino-2, the 3-nitrile dimethyl, make its final concentration be about 0.075M, at 10 ℃, react under the condition of rotating speed 180rpm.Add 10ml 2-amino-2 every 20min, the 3-nitrile dimethyl, accumulative total adds 10 times.Product 2-amino-2 in sampling vapor detection conversion fluid after reaction finishes, the concentration of 3-amide dimethyl butyrate.Detected result 2-amino-2,3-amide dimethyl butyrate concentration 0.638M, productive rate is 85.1%.
Embodiment 9:
What according to the method for embodiment 8, obtain contains 2-amino-2, the conversion fluid of 3-amide dimethyl butyrate, and the centrifugal 10min of 12000rpm, collect the supernatant liquor containing ADBA; Add Powdered Activated Carbon (0.5%, m/v), heated and stirred decolouring 30min; Proceed to vacuum filtration device suction filtration, obtain containing ADBA decolouring clear liquid; The decolouring clear liquid proceeds to Rotary Evaporators, at vacuum tightness-0.1Mpa, and 40～60 ℃ of water bath heating temperatures, rotating speed 60～100rpm reduction vaporization is removed substrate 2-amino-2,3-nitrile dimethyl and water, cooling 2-amino-2 that obtain, 3-amide dimethyl butyrate crude product.This crude product is dissolved in hot ethyl acetate, adds 10% (v/v) salting-out agent normal hexane, in 0～4 ℃ of insulation crystallization; Filter, the normal hexane washing, drying obtains 2-amino-2,3-amide dimethyl butyrate crystal (yield 90.5%, purity 98.5%).The gained crystal is done
1h NMR, infrared spectra and mass spectrometry results are as follows:
FT-IR：3521/3372/1602(v
NH)，2973(v
CH)，1675(acyl，v
C＝O)，1399(v
C-N)，708 (amino，v
NH)；
1H NMR：δ
H(500MHz；CDCl
3)0.86(3H，d，Me)，0.90(3H，d，Me)，1.3(3H，s，Me)，2.2(1H，m，CH)，5.5(1H，br s，NH)，7.4(1H，br s，NH)；ESI-MS：m/z 283(100％，[2M+Na]
+)，261(82，[2M+H]
+)，131(6.4，M
+)。
Claims (2)
1. a microorganism catalysis legal system is for 2-amino-2, the method of 3-amide dimethyl butyrate, described method comprises: with 2-amino-2, the 3-nitrile dimethyl is substrate, in the reaction system that the Nitrile hydratase that rhodococcus erythropolis (Rhodococcus erythropolis) CCTCC No:M 209244 fermentations of take produce is catalyzer, carry out the catalytic hydration reaction under pH7.2,35 ℃, make described 2-amino-2, the 3-amide dimethyl butyrate;
Described Nitrile hydratase from rhodococcus erythropolis CCTCC No:M 209244, through fermentation, obtain containing the enzyme somatic cells, described containing the enzyme somatic cells in reaction system addition containing the enzyme wet cell weight, to count 15 g/L;
The described enzyme somatic cells that contains prepares by the following method:
To in the rhodococcus erythropolis CCTCC of slant activation No:M 209244 access seed culture mediums, cultivate 24 ~ 48 h under 20 ~ 40 ℃, obtain seed liquor; Described seed culture medium final concentration is composed as follows: glucose 5 ~ 20.0 g/L, yeast extract powder 2 ~ 8.0 g/L, peptone 1 ~ 4.0 g/L, KH
2pO
40.5 ~ 1.5 g/L, K
2hPO
40.5 ~ 1.5 g/L, NaCl 0.5 ~ 3.0 g/L, hexanolactam 0.5 ~ 2.0 g/L, MgSO
40.05 ~ 0.2 g/L, CoCl
20.01 ~ 0.05 g/L, FeSO
40.01～0.05 g/L, solvent is water, and pH 6.0 ~ 8.0;
Seed liquor, with in 1 ~ 10% volume ratio access culture medium, is cultivated to 48 ~ 96 h under 20 ~ 40 ℃, and the fermented liquid that cultivation obtains, through centrifugal or membrane sepn, must contain the enzyme somatic cells; Described culture medium final concentration is composed as follows: sucrose 5.0 ~ 10.0 g/L, Trisodium Citrate 2.0 ~ 5.0 g/L, extractum carnis 2.0 ~ 8.0 g/L, yeast powder 2.0 ~ 8.0 g/L, sodium-chlor 0.5 ~ 2.0 g/L, potassium primary phosphate 0.5 ~ 2.5 g/L, cobalt chloride 0.005 ~ 0.01 g/L, iron(ic) chloride 0.005 ~ 0.01 g/L, Manganous chloride tetrahydrate 0.005 ~ 0.01 g/L, inductor 1.0 ~ 3.0 g/L, solvent is water, pH 6.0 ~ 8.0; Described inductor is hexanolactam;
Described catalytic hydration reaction is:
By described containing the enzyme somatic cells with after physiological saline washing, be suspended in the phosphate buffered saline buffer of water or pH7.2, add substrate 2-amino-2,3-nitrile dimethyl to concentration of substrate is 0.15 M, reaction 60 min under 35 ℃, 150 rpm; After reaction finishes, conversion fluid obtains described 2-amino-2,3-amide dimethyl butyrate through separation and purification.
2. the method for claim 1, is characterized in that described separation purification method is as follows: after the reaction end, get centrifugal 10 min of conversion fluid 12000 rpm, collect supernatant liquor; Add Powdered Activated Carbon, heated and stirred is decoloured after 30 min and is proceeded to vacuum filtration device suction filtration, obtains the clear liquid that decolours; The decolouring clear liquid proceeds to Rotary Evaporators, at vacuum tightness-0.1 Mpa, under 40 ~ 60 ℃ of water bath heating temperatures, rotating speed 60 ~ 100 rpm conditions, reduction vaporization is removed substrate 2-amino-2,3-nitrile dimethyl and water, cooling 2-amino-2 that obtain, 3-amide dimethyl butyrate crude product; By 2-amino-2,3-amide dimethyl butyrate crude product is dissolved in the ethyl acetate of 20 ~ 65 ℃, and adding volume is ethyl acetate volume 10% salting-out agent normal hexane, in 0 ~ 4 ℃ of insulation crystallization; Filter, get filter residue and wash with normal hexane, drying obtains 2-amino-2,3-amide dimethyl butyrate crystal.
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| Title |
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| Hua-Ping Dong et al..Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate by Rhodococcus erythropolis..《Appl Microbiol Biotechnol》.2010,1335-1345. |
| Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate by Rhodococcus erythropolis.;Hua-Ping Dong et al.;《Appl Microbiol Biotechnol》;20100415;1335-1345 * |
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