CN104749273A - Method for detecting azide ions or cyanide ions in water - Google Patents
Method for detecting azide ions or cyanide ions in water Download PDFInfo
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Abstract
The invention provides a method for detecting azide ions or cyanide ions in water. According to the method, halohydrin dehalogenase is utilized for catalyzing azide ions or cyanide ions in water to be reacted with epoxide to generate corresponding 4-azide-3-hydroxyl butanol or 4-cyano-3-hydroxyl butanol, a gas chromatography is adopted to quantitatively analyze the content of the generated 4-azide-3-hydroxyl butanol or 4-cyano-3-hydroxyl butanol according to the standard curve, and the concentration of the azide ions or the cyanide ions in the sample can be determined. According to the method, the accuracy is high, and azide ions and cyanide ions, which have the minimum concentrations up to 0.1mM and 0.3mM can be detected respectively; and besides, the method has good repeatability and high sensitivity, R2 of standard curves of the measured concentrations of the azide ions and the cyanide ions is 0.997 and 0.995 respectively; the method can be used for measuring the concentrations of azide ions or cyanide ions in various samples such as environment wastewater, industrial wastewater and drinking water.
Description
(1) technical field
The present invention relates to the assay method of azides ion and cryanide ion in water, in particular to the azides ion utilized in halide alcohol dehalogenase catalytic water and cryanide ion and epoxide reaction, produce corresponding substituted alcohols, the amount of the substituted alcohols generated by gas chromatographic analysis determines the content of azides ion and cryanide ion in water, and the method is applicable to the detection of azides ion and cryanide ion in water.
(2) background technology
Triazo-compound and prussiate occupy important effect in organic synthesis, and they are usually used to the synthesis of some medicine intermediates and fine chemicals, and wherein sodium azide is also for the manufacture of the air bag of automobile.But azides ion and cryanide ion are all deadly poisonous compounds, can be combined with the haemoglobin of human body, reduce the ability of its transports oxygen, cause people to be choked to death.Therefore, the azides ion in the water of analytical approach detection fast and accurately and cryanide ion is needed.
At present, the method detecting azides ion and cryanide ion in water has spectrophotometric method, chromatography of ions, GC-MS analysis method etc.Wherein spectrophotometric method receives extraneous interference comparatively greatly, and along with the prolongation of time, the numerical fluctuations of reading is comparatively large, and degree of accuracy is not high.Chromatography of ions and gas phase-mass spectrography are comparatively accurate, but complex operation, high to equipment requirement.
(3) summary of the invention
The invention provides a kind of method of azides ion and cryanide ion in indirect detection water, the method is low for equipment requirements, and operational stability is good, simultaneously analyze thus time short, can analyze multiple sample fast.
Technical scheme of the present invention is:
The invention provides a kind of method detecting azide ion ion or cryanide ion in water, described method is: the supernatant of the wet thallus obtained through Fiber differentiation with the recombination engineering bacteria containing halide alcohol dehalogenase encoding gene after ultrasonication is for catalyzer, take epoxide as auxiliary agent, using water sample to be measured as raw material, at 40 ~ 45 DEG C, 30 ~ 60min is reacted under 500rpm condition, extract reaction solution and be extracted with ethyl acetate, get upper organic phase after anhydrous sodium sulfate drying, adopt gas chromatographic detection 4-nitrine-3-butylated hydroxy or 4-cyano-3-hydroxy butanols peak area, according to 4-nitrine-3-butylated hydroxy or 4-cyano-3-hydroxy butanols peak area for ordinate, the sodium azide typical curve made for horizontal ordinate with sodium azide or sodium cyanide concentration or sodium cyanide typical curve, determine the concentration of azide ion ion or cryanide ion in water sample to be measured, the consumption of described catalyzer counts 5-20mg/mL water sample to be measured (preferred 10mg/mL) with the recombination engineering bacteria wet thallus weight before ultrasonication containing halide alcohol dehalogenase encoding gene, and the consumption of epoxide is 0.05 ~ 0.2mol/L water sample to be measured (preferred 0.18mol/L).
Further, the amino acid sequence of described halide alcohol dehalogenase is for shown in SEQ ID NO.2.
Further, gas chromatographic detection condition is: adopt Japanese Shimadzu gas phase GC-14, chromatographic column Astec CHIRALDEX
tMg-TA, carrier gas is helium, and split ratio is 20:1, and the temperature of injection port and detecting device is 220 DEG C, and GC program is 120 DEG C and retains 5min, and 5 DEG C/min is warming up to 140 DEG C, retains 2min.
Further, typical curve is prepared as follows: with the aqueous sodium azide of distilled water preparation 0.2 ~ 2.0mM, respectively to the aqueous sodium azide adding 500 μ l variable concentrations in 2ml EP pipe, 450 μ l epoxy butane solution and 50 μ l halide alcohol dehalogenase crude enzyme liquids, at 40 DEG C, 500rpm, reaction 30min, extract respectively to adding 1ml ethyl acetate in EP pipe, get upper organic phase 800 μ l, the peak area of vapor detection 4-nitrine-3-butylated hydroxy is adopted after anhydrous sodium sulfate drying, with sodium azide concentration for horizontal ordinate, with the peak area of 4-nitrine-3-butylated hydroxy for ordinate, obtain sodium azide typical curve, described halide alcohol dehalogenase crude enzyme liquid is the supernatant of halide alcohol dehalogenase after the wet thallus ultrasonication that fermented and cultured obtains, and the concentration of described crude enzyme liquid counts 0.1g/ml with wet thallus weight before ultrasonication, described epoxy butane solution is the 200mM epoxy butane solution of the PBS buffer with 200mM, pH 7.5, and the making of sodium cyanide typical curve is with sodium azide typical curve.
Further, epoxide is epoxy butane or octylene oxide.
Further, the consumption of epoxide is 0.1mol/L water sample.
Further, the preparation method of catalyzer is: be seeded in the LB nutrient culture media containing the ampicillin of final concentration 50 μ g/ml by the recombination engineering bacteria containing halogenohydrin dehalogenation alcohol encoding gene, be placed in 37 DEG C of shaking tables and be cultured to OD
600when reaching 0.6 ~ 0.8, add final concentration 0.2mM isopropylthiogalactoside, induce 12-14 hour, 9000rpm centrifugal 10 minutes at 28 DEG C of shaking tables, abandon supernatant, the wet thallus of centrifugal acquisition is suspended in the phosphate buffer of 100mM according to the ratio of 0.1g/L, the broken 30min of 50% power ultrasonic, broken mixed liquor centrifugal 20 minutes at 12000rpm, collects supernatant, obtain the crude enzyme liquid containing halide alcohol dehalogenase, be catalyzer.
Detection method is the content that will measure azides ion and cryanide ion, as long as so ensure enough halide alcohol dehalogenase, azides ion and cryanide ion are transformed completely just passable, in crude enzyme liquid of the present invention, the pure enzyme quality of halide alcohol dehalogenase accounts for about 30%.
The quantitative detecting method of azides ion of the present invention and cryanide ion comprises: (1) utilizes the azides ion in halide alcohol dehalogenase catalyzed samples or cryanide ion and epoxide (preferred epoxy butane) to react, and azides ion or cryanide ion is converted into corresponding 4-nitrine-3-butylated hydroxy or 4-cyano-3-hydroxy butanols; (2) utilize gas chromatography quantitatively to detect 4-nitrine-3-butylated hydroxy or 4-cyano-3-hydroxy butanol content, and then determine the content of azides ion and cryanide ion in water.
The present invention utilizes biological catalysis, and the inorganic ions (azides ion or cryanide ion) in water is converted into the organic compound of available gas phase analysis, its reaction principle is shown below:
In formula, HHDH represents halide alcohol dehalogenase (halohydrin dehalogenase).
By ensure the accuracy of survey data, the process measured from Specification Curve of Increasing to solution concentration to be measured, for typical curve solution and the solution to be measured of variable concentrations, in its mensuration process, each step operating parameter is identical.
Survey solution concentration to be measured need in drawn typical curve concentration range, data measured is only accurately, if solution concentration to be measured exceeds the concentration range of typical curve, then likely do not meet its linear relationship, now can by solution dilution to be measured or concentrated after detect, till measured concentration is in typical curve concentration range.
Effective effect of the present invention is mainly reflected in: the inventive method accuracy is good, and the least concentration that can detect azides ion and cryanide ion is respectively 0.1mM and 0.3mM; Meanwhile, this inventive method favorable reproducibility, highly sensitive, the R of the typical curve of its azides ion measured and cyanide ion concentration
2be respectively 0.997 and 0.995, this inventive method can be used for environmental wastewater, the azides ion in the various sample such as industrial waste water and potable water or the concentration determination of cryanide ion.
(4) accompanying drawing explanation
Fig. 1 is the typical curve of 4-nitrine-3-butylated hydroxy and sodium azide.
Fig. 2 is the typical curve of 4-cyano-3-hydroxy butanols and sodium cyanide.
Fig. 3 is the impact that chlorion and bromide ion detect azides ion and cryanide ion.
Fig. 4 is the impact that Thiocyanate ion detects azides ion and cryanide ion.
Fig. 5 is the impact that cyanic acid ion detects azides ion and cryanide ion.
Fig. 6 is the impact that nitrite ion detects azides ion and cryanide ion.
(5) 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: preparation halide alcohol dehalogenase
Halide alcohol dehalogenase genes of SEQ ID NO.1, utilizes technique for gene engineering, realizes it and expresses in e. coli bl21 (DE3), the amino acid sequence of this halogenohydrin dehalogenation alcohol is that (constructing plan is shown in RSC Adv. to SEQ ID NO.2,2014,4,64027-64031).Recombination engineering bacteria containing halide alcohol dehalogenase gene shown in SEQ ID NO.1 is seeded in the LB nutrient culture media containing the ampicillin of final concentration 50 μ g/ml, is placed in 37 DEG C of shaking tables and cultivates, treat OD
600when reaching 0.6 ~ 0.8, add 0.2mM isopropylthiogalactoside, be transferred to 28 DEG C of shaking table induction 12-14 hour.Centrifugal 9000rpm × 10 minute, abandon supernatant.The wet thallus of centrifugal acquisition adds the phosphate buffer of 100mM according to 1/10 (wt/vol) ratio.The broken 30min of 50% power ultrasonic, centrifugal 12000rpm × 20 minute, collect supernatant, be the crude enzyme liquid (every milliliter of crude enzyme liquid obtains after being equivalent to the fragmentation of 0.1g wet thallus) containing halide alcohol dehalogenase, can be placed in refrigerator-4 DEG C of preservations, its vigor does not almost lose when frozen.
Embodiment 2: preparation 4-nitrine-3-butylated hydroxy and 4-cyano-3-hydroxy butanols
In order to determine that azides ion and cryanide ion can form corresponding 4-nitrine-3-butylated hydroxy and 4-cyano-3-hydroxy butanols through the open loop of halide alcohol dehalogenase catalysis epoxy butane, first we utilize halide alcohol dehalogenase catalysis to prepare 4-nitrine-3-butylated hydroxy and 4-cyano-3-hydroxy butanols.
Preparation 4-nitrine-3-butylated hydroxy: add 100ml PBS (200mM in 250ml reactor, pH7.5), 1ml (0.017mol) epoxy butane, 2g (0.03mol) sodium azide and 8g halide alcohol dehalogenase wet thallus (prepared by embodiment 1).The temperature of system is risen to 40 DEG C, stirs 200rpm.Use gas phase monitoring reaction course, when epoxy butane conversion ratio is greater than 99%, stop reaction.Centrifugal 9000rpm × 10 minute, supernatant with 200ml extraction into ethyl acetate once, be separated organic phase after anhydrous sodium sulfate drying, decompression distillation is flowed out to not having solvent, acquisition yellow liquid material 4-nitrine-3-butylated hydroxy.NMR characterizes:
1h NMR (500MHz, CDCl
3) δ 3.71-3.37 (dd, J=5.8,3.3Hz, 1H), 3.29 (dd, J=12.4,3.3Hz, 1H), 3.27-3.25 (dd, J=12.4,7.4Hz, 1H), 2.05 (s, 1H), 1.55-1.52 (m, 2H), (1.00-0.97 t, J=7.5Hz, 3H).
13C NMR(126MHz,CDCl
3)δ72.28,56.71,56.71,27.30,9.63。
Preparation 4-cyano-3-hydroxy butanols: add 100ml PBS (200mM in 250ml reactor, pH7.5), 1ml (0.017mol) epoxy butane, 1.5g (0.03mol) sodium azide and 8g halide alcohol dehalogenase wet thallus (prepared by embodiment 1).The temperature of system is risen to 40 DEG C, stirs 200rpm.Use gas phase monitoring reaction course, when epoxy butane conversion ratio is greater than 99%, stop reaction.Centrifugal 9000rpm × 10 minute, supernatant with 200ml extraction into ethyl acetate once, be separated organic phase after anhydrous sodium sulfate drying, decompression distillation is flowed out to not having solvent, acquisition yellow liquid material 4-cyano-3-hydroxy butanols.NMR characterizes:
1h NMR (500MHz, CDCl
3) δ 3.89-3.86 (dd, J=6.1,5.0Hz, 1H), 2.56-2.47 (m, 3H), 1.64-1.61 (m, 2H), 1.00-0.97 (t, J=7.5Hz, 3H).
13C NMR(126MHz,CDCl
3)δ117.92,69.14,29.28,25.57,9.90。
Vapor detection epoxy butane: Agilent GC-7890A, chromatographic column HP-5, carrier gas is nitrogen, and the temperature of injection port and detecting device is respectively 230 DEG C and 250 DEG C, and GC program is, 60 DEG C retain 4min, and 20 DEG C/min is warming up to 120 DEG C.The retention time of epoxy butane is 2.7min.
The foundation of embodiment 3:4-nitrine-3-butylated hydroxy and 4-cyano-3-hydroxy butanols vapor detection method
4-nitrine-3-butylated hydroxy and 4-cyano-3-hydroxy butanols adopt Japanese Shimadzu gas phase GC-14, chromatographic column Astec CHIRALDEX
tMg-TA, carrier gas is helium, and split ratio is 20:1, and the temperature of injection port and detecting device is 220 DEG C, and GC program is 120 DEG C and retains 5min, and 5 DEG C/min is warming up to 140 DEG C, retains 2min.The retention time of 4-nitrine-3-butylated hydroxy and 4-cyano-3-hydroxy butanols is respectively 4.62min and 7.56min.
Embodiment 4: the typical curve drawing azides ion and cryanide ion
The key of the inventive method to ensure that azides ion in testing sample and cryanide ion will be converted into corresponding substituted alcohols completely, therefore we will consider the following aspects: (1) needs excessive epoxy butane substrate, (2) enough halide alcohol dehalogenase, the top condition of (3) halide alcohol dehalogenase catalysis.In view of some consideration above, we establish following mensuration scheme:
Prepare the epoxy butane solution of 200mM, prepare as solvent with 200mM (pH 7.5) PBS damping fluid.
Prepare halide alcohol dehalogenase crude enzyme liquid, the halide alcohol dehalogenase crude enzyme liquid prepared by embodiment 1.
(1) 4-nitrine-3-butylated hydroxy typical curve: the 4-nitrine-3-butylated hydroxy aqueous solution preparing 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM and 1.0mM with distilled water, get each 1ml of 4-nitrine-3-butylated hydroxy aqueous solution of variable concentrations, join respectively in different 2ml EP pipes, add 1ml ethyl acetate subsequently respectively and extract.Get upper organic phase 800 μ l, after anhydrous sodium sulfate drying, carry out vapor detection (testing conditions is with embodiment 3), with 4-nitrine-3-butylated hydroxy concentration for horizontal ordinate, with 4-nitrine-3-butylated hydroxy peak area for ordinate, 4-nitrine-3-butylated hydroxy typical curve is y=3375.33x-42.03 (R
2=0.996), Fig. 1 is seen.
(2) sodium azide typical curve: prepare the aqueous sodium azide of 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM and 2.0mM with distilled water as water sample to be measured, respectively to the aqueous sodium azide adding 500 μ l variable concentrations in 2mlEP pipe, the halide alcohol dehalogenase crude enzyme liquid of 450 μ l epoxy butane solution and 50 μ l.Be placed in Thermomixer reactor, 40 DEG C, 500rpm, reaction 30min.Add 1ml extraction into ethyl acetate respectively in EP pipe, get upper organic phase 800 μ l after anhydrous sodium sulfate drying, vapor detection 4-nitrine-3-butylated hydroxy peak area (conditions see example 3).With 4-nitrine-3-butylated hydroxy peak area for ordinate, with sodium azide concentration for horizontal ordinate, make the typical curve y=3306.37x-20.11 (R of sodium azide
2=0.997) (see Fig. 1).
(3) 4-cyano-3-hydroxy butanols typical curve: the 4-cyano-3-hydroxy butanols aqueous solution preparing 0.3mM, 0.6mM, 0.9mM, 1.2mM, 1.5mM and 2.0mM with distilled water, get each 1ml of 4-cyano-3-hydroxy butanols aqueous solution of variable concentrations, join respectively in different 2ml EP pipes, add 1ml ethyl acetate subsequently respectively and extract.Get upper organic phase 800 μ l, after anhydrous sodium sulfate drying, carry out vapor detection (see embodiment 3).With the concentration of 4-cyano-3-hydroxy butanols for horizontal ordinate, take peak area as ordinate, the typical curve of 4-cyano-3-hydroxy butanols is y=1518.46x-67.52 (R
2=0.993) (see Fig. 2).
(4) sodium cyanide typical curve: the sodium cyanide solution preparing 0.6mM, 1.2mM, 1.8mM, 2.4mM, 3.0mM and 4.0mM with distilled water, the sodium cyanide solution of 500 μ l variable concentrations is added, the halide alcohol dehalogenase crude enzyme liquid of 450 μ l epoxy butane solution and 50 μ l in 2ml EP pipe.Be placed in Thermomixer reactor, 40 DEG C, 500rpm, reaction 30min.In EP pipe, add 1ml extraction into ethyl acetate, get upper organic phase 800 μ l after anhydrous sodium sulfate drying, carry out vapor detection 4-cyano-3-hydroxy butanols peak area (see embodiment 3).Take sodium cyanide concentration as horizontal ordinate, with 4-cyano-3-hydroxy butanols peak area for ordinate, the typical curve of sodium cyanide is y=1371.50x-40.75 (R
2=0.995) (see Fig. 2).
Fig. 2 can find out, the typical curve of sodium cyanide is similar to the linear relationship of the typical curve of 4-cyano-3-hydroxy butanols, illustrates that the typical curve of the sodium cyanide set up is accurately feasible.
Embodiment 5: utilize halide alcohol dehalogenase to measure azides ion and cryanide ion
With sodium azide solution or the sodium cyanide solution 10mL of distilled water random arrangement unknown concentration, as azides ion sample to be measured or cryanide ion sample.
Mensuration scheme: respectively testing sample pure water is diluted 2 times, 5 times and 1 times, 500 μ l dilute sample solution are added, 450 μ l epoxy butane solution (embodiment 4 is prepared) and 50 μ l halide alcohol dehalogenase crude enzyme liquids (embodiment 1) in 2ml EP pipe.Be placed in Thermomixer reactor, 40 DEG C, 500rpm, reaction 30min.In EP pipe, add 1ml extraction into ethyl acetate, get upper organic phase after anhydrous sodium sulfate drying, carry out gas phase analysis.4-nitrine-3-the butylated hydroxy obtained or the gas phase peak area of 4-cyano-3-hydroxy butanols and sodium cyanide typical curve or sodium azide typical curve compare, select the extension rate of peak area within the scope of respective standard curve peak area, obtain the concentration of azides ion or cryanide ion in water sample to be measured, result is as shown in table 1.
The mensuration of table 1 azides ion and cyanide ion concentration
[a]actual concentrations is with the calculated by peak area of diluting 10, and the peak area diluting 5 times and 2 times all exceeds the typical curve of sodium azide, and computing formula (1) is:
X
[actual concentrations]=((Y
[peak area]+ 20.11)/3306.37) × 2
[multiple of reaction dilution]× 10
[multiple of Sample Dilution]
X in formula (1)
[actual concentrations]for the actual concentrations measured, Y
[peak area]for 4-nitrine-3-butylated hydroxy peak area, 2
[multiple of reaction dilution]refer to 500ul water sample, reaction final volume is 1mL, namely dilutes 2 times.
[b]actual concentrations, with the calculated by peak area of diluting 10, also can dilute the calculated by peak area of 5 times, and the peak area diluting 2 times exceeds the typical curve of sodium cyanide.Computing formula (2) is:
X
[actual concentrations]=((Y
[peak area]+ 67.52)/1515.46) × 2
[multiple of reaction dilution]× 10
[multiple of Sample Dilution]
X in formula (2)
[actual concentrations]for the actual concentrations measured, Y
[peak area]for 4-cyano-3-hydroxy butanols peak area, 2
[multiple of reaction dilution]refer to 500ul water sample, reaction final volume is 1mL, namely dilutes 2 times.
Embodiment 6: metallic ion and detergent are on the impact of halide alcohol dehalogenase vigor
Consider in detected sample can there is some metallic ions and detergent as Tween 80 and Tween 20, the present invention has also investigated part metals ion and detergent to the impact of halide alcohol dehalogenase catalytic reaction.Do not allow 1, the 3-bis-chloro-2-aqueous propanol solution of the reagent solution of concentration (see table 1) and 50mM with distilled water preparation, do catalytic reaction with halide alcohol dehalogenase crude enzyme liquid (embodiment 1).500 μ l reagent solutions are added, 1, the 3-bis-chloro-2-aqueous propanol solution of 450 μ l 50mM and 50 μ l halide alcohol dehalogenase crude enzyme liquids in 2ml EP pipe.Be placed in Thermomixer reactor, 40 DEG C, 500rpm, reaction 10min.Control reaction 500 μ l distilled water replace reagent solution.The relative activity under each condition is calculated with the turnout of product epichlorokydrin.Enzyme is lived and is defined as at 40 DEG C, and under the reaction conditions of pH 7.5, the enzyme amount required for catalysis per minute 1 μm of ol epichlorokydrin synthesis is 1 Ge Meihuo unit.
As shown in table 2, Fe
3+, Ba
2+, Al
3+and Cu
2+have stronger inhibiting effect to the vigor of halide alcohol dehalogenase, and EDTA only has slight impact to halide alcohol dehalogenase vigor.Therefore, in the process of practical measurement, in order to ensure efficient halide alcohol dehalogenase vigor, metallic ion can be removed with edta reagent, so can ensure the accuracy of assaying reaction.The gas phase process of the vapor detection embodiment 2 of the chloro-2-propyl alcohol of 1,3-bis-and epichlorokydrin, the retention time of the chloro-2-propyl alcohol of 1,3-bis-and epichlorokydrin is respectively 4.04min and 6.41min.
Table 2. metallic ion and detergent are on the impact of halide alcohol dehalogenase catalysis activity
| Reagent | Concentration | Relative activity (%) |
| Contrast | - | 100.0 |
| Fe 2+ | 5mM | 69.02±0.34 |
| Ni 2+ | 5mM | 59.7±1.92 |
| Cu 2+ | 5mM | 23.74±3.56 |
| Ca 2+ | 5mM | 98.04±1.23 |
| Mn 2+ | 5mM | 128.34±0.41 |
| Zn 2+ | 5mM | 76.60±3.78 |
| Mg 2+ | 5mM | 101.05±6.34 |
| Al 3+ | 5mM | 13.56±0.98 |
| Fe 3+ | 5mM | 0±0.0 |
| Ba 2+ | 1mM | 0±0.0 |
| EDTA | 5mM | 79.86±0.86 |
| Tween 80 | 2％(V/V) | 92.70±0.66 |
| Tween 20 | 2％(V/V) | 70.98±0.78 |
Embodiment 7: chlorion, bromide ion, nitrite anions, thiocyanate and cyanate radical are on the impact detected
Consider that halide alcohol dehalogenase also can catalysis chlorion, bromide ion, nitrite anions, thiocyanate and cyanate radical and epoxide be reacted, therefore, the present invention has also investigated the impact that chlorion, bromide ion, nitrite anions, thiocyanate and cyanate radical detect azides ion and cryanide ion.
Prepare the sodium chloride of 20mM, sodium bromide, sodium nitrite, sodium sulfocynanate and Zassol aqueous solution respectively as sample solution, 500 μ l each sample solution above-mentioned is added in 2ml EP pipe, the halide alcohol dehalogenase crude enzyme liquid (embodiment 1) of 450 μ l epoxy butane solution (200mM, embodiment 4 is prepared) and 50 μ l.Be placed in Thermomixer reactor, 40 DEG C, 500rpm, reaction 30min.In EP pipe, add 1ml extraction into ethyl acetate, get upper organic phase through anhydrous sodium sulfate drying.In organic phase, adding a certain amount of 4-nitrine-3-butylated hydroxy or 4-cyano-3-hydroxy butanols respectively, (addition is how many on this experiment not impact, fundamental purpose judges whether compound that other nucleopilic reagent is formed affects the detection of the compound that azides ion or cryanide ion are formed, the present embodiment addition is about in 500 μ l ethyl acetate and adds 1 μ l 4-nitrine-3-butylated hydroxy or 4-cyano-3-hydroxy butanols), carry out gas phase analysis.
Whether the product that observation sodium chloride, sodium bromide, sodium nitrite, sodium sulfocynanate and Zassol and epoxy butane react detects impact to 4-nitrine-3-butylated hydroxy and 4-cyano-3-hydroxy butanols.Wherein chlorion and the detection of bromide ion on azides ion and cryanide ion do not affect (Fig. 3), the detection of Thiocyanate ion on azides ion and cryanide ion does not affect (Fig. 4), the detection of cyanic acid ion azides ion and cryanide ion does not affect (Fig. 5), and but nitrite ion does not affect the detection of azides ion affects the detection (Fig. 6) of cryanide ion.
Claims (7)
1. one kind is detected the method for azide ion ion or cryanide ion in water, it is characterized in that described method is: with containing supernatant after the wet thallus ultrasonication that Fiber differentiation obtains of the recombination engineering bacteria of halide alcohol dehalogenase encoding gene for catalyzer, take epoxide as auxiliary agent, using water sample to be measured as raw material, at 40 ~ 45 DEG C, 30 ~ 60min is reacted under 500rpm condition, extract reaction solution and be extracted with ethyl acetate, get upper organic phase after anhydrous sodium sulfate drying, adopt gas chromatographic detection 4-nitrine-3-butylated hydroxy or 4-cyano-3-hydroxy butanols peak area, according to 4-nitrine-3-butylated hydroxy or 4-cyano-3-hydroxy butanols peak area for ordinate, the sodium azide typical curve made for horizontal ordinate with sodium azide or sodium cyanide concentration or sodium cyanide typical curve, determine the concentration of azide ion ion or cryanide ion in water sample to be measured, the consumption of described catalyzer counts 5 ~ 20mg/mL water sample to be measured with the recombination engineering bacteria wet thallus weight before ultrasonication containing halide alcohol dehalogenase encoding gene, and the consumption of epoxide is 0.05 ~ 0.2mol/L water sample to be measured.
2. detect the method for azide ion ion or cryanide ion in water as claimed in claim 1, it is characterized in that the amino acid sequence of described halide alcohol dehalogenase is for shown in SEQ ID NO.2.
3. detect the method for azide ion ion or cryanide ion in water as claimed in claim 1, it is characterized in that gas chromatographic detection condition is: adopt Japanese Shimadzu gas phase GC-14, chromatographic column AstecCHIRALDEX
tMg-TA, carrier gas is helium, and split ratio is 20:1, and the temperature of injection port and detecting device is 220 DEG C, and GC program is 120 DEG C and retains 5min, and 5 DEG C/min is warming up to 140 DEG C, retains 2min.
4. detect the method for azide ion ion or cryanide ion in water as claimed in claim 1, it is characterized in that typical curve is prepared as follows: with the aqueous sodium azide of distilled water preparation 0.2 ~ 2.0mM, respectively to the aqueous sodium azide adding 500 μ l variable concentrations in 2ml EP pipe, 450 μ l epoxy butane solution and 50 μ l halide alcohol dehalogenase crude enzyme liquids, at 40 DEG C, 500rpm, reaction 30min, extract respectively to adding 1ml ethyl acetate in EP pipe, get upper organic phase 800 μ l, the peak area of vapor detection 4-nitrine-3-butylated hydroxy is adopted after anhydrous sodium sulfate drying, with sodium azide concentration for horizontal ordinate, with the peak area of 4-nitrine-3-butylated hydroxy for ordinate, obtain sodium azide typical curve, described halide alcohol dehalogenase crude enzyme liquid is the supernatant of halide alcohol dehalogenase after the wet thallus ultrasonication that fermented and cultured obtains, and the concentration of described crude enzyme liquid counts 0.1g/ml with wet thallus weight before ultrasonication, described epoxy butane solution is the 200mM epoxy butane solution of the PBS buffer with 200mM, pH 7.5, and the making of sodium cyanide typical curve is with sodium azide typical curve.
5. detect the method for azide ion ion or cryanide ion in water as claimed in claim 1, it is characterized in that epoxide is epoxy butane or octylene oxide.
6. detect the method for azide ion ion or cryanide ion in water as claimed in claim 1, it is characterized in that the consumption of epoxide is 0.1mol/L water sample.
7. detect the method for azide ion ion or cryanide ion in water as claimed in claim 1, it is characterized in that the preparation method of catalyzer is: be seeded in the LB nutrient culture media containing the ampicillin of final concentration 50 μ g/ml by the recombination engineering bacteria containing halogenohydrin dehalogenation alcohol encoding gene, be placed in 37 DEG C of shaking tables and be cultured to OD
600when reaching 0.6 ~ 0.8, add final concentration 0.2mM isopropylthiogalactoside, induce 12-14 hour, 9000rpm centrifugal 10 minutes at 28 DEG C of shaking tables, abandon supernatant, the wet thallus of centrifugal acquisition is suspended in the phosphate buffer of 100mM according to the ratio of 0.1g/L, the broken 30min of 50% power ultrasonic, broken mixed liquor centrifugal 20 minutes at 12000rpm, collects supernatant, obtain the crude enzyme liquid containing halide alcohol dehalogenase, be catalyzer.
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