CN104749273A - Method for detecting azide ions or cyanide ions in water - Google Patents

Abstract

The invention provides a method for detecting azide ions or cyanide ions in water. The method uses halohydrin dehalogenase to catalyze the reaction of azide ions or cyanide ions in water with epoxides to produce the corresponding 4-azido- 3-hydroxybutanol or 4-cyano-3-hydroxybutanol, according to the standard curve, use gas chromatography to quantitatively analyze the content of 4-azido-3-hydroxybutanol or 4-cyano-3-hydroxybutanol , and then determine the concentration of azide ion or cyanide ion in the sample; the accuracy of the inventive method is good, and the minimum concentration that can detect azide ion and cyanide ion is respectively 0.1mM and 0.3mM; Simultaneously, this inventive method reproducibility is good, The sensitivity is high, and the ^R2 of the standard curve of the azide ion and the cyanide ion concentration of its determination are respectively 0.997 and 0.995. concentration determination.

Description

A method for detecting azide ion or cyanide ion in water

(1) Technical field

The invention relates to a method for measuring azide ions and cyanide ions in water, in particular to the use of halohydrin dehalogenase to catalyze the reaction of azide ions and cyanide ions in water with epoxides to produce corresponding substituted alcohols, which are analyzed by gas chromatography. The amount of alcohol is used to determine the content of azide ion and cyanide ion in water. This method is suitable for the detection of azide ion and cyanide ion in water.

(2) Background technology

Azides and cyanides play an important role in organic synthesis reactions. They are often used in the synthesis of some pharmaceutical intermediates and fine chemicals. Among them, sodium azide is also used in the manufacture of automobile airbags. However, azide ions and cyanide ions are highly toxic compounds that can combine with human hemoglobin to reduce its ability to transport oxygen and cause people to suffocate to death. Therefore, a fast and accurate analytical method is needed for the detection of azide and cyanide ions in water.

At present, methods for detecting azide ions and cyanide ions in water include spectrophotometry, ion chromatography, gas chromatography-mass spectrometry and so on. Among them, spectrophotometry is more subject to external interference, and as time goes on, the read value fluctuates greatly, and the accuracy is not high. Ion chromatography and gas chromatography-mass spectrometry are more accurate, but the operation is cumbersome and requires high equipment.

(3) Contents of the invention

The invention provides a method for indirect detection of azide ions and cyanide ions in water. The method has low equipment requirements, good operation stability, short time for simultaneous analysis, and can rapidly analyze multiple samples.

Technical scheme of the present invention is:

The invention provides a method for detecting azide ions or cyanide ions in water. The method is: using the recombinant genetically engineered bacteria containing the halohydrin dehalogenase coding gene to obtain the wet bacterium obtained through ultrasonic crushing. The clear liquid is used as a catalyst, epoxy is used as an auxiliary agent, and the water sample to be tested is used as a raw material. The reaction is carried out at 40-45°C and 500 rpm for 30-60 minutes. Use gas chromatography to detect the peak area of 4-azido-3-hydroxybutanol or 4-cyano-3-hydroxybutanol after drying over sodium sulfate in water, according to 4-azido-3-hydroxybutanol or 4-cyano -3-Hydroxybutanol peak area is the ordinate, and the sodium azide or sodium cyanide concentration is the abscissa standard curve of sodium azide or sodium cyanide standard curve, to determine the azide ion in the water sample to be tested Or the concentration of cyanide ion; The consumption of described catalyzer is 5-20mg/mL water sample to be tested (preferably 10mg/mL) by the recombinant genetically engineered bacterium that contains halohydrin dehalogenase coding gene through sonication before wet thallus weight meter , the amount of epoxide is 0.05-0.2 mol/L water sample to be tested (preferably 0.18 mol/L).

Further, the amino acid sequence of the halohydrin dehalogenase is shown in SEQ ID NO.2.

Further, the gas chromatographic detection conditions are as follows: Shimadzu gas phase GC-14, chromatographic column Astec CHIRALDEX ^TM G-TA, carrier gas is helium, split ratio is 20:1, and the temperature of the inlet and detector is 220°C. ℃, the GC program is 120°C for 5 minutes, 5°C/min to 140°C, and hold for 2 minutes.

Further, the standard curve is prepared as follows: prepare 0.2-2.0 mM sodium azide aqueous solution with distilled water, add 500 μl of sodium azide aqueous solution of different concentrations, 450 μl of butylene oxide solution and 50 μl of halohydrin into 2ml EP tubes Dehalogenase crude enzyme solution, at 40°C, 500rpm, react for 30min, add 1ml of ethyl acetate to the EP tube for extraction, take 800μl of the upper organic phase, dry it with anhydrous sodium sulfate, and use gas phase to detect 4-azide- The peak area of 3-hydroxybutanol, taking the concentration of sodium azide as the abscissa, and the peak area of 4-azido-3-hydroxybutanol as the ordinate, obtains the sodium azide standard curve; The crude enzyme liquid of halogenase is the supernatant liquid obtained by fermenting and culturing wet cells of halohydrin dehalogenase after ultrasonic crushing, and the concentration of the crude enzyme liquid is 0.1 g/ml based on the weight of wet cells before ultrasonic crushing; Described butylene oxide solution is the 200mM butylene oxide solution prepared with the PBS damping fluid of 200mM, pH 7.5, and the making of sodium cyanide standard curve is the same as sodium azide standard curve.

Further, the epoxide is butylene oxide or octene oxide.

Further, the dosage of epoxide is 0.1mol/L water sample.

Further, the preparation method of the catalyst is as follows: inoculate the recombinant genetically engineered bacteria containing the coding gene of halohydrin dehalohydrin in LB medium containing ampicillin at a final concentration of 50 μg/ml, and culture it in a shaker at 37°C to OD ₆₀₀ When it reaches 0.6-0.8, add the final concentration of 0.2mM isopropyl thiogalactopyranoside, induce on a shaker at 28°C for 12-14 hours, centrifuge at 9000rpm for 10 minutes, discard the supernatant, and centrifuge the wet cells obtained at a rate of 0.1g/ The ratio of L was suspended in 100 mM phosphate buffer, ultrasonically crushed at 50% power for 30 minutes, the crushed mixture was centrifuged at 12000 rpm for 20 minutes, and the supernatant was collected to obtain a crude enzyme solution containing halohydrin dehalogenase, which was the catalyst.

The detection method of the present invention is to determine the content of azide ions and cyanide ions, so as long as sufficient halohydrin dehalogenase is ensured to completely convert azide ions and cyanide ions, the halohydrin dehalogenation in the crude enzyme liquid of the present invention The quality of pure enzyme accounts for about 30%.

The quantitative detection method of azide ion and cyanide ion of the present invention comprises: (1) utilize halohydrin dehalogenase to catalyze the reaction of azide ion or cyanide ion in the sample and epoxide (preferably butylene oxide), and azide ion Or cyanide ion is converted into corresponding 4-azido-3-hydroxybutanol or 4-cyano-3-hydroxybutanol; (2) Utilize gas chromatography to quantitatively detect 4-azido-3-hydroxybutanol or 4- The content of cyano-3-hydroxybutanol, and then determine the content of azide ion and cyanide ion in water.

The present invention utilizes biocatalysis to convert inorganic ions (azide ions or cyanide ions) in water into organic compounds that can be analyzed by gas phase. The reaction principle is shown in the following formula:

In the formula, HHDH represents halohydrin dehalogenase (halohydrin dehalogenase).

In order to ensure the accuracy of the measured data, during the process from the drawing of the standard curve to the determination of the concentration of the solution to be tested, for the standard curve solution and the solution to be tested with different concentrations, the operating parameters of each step in the determination process are exactly the same.

The measured concentration of the solution to be tested must be within the concentration range of the standard curve drawn, and the measured data is accurate. If the concentration of the solution to be tested exceeds the concentration range of the standard curve, it may not conform to its linear relationship. At this time, the After the solution to be tested is diluted or concentrated, it is detected until the measured concentration is within the concentration range of the standard curve.

The effective effect of the present invention is mainly reflected in: the method of the present invention has good accuracy, and the minimum concentrations that can detect azide ion and cyanide ion are respectively 0.1mM and 0.3mM; meanwhile, the method of the invention has good reproducibility and high sensitivity, and its determination The ^R2 of the standard curve of the azide ion and cyanide ion concentration is 0.997 and 0.995 respectively, and the inventive method can be used for the concentration determination of the azide ion or cyanide ion in various samples such as environmental sewage, industrial waste water and drinking water.

(4) Description of drawings

Figure 1 is a standard curve of 4-azido-3-hydroxybutanol and sodium azide.

Fig. 2 is the standard curve of 4-cyano-3-hydroxybutanol and sodium cyanide.

Figure 3 shows the effect of chloride and bromide ions on the detection of azide and cyanide ions.

Figure 4 shows the effect of thiocyanate ion on the detection of azide ion and cyanide ion.

Figure 5 shows the effect of cyanate ion on the detection of azide ion and cyanide ion.

Figure 6 shows the effect of nitrite ions on the detection of azide ions and cyanide ions.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Embodiment 1: Preparation of halohydrin dehalogenase

The halohydrin dehalogenase gene SEQ ID NO.1 is expressed in Escherichia coli BL21 (DE3) by using genetic engineering technology. The amino acid sequence of the halohydrin dehalohydrin is SEQ ID NO.2 (see RSC Adv ., 2014, 4, 64027-64031). Inoculate the recombinant genetically engineered bacteria containing the halohydrin dehalogenase gene shown in SEQ ID NO.1 in LB medium containing ampicillin at a final concentration of 50 μg/ml, and culture it in a shaker at 37°C until the OD ₆₀₀ reaches At 0.6 to 0.8, add 0.2 mM isopropyl thiogalactoside and transfer to a shaker at 28°C for induction for 12-14 hours. Centrifuge at 9000 rpm for 10 minutes and discard the supernatant. The wet cells obtained by centrifugation were added with 100 mM phosphate buffer at a ratio of 1/10 (wt/vol). Sonicate at 50% power for 30 minutes, centrifuge at 12000 rpm × 20 minutes, collect the supernatant, which is the crude enzyme solution containing halohydrin dehalogenase (each milliliter of crude enzyme solution is equivalent to 0.1g of wet bacterial cell crushing), which can be placed in Stored in the refrigerator at -4°C, there is almost no loss of its activity in the case of frozen storage.

Embodiment 2: Preparation of 4-azido-3-hydroxybutanol and 4-cyano-3-hydroxybutanol

In order to confirm that the azide ion and cyanide ion will form the corresponding 4-azido-3-hydroxybutanol and 4-cyano-3-hydroxybutanol through halohydrin dehalogenase-catalyzed ring opening of butylene oxide, first we use the halogen Alcohol dehalogenase catalyzes the production of 4-azido-3-hydroxybutanol and 4-cyano-3-hydroxybutanol.

Preparation of 4-azido-3-hydroxybutanol: Add 100ml PBS (200mM, pH7.5), 1ml (0.017mol) butylene oxide, 2g (0.03mol) sodium azide and 8g halide to a 250ml reactor Alcohol dehalogenase wet cells (prepared in Example 1). The temperature of the system was raised to 40° C. and stirred at 200 rpm. The reaction process is monitored by gas phase, and the reaction is stopped when the conversion rate of butylene oxide is greater than 99%. Centrifuge at 9000rpm for 10 minutes, extract the supernatant once with 200ml ethyl acetate, separate the organic phase and dry it over anhydrous sodium sulfate, distill under reduced pressure until no solvent flows out, and obtain the yellow liquid substance 4-azido-3-hydroxybutanol . NMR characterization: ¹ H NMR (500MHz, CDCl ₃ ) δ3.71-3.37 (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). ¹³ C NMR (126 MHz, CDCl ₃ ) δ 72.28, 56.71, 56.71, 27.30, 9.63.

Preparation of 4-cyano-3-hydroxybutanol: Add 100ml PBS (200mM, pH7.5), 1ml (0.017mol) butylene oxide, 1.5g (0.03mol) sodium azide and 8g Halohydrin dehalogenase wet cells (prepared in Example 1). The temperature of the system was raised to 40° C. and stirred at 200 rpm. The reaction process is monitored by gas phase, and the reaction is stopped when the conversion rate of butylene oxide is greater than 99%. Centrifuge at 9000rpm for 10 minutes, extract the supernatant once with 200ml ethyl acetate, separate the organic phase and dry it over anhydrous sodium sulfate, distill under reduced pressure until no solvent flows out, and obtain the yellow liquid substance 4-cyano-3-hydroxybutanol . NMR characterization: ¹ H NMR (500MHz, CDCl ₃ ) δ3.89-3.86 (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). ¹³ C NMR (126 MHz, CDCl ₃ ) δ 117.92, 69.14, 29.28, 25.57, 9.90.

Gas phase detection of butylene oxide: Agilent GC-7890A, chromatographic column HP-5, carrier gas is nitrogen, the temperature of the inlet and detector is 230°C and 250°C respectively, the GC program is 60°C for 4min, 20°C /min to 120°C. The retention time of butylene oxide is 2.7min.

Example 3: Establishment of gas phase detection method for 4-azido-3-hydroxybutanol and 4-cyano-3-hydroxybutanol

4-Azido-3-hydroxybutanol and 4-cyano-3-hydroxybutanol were used Shimadzu gas phase GC-14, the chromatographic column Astec CHIRALDEX ^TM G-TA, the carrier gas was helium, and the split ratio was 20:1 , the temperature of the injection port and the detector is 220°C, the GC program is 120°C for 5 minutes, 5°C/min to 140°C, and hold for 2 minutes. The retention times of 4-azido-3-hydroxybutanol and 4-cyano-3-hydroxybutanol were 4.62min and 7.56min, respectively.

Embodiment 4: draw the standard curve of azide ion and cyanide ion

The key of the inventive method is to guarantee that the azide ion and the cyanide ion in the sample to be tested will be completely converted into corresponding substituted alcohols, so we will consider the following aspects: (1) need excessive butylene oxide substrate, (2) Sufficient amount of halohydrin dehalogenase, (3) optimal conditions for the catalysis of the halohydrin dehalogenase. In view of the above considerations, we have established the following measurement scheme:

Prepare 200mM butylene oxide solution and use 200mM (pH 7.5) PBS buffer as solvent.

Prepare the crude halohydrin dehalogenase enzyme liquid, using the crude halohydrin dehalogenase enzyme liquid prepared in Example 1.

(1) 4-azido-3-hydroxybutanol standard curve: prepare 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM and 1.0mM 4-azido-3-hydroxybutanol aqueous solution with distilled water, take 1ml of 4-azido-3-hydroxybutanol aqueous solutions with different concentrations were added to different 2ml EP tubes, and then 1ml of ethyl acetate was added for extraction. Take 800 μ l of the upper organic phase, dry it over anhydrous sodium sulfate, and carry out gas phase detection (detection conditions are the same as in Example 3), with the concentration of 4-azido-3-hydroxybutanol as the abscissa and the concentration of 4-azido-3- The peak area of hydroxybutanol is the ordinate, and the standard curve of 4-azido-3-hydroxybutanol is y=3375.33x-42.03 (R ² =0.996), see FIG. 1 .

(2) Sodium azide standard curve: prepare 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM and 2.0mM sodium azide aqueous solutions with distilled water as the water samples to be tested, and add 500μl of different concentration of sodium azide aqueous solution, 450 μl of butylene oxide solution and 50 μl of halohydrin dehalogenase crude enzyme solution. Place in a Thermomixer reactor at 40°C, 500rpm, and react for 30min. Add 1 ml of ethyl acetate to the EP tubes for extraction, take 800 μl of the upper organic phase and dry it over anhydrous sodium sulfate, and detect the peak area of 4-azido-3-hydroxybutanol by gas phase (see Example 3 for the conditions). Taking the peak area of 4-azido-3-hydroxybutanol as the ordinate and the concentration of sodium azide as the abscissa, make the standard curve y=3306.37x-20.11 (R ² =0.997) of sodium azide (see figure 1).

(3) 4-cyano-3-hydroxybutanol standard curve: prepare 0.3mM, 0.6mM, 0.9mM, 1.2mM, 1.5mM and 2.0mM 4-cyano-3-hydroxybutanol aqueous solution with distilled water, take 1ml of 4-cyano-3-hydroxybutanol aqueous solutions with different concentrations were added to different 2ml EP tubes, and then 1ml of ethyl acetate was added for extraction. Take 800 μl of the upper organic phase, dry it over anhydrous sodium sulfate, and perform gas phase detection (see Example 3). Taking the concentration of 4-cyano-3-hydroxybutanol as the abscissa and the peak area as the ordinate, the standard curve of 4-cyano-3-hydroxybutanol is y=1518.46x-67.52 (R ² =0.993) (See Figure 2).

(4) Sodium cyanide standard curve: Prepare 0.6mM, 1.2mM, 1.8mM, 2.4mM, 3.0mM and 4.0mM sodium cyanide aqueous solutions with distilled water, add 500μl sodium cyanide aqueous solutions of different concentrations to 2ml EP tubes, 450 μl of butylene oxide solution and 50 μl of halohydrin dehalogenase crude enzyme solution. Place in a Thermomixer reactor at 40°C, 500rpm, and react for 30min. Add 1ml of ethyl acetate to the EP tube for extraction, take 800 μl of the upper organic phase and dry it over anhydrous sodium sulfate, then perform gas phase detection of the peak area of 4-cyano-3-hydroxybutanol (see Example 3). Taking the sodium cyanide concentration as the abscissa and the 4-cyano-3-hydroxybutanol peak area as the ordinate, the standard curve of sodium cyanide is y=1371.50x-40.75 (R ² =0.995) (see Figure 2) .

As can be seen from Fig. 2, the linear relationship between the standard curve of sodium cyanide and the standard curve of 4-cyano-3-hydroxybutanol is similar, indicating that the standard curve of sodium cyanide established is accurate and feasible.

Embodiment 5: Determination of azide ion and cyanide ion by halohydrin dehalogenase

Randomly configure 10 mL of sodium azide solution or sodium cyanide solution of unknown concentration with distilled water as the azide ion sample or cyanide ion sample to be tested.

Determination scheme: Dilute the sample to be tested by 2 times, 5 times and 1 time with pure water respectively, add 500 μl of diluted sample solution, 450 μl of butylene oxide solution (prepared in Example 4) and 50 μl of haloalcohol dehalogenation to a 2ml EP tube Enzyme crude enzyme liquid (embodiment 1). Place in a Thermomixer reactor at 40°C, 500rpm, and react for 30min. Add 1ml of ethyl acetate to the EP tube for extraction, take the upper organic phase and dry it over anhydrous sodium sulfate for gas phase analysis. The gas phase peak area of the obtained 4-azido-3-hydroxybutanol or 4-cyano-3-hydroxybutanol is compared with the sodium cyanide standard curve or the sodium azide standard curve, and the selected peak area is in the corresponding standard curve. The dilution factor within the peak area range is used to obtain the concentration of azide ion or cyanide ion in the water sample to be tested, and the results are shown in Table 1.

Table 1 Determination of azide ion and cyanide ion concentration

^[a] The actual concentration is calculated based on the peak area diluted by 10, and the peak areas diluted by 5 times and 2 times all exceed the standard curve of sodium azide, and the calculation formula (1) is:

X _{[actual concentration]} = ((Y _{[peak area]} +20.11)/3306.37) × 2 _{[multiple of reaction dilution]} × 10 _{[multiple of sample dilution]}

In the formula (1), X _{[actual concentration]} is the actual concentration measured, Y _{[peak area]} is the peak area of 4-azido-3-hydroxybutanol, 2 _{[multiple of reaction dilution]} refers to 500ul water sample, and the final volume of reaction It is 1 mL, that is, it is diluted 2 times.

^[b] The actual concentration is calculated based on the peak area diluted 10, or the peak area diluted 5 times, and the peak area diluted 2 times exceeds the standard curve of sodium cyanide. Calculation formula (2) is:

X _{[actual concentration]} = ((Y _{[peak area]} +67.52)/1515.46) × 2 _{[multiple of reaction dilution]} × 10 _{[multiple of sample dilution]}

In the formula (2), X _{[actual concentration]} is the actual concentration measured, Y _{[peak area]} is the peak area of 4-cyano-3-hydroxybutanol, 2 _{[multiple of reaction dilution]} refers to 500ul water sample, and the final volume of reaction It is 1 mL, that is, it is diluted 2 times.

Example 6: Effects of metal ions and detergents on the activity of halohydrin dehalogenase

Considering that there will be some metal ions and detergents such as Tween 80 and Tween 20 in the sample to be detected, the present invention also investigates the influence of some metal ions and detergents on the catalyzed reaction of halohydrin dehalogenase. Distilled water was used to prepare a reagent solution (see Table 1) and a 50 mM 1,3-dichloro-2-propanol aqueous solution with distilled water, and a crude halohydrin dehalogenase solution (Example 1) was used to catalyze the reaction. Add 500μl reagent solution, 450μl 50mM 1,3-dichloro-2-propanol aqueous solution and 50μl halohydrin dehalogenase crude enzyme solution to 2ml EP tube. Place in a Thermomixer reactor at 40° C., 500 rpm, and react for 10 minutes. For control reactions, 500 μl of distilled water was used instead of the reagent solution. The relative activity under each condition was calculated using the production amount of the product epichlorohydrin. Enzyme activity is defined as the amount of enzyme required to catalyze the synthesis of 1 μmol of epichlorohydrin per minute under the reaction conditions of 40°C and pH 7.5, which is 1 enzyme activity unit.

As shown in Table 2, Fe ³⁺ , Ba ²⁺ , Al ³⁺ and Cu ²⁺ have a strong inhibitory effect on the activity of halohydrin dehalogenase, while EDTA has only a slight effect on the activity of halohydrin dehalogenase. Therefore, in the actual determination process, in order to ensure the high-efficiency halohydrin dehalogenase activity, EDTA reagent can be used to remove metal ions, so as to ensure the accuracy of the determination reaction. 1,3-dichloro-2-propanol and epichlorohydrin gas-phase detection with the gas-phase method of embodiment 2, the retention times of 1,3-dichloro-2-propanol and epichlorohydrin are respectively 4.04min and 6.41 min.

Table 2. Effects of metal ions and detergents on the catalytic activity of halohydrin dehalogenases

Reagent concentration Relative activity (%) control - 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: the impact of chloride ion, bromide ion, nitrite, thiocyanate and cyanate on detection

Considering that halohydrin dehalogenase can also catalyze the reaction of chloride ion, bromide ion, nitrite, thiocyanate and cyanate with epoxide, therefore, the present invention also investigates chloride ion, bromide ion, nitrite ion, The effect of thiocyanate and cyanate on the detection of azide and cyanide ions.

Prepare 20mM aqueous solutions of sodium chloride, sodium bromide, sodium nitrite, sodium thiocyanate and sodium cyanate as sample solutions, add 500 μl of the above-mentioned sample solutions, 450 μl of butylene oxide solution (200 mM, Example 4) and 50 μl of halohydrin dehalogenase crude enzyme solution (Example 1). Place in a Thermomixer reactor at 40°C, 500rpm, and react for 30min. Add 1ml of ethyl acetate to the EP tube for extraction, and take the upper organic phase and dry it over anhydrous sodium sulfate. Add a certain amount of 4-azido-3-hydroxybutanol or 4-cyano-3-hydroxybutanol to the organic phase (the amount of addition has no effect on this experiment, the main purpose is to determine the compound formed by other nucleophiles) Whether it affects the detection of compounds formed by azide ions or cyanide ions, the amount added in this example is about 500 μl of ethyl acetate and 1 μl of 4-azido-3-hydroxybutanol or 4-cyano-3-hydroxybutanol) , for gas phase analysis.

Observe whether the reaction products of sodium chloride, sodium bromide, sodium nitrite, sodium thiocyanate and sodium cyanate with butylene oxide are sensitive to 4-azido-3-hydroxybutanol and 4-cyano-3-hydroxybutanol Alcohol detection is affected. Among them, chloride ion and bromide ion have no effect on the detection of azide ion and cyanide ion (Fig. 3), thiocyanate ion has no effect on the detection of azide ion and cyanide ion (Fig. The detection of ions has no effect (Figure 5), and the nitrite ion does not affect the detection of azide ion but affects the detection of cyanide ion (Figure 6).

Claims (7)

1. A method for detecting azide ions or cyanide ions in water, characterized in that the method is: after the ultrasonic crushing of wet thallus obtained by induced culture with recombinant genetically engineered bacteria containing halohydrin dehalogenase coding gene The supernatant is used as a catalyst, epoxy is used as an auxiliary agent, and the water sample to be tested is used as a raw material, and the reaction is carried out at 40-45 °C and 500 rpm for 30-60 minutes. The reaction solution is extracted with ethyl acetate, and the upper organic phase is washed After drying over anhydrous sodium sulfate, use gas chromatography to detect the peak area of 4-azido-3-hydroxybutanol or 4-cyano-3-hydroxybutanol. Base-3-hydroxybutanol peak area is the ordinate, the sodium azide standard curve or sodium cyanide standard curve made with the concentration of sodium azide or sodium cyanide as the abscissa, to determine the azide radical in the water sample to be tested The concentration of ions or cyanide ions; the amount of the catalyst is 5 to 20 mg/mL of the water sample to be tested based on the weight of the recombinant genetically engineered bacteria containing the halohydrin dehalogenase coding gene before ultrasonic crushing. The dosage is 0.05-0.2mol/L water sample to be tested. 2. the method for detecting azide ion or cyanide ion in water as claimed in claim 1 is characterized in that the aminoacid sequence of described halohydrin dehalogenase is shown in SEQ ID NO.2. 3. detect the method for azide ion or cyanide ion in water as claimed in claim 1, it is characterized in that gas chromatography detection condition is: adopt Japan Shimadzu gas phase GC-14, chromatographic column ^{AstecCHIRALDEX} G-TA, carrier gas is helium Gas, the split ratio is 20:1, the temperature of the injection port and the detector are both 220°C, the GC program is 120°C for 5 minutes, 5°C/min to 140°C, and hold for 2 minutes. 4. detect the method for azide ion or cyanide ion in water as claimed in claim 1, it is characterized in that standard curve is prepared by the following method: prepare the sodium azide aqueous solution of 0.2～2.0mM with distilled water, respectively in 2ml EP tube Add 500 μl of sodium azide aqueous solution of different concentrations, 450 μl of butylene oxide solution and 50 μl of halohydrin dehalogenase crude enzyme solution, react at 40°C, 500 rpm for 30 minutes, add 1ml of ethyl acetate to the EP tube for extraction, Take 800 μl of the upper organic phase, dry it with anhydrous sodium sulfate, and detect the peak area of 4-azido-3-hydroxybutanol by gas phase, take the concentration of sodium azide as the abscissa, and take the The peak area of the alcohol is the ordinate, and the standard curve of sodium azide is obtained; the crude enzyme liquid of the halohydrin dehalogenase is the supernatant after the sonication of wet cells obtained by the fermentation of the halohydrin dehalogenase, and the The concentration of crude enzyme liquid is 0.1g/ml on the basis of wet thalline weight before sonication; Described butylene oxide solution is the 200mM butylene oxide solution prepared with the PBS damping fluid of 200mM, pH 7.5, sodium cyanide standard The curve was made with the sodium azide standard curve. 5. the method for detecting azide ion or cyanide ion in water as claimed in claim 1 is characterized in that epoxide is butylene oxide or octane oxide. 6. the method for detecting azide ion or cyanide ion in water as claimed in claim 1 is characterized in that the consumption of epoxide is 0.1mol/L water sample. 7. The method for detecting azide ions or cyanide ions in water as claimed in claim 1 is characterized in that the preparation method of the catalyst is: inoculating the recombinant genetically engineered bacteria containing the halohydrin dehalohydrin coding gene at a concentration of 50 μg/ In LB medium of ampicillin, place it in a shaker at 37°C and culture it until the _OD600 reaches 0.6-0.8, add a final concentration of 0.2mM isopropyl thiogalactoside, and induce it in a shaker at 28°C for 12-14 hour, 9000rpm centrifugation for 10 minutes, discard the supernatant, the wet thallus obtained by centrifugation was suspended in the phosphate buffer solution of 100mM according to the ratio of 0.1g/L, 50% power sonicated for 30min, and the broken mixture was centrifuged at 12000rpm for 20 minutes, The supernatant is collected to obtain a crude enzyme solution containing halohydrin dehalogenase, which is the catalyst.